Simultaneous detection of Cryptosporidium oocysts and Giardia cysts in environmental samples using the polymerase chain reaction

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Simultaneous detection of Cryptosporidium oocysts and Giardia cysts in environmental samples using the polymerase chain reaction

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Title:
Simultaneous detection of Cryptosporidium oocysts and Giardia cysts in environmental samples using the polymerase chain reaction
Creator:
Griffin, Dale Warren
Place of Publication:
Tampa, Florida
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University of South Florida
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English
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viii, 110 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Cryptosporidiosis -- Transmission ( lcsh )
Giardiasis -- Transmission ( lcsh )
Polymerase chain reaction ( lcsh )
Polymerase Chain Reaction ( mesh )
Giardiasis -- transmission ( mesh )
Cryptosporidiosis -- transmission ( mesh )
Dissertations, Academic -- Public Health -- Masters -- USF ( fts )

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General Note:
Thesis (M.S.P.H.)--University of South Florida, 1994. Includes bibliographical references (leaves 105-110).

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University of South Florida
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Universtity of South Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
021774773 ( ALEPH )
33349197 ( OCLC )
F51-00110 ( USFLDC DOI )
f51.110 ( USFLDC Handle )

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SIMULTANEOUS DETECTION OF CRYPTOSPORIDiu.M OOCYSTS AND GIARDIA CYSTS IN ENVIRONMENTAL SAMPLES USING THE POLYMERASE CHAIN REACTION by DALE WARREN GRIFFIN A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Environmental and Occupational Health University of South Florida December 1994 Major Professor: Joan B. Rose, Ph.D.

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Graduate School University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Master's Thesis This is to certify that the Master's Thesis of DALE WARREN GRIFFIN with a major in Environmental and Occupational Health has been approved by the Examining Committee on October 25, 1994 as satisfactory for the thesis requirement for the Master of Science in Public Health degree Examining Joan B. Rose, Ph.D. Member: My L. Dao , ,Ph.D._ Member: Boo H. Kwa, Ph.D.

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ACKNOWLEDGMENTS I would like to thank Dr. Joan B. Rose for the opportunity, support, and advise, which has made all of this and more possible. Thanks!!!!!!!!!!!!!!!!!! I would also like to acknowledge and thank Dr My L. Dao and Dr Boo H. Kwa for their patience and advice at various points of this research project. Thanks are also due to Dr. David W. Johnson for a competent introduction to molecular biology, and John Lisle for the advise and never ending supply of references and articles. Special thanks to my family for all of the support and help. Damn the Torpedoes

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TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT vi 1. INTRODUCTION 1 Giardia Lifecycle 3 Cryptosporidium Lifecycle 4 Methods for Detection of Cysts and Oocysts in Environmental Samples 5 Polymerase Chain Reaction 10 Surface Water Treatment Rule Guidelines 13 2. OBJECTIVE 15 3. MATERIAL AND METHODS 16 Preparation of Oocysts and Cysts for Seeded Trials 16 PCR Amplification Profile 17 Master Mix Recipes/Primers and Probes 18 Detection of PCR Product 21 Cryptosporidium Probe Protocol 22 Giardia Probe Protocol 23 Standard Cryptosporidium PCR Protocol for Detection of Oocysts in Environmental Samples 25 Preliminary Inhibition Studies 27 Chelex 100 Cryptosporidium PCR Protocol 32 Simultaneous Detection Protocol 35 Multiplex Protocol 35 Separate Master Mix Protocol 38 Magnetic-Antibody Capture Detection Protocol 42 4. RESULTS 48 Standard Cryptosporidium PCR Protocol/ Preliminary Inhibition Studies 49 Chelex 100 Cryptosporidium PCR Protocol 57 Simultaneous Detection Protocol 63 Multiplex Protocol 63 Separate Master Mix Protocol 72 Antibody Capture Detection Protocol 78 i

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5. DISCUSSION 6. CONCLUSION REFERENCES ii 86 102 105

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Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9 Table 10. Table 11. Table 12. LIST OF TABLES Currently Known Species of Cryptosporidium and Giardia Current Detection Methods Cryptosporidium PCR Profile Cryptosporidium and Giardia Primer and Probe Sequences Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition (PCR 141) Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition (PCR 143) Analysis of Three Purified Calf Oocysts Isolates using the Chelex 100 Cryptosporidium Protocol (PCR 196) Simultaneous Detection in Seeded Environmental Samples using Multiplex PCR (PCR 184) Simultaneous Detection in a Purified Sample using Multiplex PCR with the Giardian Gene Primer Set (PCR 217) Simultaneous Detection using Split Master Mix PCR (PCR 190) Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR (PCR 200) Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR (PCR 202) iii 2 7 18 20 50 53 58 67 70 76 79 83

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Figure 1. Figure 2. Figure 3. Figure 4. Figure 5 Figure 6 Figure 7 Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. LIST OF FIGURES Protocol Development Outline 17 Flow Chart of Standard Cryptosporidium PCR Detection Protocol 26 Flow Chart of Sample Pretreatment (PCR Inhibition Studies) Experiments 28 Flow Chart of the Modified Cryptosporidium PCR Detection Protocol 33 Flow Chart for Simultaneous Detection using Multiplex PCR 37 Flow Chart for Simultaneous Detection using Split Master Mix PCR 39 Flow Chart for Magnetic-Antibody Capture PCR 43 Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition (PCR 143, gel) 54 Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition (PCR 143, blo t ) 55 Analysis of Three Purified Calf Oocyst Isolates using the Chelex 100 Cryptosporidium Protocol (PCR 196, gel) 60 Analysis of Three Purified Calf Oocyst Isolates using the Chelex 100 Cryptosporidium Protocol ( PCR 196, blot) 61 Simultaneous Detection of Purified Oocysts and Cysts using Multiplex PCR (PCR 175, gel) 64 iv

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Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Simultaneous Detection of Purified Oocysts and Cysts using Multiplex PCR (PCR 205, gel) Simultaneous Detection in Seeded Environmental Samples using Multiplex PCR (PCR 184, blot) Simultaneous Detection in a Purified Sample using Multiplex PCR with the Giardian Gene Primer Set (PCR 217, blot) Simultaneous Detection using Split Master Mix PCR (PCR 180, gel) Dot Blot Hybridization with Giardia 163bp Probe of an Environmental Sample Seeded with Oocysts and Cysts (PCR 180, blot) Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR (PCR 200, gel) Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR (PCR 202, gel) Optimization of Master Mix Reagents based on Reagent Concentrations v 65 68 71 73 75 80 84 87

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SIMULTANEOUS DETECTION OF CRYPTOSPORIDiu.M OOCYSTS AND GIARDIA CYSTS IN ENVIRONMENTAL SAMPLES USING THE POLYMERASE CHAIN REACTION by DALE WARREN GRIFFIN An Abstract Of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Environmental and Occupational Health University of South Florida December 1994 Major Professor: Joan B. Rose, Ph.D. vi

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Giardia and Cryptosporidiumare two protozoa which cause acute gastroenteritis and waterborne disease worldwide. The primary focus of this research was to develop a method for simultaneous detection of both protozoa in environmental samples using the polymerase chain reaction (PCR) Two PCR assays were used to simultaneously detect both protozoa in seeded environmental samples. Two approaches for simultaneous detection were analyzed, these included the use of Multiplex PCR, and the use of two separate master mixes applied to a split sample. Simultaneous detection of both protozoa was observed in seeded environmental samples using a standard multiplex detection protocol, and in seeded environmental samples using a magnetic-antibody capture/multiplex protocol. The primary obstacle to successful protocol development has been the effect of environmental inhibitors on the PCR assays. Environmental inhibitors effect the sensitivity of the assay by interfering with the amplification reaction. This observed interference results in the increased probability of false negative results by prohibiting detection of small numbers of oocysts and cysts. Throughout this study, various methods to counter the effects of the inhibitors were analyzed. Of the methods analyzed to date, magnetic-antibody capture proved to be the most effective pretreatment method for countering sample based assay inhibition. vii

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The use of magnetic-antibody capture resulted in simultaneous detection of both protozoa in environmental samples that had previously inhibited all detection assays. Abstract Major Professor: Joan B Rose, Ph.D. Assistant Professor, Department of Environmental and Occupational Health Date viii

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1 1. INTRODUCTION Two Protozoa Giardia and Cryptosporidium, have been recognized as worldwide threats to drinking water supplies. Giardia lamblia is one of the agents most associated with outbreaks of waterborne disease in the United States (Herwaldt et al., 1992, Craun, 1990, Kent et al., 1988). Cryptosporidium parvum has recently been responsible for several large outbreaks of waterborne disease in the United States (Mackenzie et. al., 1994, Leland et al., 1993, Berger et al., 1992, Smith and Rose 1990, Hayes et al., 1989, McNabb et al., 1985) Both Protozoa cause severe gastroenteritis in healthy humans, and while the diseases are usually self-limiting, cryptosporidiosis can be fatal to the immunocompromised (Soave et. al., 1986). Symptomatic infections with either protozoa result in varying degrees of diarrhea, abdominal cramps, nausea, weight loss, vomiting, and foul smelling/greasy stools. The incubation period for cryptosporidiosis is approximately three to eight days (Katz et al. 1988) and no effective drug exists for treatment. The incubation period for giardiasis ranges from one week to forty-five days (Adam, 199 1 ) and there are a number of effective drugs for treatment such as quinacrine and metronidazole.

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2 Fecal-oral transmission is the route of infection with both protozoa, with water serving as the primary media for movement in the environment. Table 1, lists the currently known species of Cryptosporidium and Giardia. Both protozoa infect humans through zoonotic transmission from other mammalian hosts (Glicker and Edwards, 1991, Casemore, 1989, Rose, 1988) The species Cryptosporidium parvum and Giardia lamblia have been found in many mammals including calves, lambs, pigs, cats, dogs and beavers; therefore both cysts and oocysts originating from animal as well as human feces have been associated with waterborne disease. Current models of risk assessment assume a zero threshold on the numbers of ingested cysts or oocysts required for infection to occur, therefore as few as one cyst or oocyst can initiate infection. (Rose, 1993, Glicker, 1991, Regli et al., 1991, Casemore, 1989, Rose, 19 88) Table 1. Currently known species of Cryptosporidium and Giardia. (Warren, 1993, Adam 1991, Current et a l., 1991, Rose, 1988, Tyzzer, 1907) Cryptosporidium spp. Cryptosporidium baileyi Cryptosporidium meleagridis Cryptosporidium muris Cryptosporidium parvum* Cryptosporidium serpentis Giardia spp. Giardia agilis Giardia muris Giardia lamblia* Giardia ondatrae Primary species associated with human disease. The other species infect a variety of hosts, including snakes, frogs, birds. and a wide range of mammals.

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3 Giardia Lifecycle Giardia lamblia is in the phylum Sarcomastigophora, order Mastigophora, class Zoomastigophora, and order Diplomonadida. Species specificity is thought to be low as several species of Giardia have demonstrated the ability to cross-infect several different host types (Adam, 1991) The infectious stage in the life cycle of Giardia lamblia is the cyst which is 8-10 urn in diameter. The dense cyst wall allows Giardia to remain viable in the environment for extended periods of time (Erlandsen et al., 1988, LeChevallier et al. 1991, LeChevallier et al., 1991, Rose et al., 1991). The cyst is also more resistant to chlorine inactivation than bacteria, which presents a significant problem to water treatment plants relying on chlorine treatment for microbial control (Craun, 1990) The cyst wall is 0.3 urn thick (Sheffield and Bjorvatn, 1977) and is composed of three layers. The outer portion of the cyst membrane is composed of proteins and sugars (Adams, 1991) and it is this cyst-wall matrix that allows Giardia to survive in the open environment and in the presence of disinfectants. Upon ingestion of the cyst, the cyst wall is degraded in the stomach. Trophozoites are then released from the ingested cyst, a process known as excystation. The trophozoites then attach to the duodenum and jejunum of the upper intestine via a sucking disk and undergo binary fission. After fission the two daughter cells then mature into two trophozoites. The

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4 trophozoites are thought to feed on intestinal contents rather than on, or through the hosts intestinal cell wall (Warren, 1993). Once the trophozoite moves or is washed into the lower intestine, it encysts and undergoes binary fission twice which produces the four nucleated cyst. The cyst is then passed into the environment in the feces. Cryptosporidium Lifecycle Cryptosporidium parvum is in the phylum Apicomplexa, subclass Coccidiasina, order Eucoccidiorida, suborder Eimeriorina, and family Eimeriidae. The infectious stage of Cryptosporidium parvum is the oocyst which is 4-6 urn in diameter and is more resistant to disinfectants such as chlorine and iodine than the Giardia cyst (Korich et. al., 1990, Peeters et. al., 1989). The oocyst wall is composed of two layers, a thick inner layer which contains a suture line that aides in sporozoite escape, and a thin outer layer (Reducker, 1985). These two layers protect the sporozoites from disinfectants used in drinking water, which in turn allows the transmission of infection through treated waters (Rob ertson et al., 1992, LeChevallier et al., 1991, Rose et al., 1991, Madore et al., 1987). The oocyst usually contains four sporozoites which are released upon ingestion, but morphological studies have shown that an oocyst may contain anywhere from zero to four sporozoites.

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5 The sporozoites, once released then infect the epithelial cells of the upper intestine. The infection is intracellular but extracytoplasmic (pseudo-external like position, Casemore, 1989), with the sporozoites maturing into trophozoites within the infected cell. The trophozoites then undergo asexual reproduction producing two different types of merozoites (Type I and Type II meronts). Type I meronts infect other epithelial cells, and ultimately give rise to other type I meronts or type II meronts. Type II meronts produce microgametes and macrogametes which produce oocysts via sexual reproduction (Warren, 1993, Rose, 1988). The oocyst is then shed into the environment through feces deposition. Methods for Detection of Cysts and Oocysts in Environmental Samples Methods for recovery of cysts and oocysts from water include collection of large volumes of water by cartridge filtration, recovery of the organisms off the filter by washing, and density gradient centrifugation (Lechevallier et al. 1991, Rose et al., 1991). The resulting pellet is then examined to determine the presence or absence of oocysts and cysts. Several methods for recovering and identifying Giardia cysts in environmental samples have been developed.

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6 Concentration and purification techniques exist such as the use of filter cartridges for concentration, and sucrose density gradient centrifugation for purification (RobertsThompson, 1979) Identification methods which are currently in use are direct microscopic examination, immunofluorescence assay (IFA) with epifluorescence microscopy, PCR, and eDNA probes (Warren, 1993, Atlas et al., 1992, Abbaszadegan et al., 1991, Mahbubani et al., 1991, Rose et al., 1989,). Identification techniques used to identify Cryptosporidium include the Ziehl-Neelsen acid-fast technique, IFA, eDNA probes, and PCR (Johnson et al., 1993, Laxer et al., 1991, Rose et al., 1989, Johnson et al., In Press,). When applied to environmental samples these methods have noted limitations. Of primary concern are loss of cysts during concentration techniques and overall efficiency with the detection methods. Table 2, lists the different detection methods, along with the advantages and disadvantages of each methodology. IFA (using monoclonal antibodies) is currently recognized as the most reliable method for detection of oocysts and cysts in environmental samples. In this case samples after clarification with density gradients are passed through membrane filters. The samples are stained with monoclonal antibodies tagged with FITC. Usually an indirect IFA is used, where by a secondary antibody caries the FITC label. The membrane is then scanned and cysts and oocyst are identified

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Table 2. Current Detection Methods. Direct microscopic examination Advantages -The method is rapid and requires no reagents. Enumeration is possible. 7 Disadvantages -Extensive training is needed to recognize the organism of interest and the organisms are easily masked by debris. The overall efficiency of the method is low due the effect of masking. Cannot determine viability. Stains Advantages -The method is rapid and few reagents are needed. Viability may be possible, and enumeration is possible. Disadvantages -Not all cells stain the same, which requires extensive training to ensure accurate counts. Debris can mask cells. Efficiency is low and viability studies are questionable. Advantages Species can be identified, and the organism stands out against background debris. Enumeration is possible. Disadvantages -Extensive training is needed to ensure accurate counts. Time is needed for staining and counting (6-Shrs). Autoflouresence can interfere with counts and debris can mask stained organisms. Efficiency of the overall assay is fair to poor. Cannot determine viability. eDNA Probes Advantages -Molecular probes can be designed species specific. Training requirements are minimal. Disadvantages Low sensitivity with low target numbers. Cannot determine viability. Time is required for hybridization (> 12hrs). Non-specific binding due to low stringency conditions can result in false positives/low efficiency. Advantages -Excellent sensitivity and specificity. Can be species specific. Can be designed to determine viability. Training requirements are minimal. Method is quick (< 6hrs). Disadvantages -Enumeration is not yet reliable. Environmental samples can inhibit the reaction. Requires time to set up experimental protocol if not already established.

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8 using epiflourescence microscopy. Fluorescence, size, and shape are criteria for identification. The membranes may be cleared with a series of ethanol washes and phase or differential interference contrast microscopy is used to identify internal features within the cysts or oocysts (LeChevallier et al., 1991). One of the advantages of the test is that the monoclonal antibodies currently used are available in a kit form (Hydroflour Combo, catalog # 240025, Meridian Diagnostics, Inc. Cincinnati, Ohio). Both cysts and oocysts can be detected and enumerated simultaneously. The limitation of this method is its efficiency in detection when examining environmental samples. Both false positives and false negative counts can occur due to dirty samples (Johnson et al., 1993) age of sample (up to 80% underestimation in regard to oocysts numbers, Rose et al., 1989), and specificity of the antibodies used. Other areas effecting the efficiency of the assay include sample concentration methods, and technician counting errors due to the lack of microscopy experience. Loss of oocysts or cysts has been demonstrated with sucrose gradient centrifugation (this becomes extremely important since the threshold for infectivity is low, and loss occurs in samples which contain relatively small number of cysts or oocysts. Johnson et. al., In Press) In addition to loss during floatation, loss can also occur during sample concentration. Loss at this step can occur due to the flow rate through the cartridge filter

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9 (sheering and destruction of both cysts and oocysts, or possible passage of cysts or oocysts through the filter), and failure to elute the captured organisms from the filter. The antibody used (monoclonals or polyclonals) can effect efficiency through low sensitivity or specificity. Sample autofluorescence or fluorescence from the use of antibodies of questionable specificity can also interfere with epifluorescent microscopy counts (Rose et al., 1991, Weber et al., 1991, Rose et al. 1989, Sterling and Arrowood, 1986). It is a common occurrence during counting, to observe an applegreen background and to see other cell types other than the target fluorescing with intensity. Fluorescing algae cells the size of oocysts, have presented a particular problem to microscopists. Fluorescing environmental debris can also mask both cysts and oocysts resulting in inaccurate counts. The other methods as listed in Table 2 (Page 7), all have advantages and disadvantages, and currently no lab exclusively utilizes these methods for monitoring environmental samples for the presence of either protozoa. Debris masking severely limits the use of direct examination, stains, and as previously mentioned effects the efficiency of IFA. eDNA probes while highly specific, demonstrated low sensitivity when cell counts were low (Johnson et al., 1992). The use of the PCR, although susceptible to inhibition, offers the possibility of development of a highly sensitive and specific assay, once the problem of inhibition is addressed.

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10 Polymerase Chain Reaction PCR is an experimental methodology that allows amplification of DNA sequences (Saiki et al., 1988, Erlich et al. 1988, Saiki et al., 1986, Mullis et al., 1986, Saiki et al., 1985). DNA is amplified through the use of a set of primers (short single strand sequences of DNA) that are complimentary and specific to a segment of nucleic acid sequences in the target genome. The primers (both located 5' -3' to the area of interest, with one primer on each initial complimentary strand) anneal to their target, and the presence of DNA polymerase and free nucleotides (along with the other master mix components) allows synthesis and amplification of the sequence of interest (Mullis et al. 1986). The use of the PCR results in exponential amplification of the sequence of DNA of interest, through a cyclic amplification profile. This technology allows the study and detection of any cell type containing DNA, and due to the amplification of the target DNA sequence, allows the study of as little as one cell. PCR has been used to detect Giardia cysts and Cryptosporidium oocysts in both purified and environmental sample s (Jonhson et al., 1993, Atlas et al., 1992, Laxer et al., 1991, Mahbubani et al., 1991, Johnson et al. In Press). The problems associated with the use of the PCR for screening environmental samples has been primer set sensitivity/ specificity and the presence of sample based inhibitors. Low

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11 primer set specificity can result in false positive results and low primer set sensitivity can result in false negative results. Sample preparation is also susceptible to contamination which is another source of false positive results (Kwok and Higuchi, 1989). Other concerns with PCR include the low volume of sample that is screened (usually limited to 100 ul), the inability to determine viability with primer sets designed to directly amplify gene segments, and the fact that the assay is not yet quantitative. The primary obstacle to the use of PCR assay for monitoring environmental samples has been the effect of sample based inhibitors on the reaction (Andersen and Omiecinski, 1992, Walsh et al., 1991). Inhibitors have been shown to be associated with the pellet fragment of the samples (Johnson et al., In Press). Several compounds such as humic acids and divalent cations have been identified as inhibitors of the PCR, but the exact identification of different particulates from sample type to sample type (soil, water, and sludge) that interfere with the reaction, have not been distinguished. Both formalin and dichromate, which are two preservatives used for storage of oocysts and cysts, have also demonstrated the ability to inhibit the reaction (Johnson et al., In Press). Of the two compounds, formalin inhibition is greater than dichromate. Different protocols to deal with the problem of inhibition have been developed. These include protocols that

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12 utilize sample pretreatments such as the use of spin columns, Chelex 100, Sephadex G-200 and G-50 columns, EDTA, sucrose gradient floatation, and PEG (polyethylene glycol), Polyvinylpyrrolidone-agarose gel electrophoresis, cell versus DNA extraction, genetic probing, and lysate protocols (Herrick et. al. 1993, Johnson et. al., 1993, Young et. al., 1993, Bruce et. al., 1992, Tsai et. al., 1992, Tsai et. al., 1992, Porteous et. al., 1991, Tsai et. al., 1991, Steffan et. al., 1988, Steffan et. al., 1988). While some of these methods have demonstrated the ability to lower the level of inhibition in certain samples, no one method has shown the ability to work every time with a broad range of sample types. Many of these methods are also subject to loss of target organism and therefore are limited in their use. Magnetic-antibody capture has recently been utilized as a means to capture an organism of particular interest from a variety of environmental and clinical sample types. Antibodies, due to thier high specificity for the antigens that they are raised against, can be employed to selectivily bind suface antigens of particular cell types. If the antibodies are tagged with an iron particle and are exposed to a magnetic field, then the tagged antibodies along with the cells that they are bound to, will migrate toward the magnetic. Cell types to include Hela cells and Giardia cysts were purified with magnetic antibody capture and analyzed by methods such as PCR and IFA (Bifulco and Schaefer, 1993,

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13 Gribben et al., 1992, Padmanabhan et al., 1989, Padmanabhan et al., 1988). This sample pretreatment allows the researcher to both purify and concentrate a sample, and thus circumnavigate the problem of assay inhibition. Two different methods for magnetic-antibody capture can be utilized, direct and indirect capture. Direct magnetic-antibody capture utilizes a target specific antibody tagged with an iron particle, and indirect utilizes a secondary antibody tagged with an iron particle which is targeted against a primary antibody. In both cases the antibodies once bound to their target, allow isolation of the target cell type. Surface Water Treatment Rule Guidelines The United States Environmental Protection through the Surface Water Treatment Rule (SWTR), sets filtration and disinfection standards Agency, currently for the removal/inactivation of Giardia sp. and viruses from surface source waters (Federal Register, 1989). Minimum removal/inactivation is currently set a 3 logs (99.9%) when source water cyst concentrations are less than 1 per 100 liters of water, with an increase to 5 logs when cyst concentrations reach 10 to 100 per 100 liters in the source water. Standards for the removal of Cryptosporidium sp. in surface waters are expected in the near future through development of the Enhanced SWTR.

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14 Monitoring guidelines for both Giardia sp. and Cryptosporidium sp. do not exist. Implementation of monitoring protocols is expected, and will be based on the p roposed Information Collection Rule (ICR, Federal Register, 1994) The ICR will require water utilities to monitor their source and potentially their finished water for cysts and oocysts, to establish treatment guidelines and improve public health protection. Monitoring as outlined by the ICR will be based on the utilization of the IFA method. As previously mentioned, the efficiency of the overall IFA protocol is questionable. Due to the short comings of IFA, improvements in the existing methodology, or the development of new techniques are needed for the detection of both protozoa which i s more rapid, s ensitive, and specific than the IFA procedure.

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15 2. OBJECTIVE This research addressed the development of a protocol for simultaneous detection of both Cryptosporidium oocysts and Giardia cysts in environmental samples by PCR assay, and development of a pretreatment that allowed detection in samples that had previously demonstrated inhibition of the PCR assay. Prior to this research no protocol existed for simultaneous detection of both protozoa in environmental samples using a PCR assay. The objectives of this research were twofold as follows. 1. To develop a rapid, sensitive, and specific method to simultaneously detect Giardia cysts and Cryptosporidium oocysts in environmental samples using a PCR assay. 2. To address the problem of assay inhibition by analyzing various pretreatment methodologies.

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1.6 3 MATERIALS AND METHODS Figure 1, represents the experimental protocol development of this research. Starting with a protocol used for Cryptosporidium oocyst detection (Johnson et al. In Press), modifications were made and tested for sensitivity and simultaneous detection of cysts and oocysts, and to limit the effects of environmental inhibitors. Preparation of Oocysts and Cysts for Seeded Trials Cryptosporidium parvum oocysts from calf feces, which were used in seeded experiments were obtained from Alexon, Inc. (Mountain View, California). Oocysts were also obtained from calves at the University of Arizona. Generally oocysts were received as purified or semi-purified stocks via sieving and percol density gradient centrifugation. The stock concentrations were in the range of 1 x 1.07 oocysts per ml, and these numbers were verified by the IFA procedure (LeChevallier et al., 1991, Rose et al., 1991). The Giardia lamblia cysts which were used in seeded experiments were obtained from Swabby Gerbec Inc. (Fort Collins, Colorado) and

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17 were concentrated from gerbil feces. The final cyst concentration was 4 x 104 cysts per ml. Both oocysts and cysts were suspended in 2 5% dichromate and stored at 4 degrees celsius. Prior to use, the dichromate which inhibits PCR assay was removed by centrifugation washing. Standard Cryptosporidium PCR Protocol ( 6 experiments) Preliminary Inhibition Studies (10 experiments) Modified Cryptosporidium PCR Protocol Chelex 100 Pretreatment (23 experiments) Simultaneous Detection Protocol Multiplex and Separate Master Mix Protocols (17 experiments) Antibody Capture Detection Protocol (6 experiments) Figure 1. Protocol Development Outline. PCR Amplification Profile The PCR amplification profile (Table 3) used for all the listed experiments, was developed for the detection of Cryptosporidium sp. (Johnson et al., In Press) Extensive

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18 testing had shown that this produced a highly specific signal with sensitivity of 1 to 10 purified oocysts with the current set of Cryptosporidium primers, and therefore the Giardia Table 3. Cryptosporidium PCR Profile. Profile 1 cycle of the following steps 80 for 5 minutes 98 for 30 seconds 39 cycles of the following steps 55 for 30 seconds 72 for 1 minute 94 for 30 seconds 1 cycle of the following steps 55 for 30 seconds 72 for 10 minutes 4 -Hold primer set (s) were incorporated into protocols using this profile. This was evaluated during the simultaneous detection experiments. Tempcyclers used included the Model 60 Tempcycler (Coy -Lab. Products Inc., Ann Arbor, MI) in early experiments, and the Model 480 Tempcycler (Perkin Elmer Corp., Norwalk, CT) in later experiments. Master Mix Recipes/Primers and Probes The master mix basic reagent concentrations remained the same throughout the following protocols, and varied only with

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19 the addition of the Giardia primer set(s) when simultaneous detection studies were initiated. Due to the incorporation of the Giardia primer set(s) using two different master mix protocols, all master mix recipes are included in each protocol section. Table 4, lists the Cryptosporidium and Giardia primer and probe (sequences) sets used in the experiments. The Cryptosporidium oligonucleotide primer set and probe sequences were obtained from Norman Pieniazek of the Center for Disease Control, Atlanta, Georgia (Johnson et. al. In Press). The Cryptosporidium primer target for amplification was a 435 base region of the 18s rRNA gene. The primer set was specific for all species of Cryptosporidium tested to date (C. baileyi, c. muris, and C. parvum). The first Giardia oligonucleotide primer set and probe sequences were obtained from Morteza Abbaszadegan of the American Water Works Service Company, Inc., Belleville, Illinois. The target for amplification for this set of Giardia primers was a 163 base region of the HSP 70 gene (Heat Shock Protein 70). This primer set was designed to be specific for Giardia lamblia and produced a 163 base PCR product. The second set of Giardia primer sequences used were obtained from D. A. Baker (Baker et al., 1988). This primer set amplified region of the giardian gene and produced a 171 base PCR product. The primer set was specific for Giardia sp .. All of Giardia and Cryptosporidium probes were biotinylated and designed to hybridize to an internal segment

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20 of the amplified products mentioned. All primer sets and biotinylated probes were synthesized at the University of Florida DNA Synthesis Laboratory, Gainesville, FL. Table 4. Cryptosporidium and Giardia Primer and probe sequences. Cryptosporidium primer and probe sequences (Pieniazek) 21 base forward primer 5'-AAGCTCGTAGTTGGATTTCTG-3' 2 1 base reverse primer 5'-TAAGGTGCTGAAGGAGTAAGG-3' 38 base biotinylated probe 5'-GGGGATCGAAGACGATCAGATA -CCGTCGTAGTCTTAAC-3' Primers are designated DJ 3 (early experiments) or JR 3 for the forward primer, and DJ 4 (early experiments), or JR 4 for the reverse primer. These designations are located in the master mix recipes in the materials and methods section. Giardia primer and probe sequences (Abbaszadegan, HSP 70 gene) 20 base forward primer 5'-GTATCTGTGACCCGTCCGAG-3' 21 base reverse primer 5'AGGGCTCCGGCATAACTTTCC-3' 17 base biotinylated probe 5'-CTTTCCAGTCAGCTTTG-3' Primers are designated JR 6 for the forward primer, and JR 7 for the reverse primer in the material and methods section. Giardia primer and probe sequences (Baker, giardian gene) 20 base primer 5'-AAGTGCGTCAACGAGCAGCT-3' 21 base primer 5'-TTAGTGCTTTGTGACCATCGA-3' 26 base biotinylated probe 5'-TCGAGGACGTCGTCTCGAAGATCCAG-3' Primers are designated JR 10 for the 20 base primer, and JR 11 for the 21 base primer.

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21 Detection of PCR Product Ten ul of PCR product was analyzed by gel electrophoresis and in some cases, by dot blot hybridization. TBE gels (1M Tris base, 1M Boric acid, 20mM EDTA-Na(2)-2H(2)0, 1.5% to 2.0% agarose) stained with ethidium bromide (0.5 ug per ml) were loaded with PCR product and electrophoresed (Power supply, model E-C 105, and minigel rig, E-C Apparatus Corp., St. Petersburg, Florida) at approximately 90 volts for 1h in 1X TBE running buffer. Results were visualized over an ultraviolet transilluminator (Model FBTIV-614, Fisher Scientific, Pittsburgh, Pennsylvania). A dot blot apparatus was also used to probe the PCR products for verification of product identification. The cited Cryptosporidiu.m and Giardia biotinylated oligonucleotide probes were utilized for the appropriate blots. All probes were biotinylated and the detection protocol used was the Southern Light Chemiluminescence Detection System(Tropix,Inc., Bedford, MA). Both nitrocellulose (used in early experiments) and positively charged nylon membranes (used in later experiments, after obtaining a ultra-violet crosslinker) were used for the blots, and each blot was run in duplicate (One blot served as a negative control which was tested without probing, to account for the possible presence of environmental sample based biotin. Without a negative control, environmental sample based biotin could produce signals which would result in falsepositive results)

PAGE 33

22 Cryptosporidium Probe Protocol Thirty ul from each PCR product sample was transferred to 1.5 ml microcentrifuge tubes. The samples were placed in a tube float rack and incubated in a boiling water bath for 10 minutes. After the boiling step, the samples were placed on ice for 3 minutes. The dot blot apparatus (BIO-RAD, model # BIO-DOT) was then set up using a MagnaGraph nylon transfer membrane (MSI, Westboro, MA). The vacuum pressure was adjusted so that the vacuum would pull one drop of buffer (Approximately 20 ul) through the membrane in approximately 8 seconds. Two-hundred ul of 2X SSPE (100 mL of 20X SSPE = 17.5 g of NaCl, 2.76 g of NaH(2)P0(4) .H(2)0, and 0.74 g of EDTA, plus H(2)0 to a 100 ml volume at a pH of 7.4) was added to each well and the vacuum was allowed to empty the wells. Ten ul of each sample was added to separate blot wells, which was repeated twice (one blot served as the negative control which was not probed but processed through the protocol) After the samples were loaded, 400 ul of 2X SSPE was added to each well. The vacuum was allowed to clear each well. The apparatus was then taken apart, and the nylon membrane was dried by placing it in an oven at 80 for 10 min. The DNA was then crosslinked to the membrane by using the optimal crosslink program in the Ultraviolet crosslinker (Fisher Scientific model # FBUVXL-1000, Pittsburgh, PA) One-hundred ml of prehybridization buffer (6X SSPE, 0.3% SDS, 1.0% NFDM) was then prepared. The

PAGE 34

23 filters (negative control and the filter to be probed) were then placed in separate bags and 20 ml of prehybridization buffer was added to each. The filters were then incubated at 65 for 1h. The prehybridization buffer was removed from each bag and replaced with 10 ml of fresh buffer. To the filter to be probed 2 ul of the Cryptosporidium biotinylated oligonucleotide probe (approximately 60 ng of probe) was added, and then both filters were incubated overnight at 37. After hybridization, the filters were washed x4 (stringency washes) in 4X SSPE at 65. The Tropix (Tropix, Inc., SouthernLight Chemiluminescent Detection System) CSPD protocol was followed for the chemiluminescent processing of the filter. The blots were visualized by exposing Kodak XAR imaging film (Kodak, Rochester, NY) to the membranes from 10 min to 1h. Giardia Probe Protocol The protocol for the Giardia probes was similar to the Cryptosporidium protocol with the exception of the prehybridization solution, hybridization solution, stringency wash solution, hybridization temperature, and stringency wash temperatures (these solutions and temperatures followed the Tropix CSPD protocol). The hybridization temperature, and the stringency wash temperature for the HSP 70 probe, were both room temperature (room temperature was used as calculated

PAGE 35

24 melting point temperatures (Tm), and temperatures significantly below the Tm failed to produce signal) The giardian gene probe was hybridized and washed at 37 and produced excellent signals. The Tropix CSPD protocol for oligonucleotide probes was used to include the temperature and solution used for prehybridization, which was one hour at 55, in hybridization buffer (1mM EDTA, 7% SDS, 0.25 M Disodium Phosphate, pH 7.2), overnight hybridization in hybridization buffer (10 ml, at the mentioned temperatures) with approximately 60 ng of probe, and stringency washes of 2X sse (25X SSC, pH 7.0 = 3.75 M Sodium Chloride, o.375 M Sodium Citrate dihydrate) 1. 0% SDS (Sodium Dodecyl Sulfate) (2x5 minutes at room temperature), 1X sse, 1.0% SDS (2x15 minutes at hybridization temperature) and 1X sse (2x5 min at room temperature) The filters were then processed through the Tropix Chemiluminescent detection protocol for biotinylated DNA, which was the same protocol used for the Cryptosporidium probe.

PAGE 36

25 standard Cryptosporidium PCR Protocol for Detection of Oocysts in. Environmental Samples The following protocol was the preliminary PCR protocol developed for the detection of Cryptosporidium sp. and is illustrated in Figure 2 (Johnson et al. In Press). One ml of environmental sample was placed in a 1.5 ml microcentrifuge tube. To remove the Potassium Dichromate (sample preservative), the sample was washed by centrifugation (12,500 rpm, for 2 min) x4 in lX PCR Buffer (lOX PCR buffer= 10 mM Tris, 50 mM KCL, pH 8 .3). The pellet was then resuspend to 1 ml in lX PCR buffer upon completion of the washes. Onehundred ul of the sample was then transferred to another 1.5 ml tube, and the remaining 900 ul of sample was saved for IFA verification counts, or discarded if not needed. Ten-fold d ilutions were made with 10 ul of sample being placed into 90 ul of lX PCR buffer. The sample and dilutions were then cycled through a freeze/thaw process x6 to extract the DNA (frozen in a crushed dry ice/ethanol bath, and thawed in a boiling water bath) After the freeze/thaw procedure, the samples were centrifuged for two minutes at 12,500 rpm, and 50 ul of the samples were transferred to separate PCR tubes. Positive and negative controls were then prepared in PCR tubes at a volume of 50 ul. The positive control for Cryptosporidium detection was a plasmid containing the PCR target gene sequence, called Al. Ten ul of a dilution of the

PAGE 37

Do dilutions to 1 0( -4) Transfer 50 uL of supernatant to PCR tubes l l =g Load PCR tubes in tempcycler and run profile Wash 1 mL of sample and wash the sample x4 to remove preservative Do freeze/thaw procedure on samples Add master mix to samples L Analyze with gel electrophoresis and dot blot 26 Figure 2. Flow Chart of Standard Cryptosporidium PCR Detection Protocol.

PAGE 38

27 plasmid concentrate was added to 40 ul of 1X PCR buffer. The negative control was 50 ul of 1X PCR buffer. The master mix was then prepared on ice. The master mix volume was calculated at 50 ul per reaction with an additional 50 ul to compensate for pipette loss (pipettes are designed to extract and retain a small volume over what is actually being dispensed) This additional 50 ul was usually increased to 100 ul when the sample number exceeded 20 samples. At 50 ul per reaction the total reaction volume was 100 ul per sample. The master mix reagent concentrations used were; 150 uM dNTPs (Perkin Elmer Corp., Norwalk, CT) 200-800 nM primers, 2. 5-3.5 roM magnesium chloride (Sigma Chemical company, St. Louis, MO), lX PCR buffer, 2 units of TAQ polymerase per reaction (Perkin Elmer Corp., Norwalk CT), and HPLC H20. Fifty ul of master mix was added to each sample. Three drops of autoclaved mineral oil was added to each sample and the tubes were capped. The tubes were then loaded in the Model 60 tempcycler and the reaction profile was started. Preliminary Inhibition Studies The following protocol was used to analyze the effect of various pretreatments on environmental samples for reversal of inhibition (Figure 3).

PAGE 39

28 Oocysts Environmental for U U Lsample seed U 1 ml of seeded env i ronmental sample Oocysts plus PCR buffer Purified oocysts untreated Split sample into four 1 00 ul subsamples l sample to remove preservative 1;1 1;J l Process through preteatments v Seeded Seeded sample sample+ untreated tAN A l Process through freeze/thaw l--Set up and run PCR l Seeded sample + 20% Chelex Do dilutions to 1 0(-4) Seeded sample + enhanced !5% Chalex Gel and dot blot --"""' analysis Figure 3 Flow Chart of Sample Pretreatment (PCR Inhibition Studies) Experiments.

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29 Five 1 ml aliquots of Cryptosporidium oocysts at concentrations of 106 oocysts per ml, were placed into separate 1. 5 ml microcentrifuge tubes. Two separate 1 ml samples of an environmental pellet (sample LA39K) were also placed into 1.5 ml microcentrifuge tubes. LA39K was a surface water sample which was taken from Los Angeles, CA, filter concentrated and stored in dichromate at 2. 5%. This sample was used because previous studies had shown PCR inhibition of as many as 10,000 oocysts. All samples were pelleted and washed x4 in 1 ml of 1X PCR buffer which contained 2.5 mM magnesium. Each of the five tubes containing the oocysts were resuspended to 0.5 ml and then mixed together in one tube (= 2.5 ml of washed oocysts, to ensure even distribution of oocysts when preparing the seeded samples) Each of the two tubes containing the LA39K pellets, were resuspended to 1 ml in 1X PCR buffer which contained 2.5 mM magnesium. The five seeded samples were then prepared for the various pretreatments (each designated by a letter) a. 0.5 ml of oocysts + 0.5 ml of 1X PCR buffer w/2.5 mM Mg. (used as a purified sample control) b. 0.5 ml of oocysts + 0.5 ml of LA39K (no pretreatment) c. 0.5 ml of oocysts + 0.5 ml of LA39K (for tRNA pretreatment) d. 0.5 ml of oocysts + 0.5 ml of LA39K (for 20% Chelex pretreatment) e. 0.5 ml of oocysts + 0.5 ml of LA39K (for enhanced Chelex pretreatment)

PAGE 41

30 For pretreatment c 10 ul of 100 ug per ml of yeast tRNA was added to the sample prior to dilutions. Sample c was then vortexed for 10 seconds and then incubated at room temperature for 10 min. Samples a, b, c, and d, were diluted ten-fold to 10-4 (100 ul into 900 ul of 1X PCR buffer w/2.5 mM Mg). To each tube and series of dilutions for pretreatment d, 200 ul of 20% Chelex 100 (Bio-Rad Laboratories, Richmond, CA) was added. The samples a, b, c, and d, were then processed through freeze/thaw x6. Dilution sets a, b, and c, were then placed on dry ice. Sample dilution set d, was boiled an additional 30 minutes after freeze/thaw, and after boiling was centrifuged at 12 x 1,000 g for 2 min. Seventy-five ul of supernatant from each dilution was then transferred to another set of 1.5 ml tubes. The centrifugation was then repeated and 50 ul of supernatant from each dilution was then transferred to a set of PCR tubes. These tubes were stored on dry ice, until prior to running the PCR profile. Sample e was used for the enhanced Chelex 100 procedure (Protocol per Norman Pieniazek, CDC) The seeded pellet was split into two 0.5 ml aliquots (this was completed because maximum capacity of each microcentrifuge tube is approximately 1.8 ml of sample). To each aliquot, 0.5 ml of 5% Chelex 100 was added at room temperature (total volume of each microcentrifuge tube thus equaled 1 ml) The samples were then slowly vortexed for 5 to 10 seconds. The two tubes were then capped and pierced with a sharp object (to allow venting of

PAGE 42

31 expanding air during the following heating process), and then incubated for 1h at 56 while being slowly vortexed every 10 min. After this step the samples were heated to 95 to 100 for 10 min. The samples (samples e) were then cooled to room temperature and vortexed to bring down the condensation. The samples (samples e) were centrifuged for 5 min at 12,500 rpm, and 750 ul of supernatant from each tube was transferred to separate 1.5 ml microcentrifuge tubes. This step was repeated (with centrifuging for only 2 min) and 500 ul of supernatant from each tube was combined into one new 1. 5 ml microcentrifuge tube. Dilutions to 10-4 were made as previously described. Fifty ul of supernatant from each of these dilutions was then transferred to PCR tubes. The frozen samples stored on dry ice were thawed at room temperature. After thawing sample dilution sets a, b, and c, were centrifuged at 12,500 rpm for 2 min. Fifty ul of supernatant from each of these tubes was then transferred to PCR tubes. The A1 plasmid used for the Cryptosporidium positive control was diluted to 10-7 10-8 and 10-9 Ten ul from each one of the dilutions was used for positive controls (each was combined with 40 ul of 1X PCR buffer w/2.5% mM Mg in separate tubes) A negative control consisting of 1X PCR buffer was also used. The master mix was prepared for 31 reactions (25 test samples and 4 controls). Thirty-one reactions x 50 ul per sample = 1550 ul of master mix.

PAGE 43

32 lOX PCR buffer 155 ul 100 mM Mg 38.7 ul DJ 5 155 ul Crypto. forward primer DJ 6 155 ul Crypto. reverse primer dNTPs 186 ul TAQ 9.3 ul HPLC H(2)0 851 ul Fifty ul of master mix was added to each sample tube, followed by the addition of three drops of autoclaved mineral oil. The tubes were then loaded into the Model 480 tempcycler and the amplification profile was started. All results were visualized by gel electrophoresis and dot blot. Chelex 100 Cryptosporidium PCR Protocol The use of Chelex 100 was adopted after the preliminary inhibition experiments, and was used in 23 experiments to analyze both purified and seeded environmental samples (Figure 4). This set of experiments was designed to examine detection of results by electrophoresis versus dot blot for sensitivity, specificity and reproducibility, using a series of purified Cryptosporidium oocyst preparations (three separate preparations of purified oocysts designated as Texas # 2 at 108 oocysts per ml, Texas # 5 at 107 oocysts per ml, and Texas-HRS at 107 oocysts per ml).

PAGE 44

Do dilutions to 1 0(-4) Do freeze/thaw procedure Purified sample containing 1 0(6) oocysts Wash sample to remove preservative x2 _Add 20 uL of 20% Chelex 1 00 to samples .C:U -Transfer 50 uL of supernatant to PCR tubes -Add master mix to samples Load tubes into the tempcycler and run PCR Probe for Cryptosporidium Figure 4. Flow Chart of the Modified Cryptosporidium PCR Detection Protocol. 33

PAGE 45

34 From the Texas # 2, and Texas # S oocyst preparation, 10 ul of each sample was transferred to separate 1. S ml microcentrifuge tubes containing 990 ul of 1X PCR buffer. Each tube was washed x2 in 1X PCR buffer, and resuspend to 1 ml in the same buffer. One-hundred ul of the resuspended samples were then transferred to a triplicate set of 1.S ml microcentrifuge tubes. Dilutions to 10-7 were then made for each sample set (10 ul of sample in 90 ul of 1X PCR buffer. Three sets per sample, each diluted to 10-7 = 36 tubes). One-hundred ul of Texas-HRS sample was washed x2 in 1X PCR buffer. Texas-HRS was then resuspended to 100 ul in the same buffer, and dilutions were completed on this sample to 10-7 (resulting in 8 tubes) To the resulting 44 tubes, 20 ul of 20% Chelex 100 was added. All samples were processed through freeze/thaw x6. After freeze/thaw, the samples were boiled an additional 30 minutes with vortexing every 10 min. The samples were then centrifuged for 3 min at 12, SOO rpm, and SO ul of sample supernatant from each tube was transferred to separate PCR tubes. The following two controls were then prepared in PCR tubes. a. + control = 10 ul of 10s A1 + 40 ul of 1X PCR buffer. b. -control SO ul of 1X PCR buffer. The master mix was then prepared for 48 reactions (44 samples + 2 controls + 100 ul for pipette loss) at SO ul per reaction 2300 ul.

PAGE 46

35 lOX PCR buffer 230 ul 100 mM Mg 168 ul JR 3 230 ul Cryptosporidium forward primer JR 4 230 ul Cryptosporidium reverse primer dNTPs 288 ul TAQ polymerase 12 ul HPLC H(2)0 1142 ul Fifty ul of master mix was then added to each PCR tube, followed by the addition of 3 drops of autoclaved mineral oil. Each PCR tube was then capped and loaded into the Model 480 tempcycler. The reaction profile was then started. In each experiment the resulting PCR product was analyzed by electrophoresis and dot blot. Sensitivity was evaluated through the use of the dilution series. Simultaneous Detection Protocol Multiplex Protocol The following protocol was tested to simultaneously detect both Giardia cysts and Cryptosporidium oocysts in purified samples, and is illustrated in Figure 5. One-hundred ul of Cryptosporidiumparvum oocysts (50,000 oocysts) and 100 ul of Giardia lamblia cysts (2,000 cysts) were placed in separate 1.5 ml microcentrifuge tubes. Cysts

PAGE 47

36 were used at lower concentrations due to the low numbers in stock solution. Fifty ul from each 100 ul seed were combined in a 1.5 ml microcentrifuge tube. Fifty ul of lX PCR buffer was then added to each of the remaining 50 ul seed aliquots. Three prepared samples (oocysts alone, cysts alone, and oocysts and cysts combined) were washed x3 in lX PCR buffer and resuspended to 100 ul in the same buffer. Twenty ul of 20% Chelex 100 was then added to each tube. The samples were then processed through freeze/thaw x6. After freeze/thaw the samples were boiled for an additional thirty min while being vortexed every 10 min. All of the tubes were then centrifuged for 3 min at 12 x 1,000 g, and 50 ul of supernatant was transferred to PCR tubes. A negative control was prepared by adding 50 ul of lX PCR buffer to a PCR tube. The following master mix was then prepared. Master Mix lOX PCR buffer 25 ul 100 mM Mg 17.5 ul JR 3 25 ul Cryptosporidium forward primer JR 4 25 ul Cryptosporidium reverse primer JR 6 25 ul Giardia forward primer JR 7 25 ul Giardia reverse primer d.NTPs 30 ul TAQ polymerase 1.25 ul HPLC H(2) 76.25 ul

PAGE 48

1J Do dilutions to 1 0(-4) -Do freeze/thaw -procedure -Seed environmental sample with both oocysts and cysts -Wash environmental sample to remove preservative -Add 20 uL of 20% Chelex 100 to samples -Transfer so uL of supernatant to PCR tubes -Add master mix containing both protozoa primer sets Load tubes into the tempcycler and run PCR Gel analysis Probe for Giardia Probe for Cryptosporidium Figure 5. Flow Chart for Simultaneous Detection using Multiplex PCR. 37

PAGE 49

38 Fifty ul of master mix was added to each tube followed by the addition of three drops of autoclaved mineral oil. Each tube was then closed and loaded into the Model 480 tempcycler. The amplification profile was then started. PCR product was detected by gel electrophoresis and dot blot. Separate Master Mix Protocol The following experimental outline was developed for simultaneous detection of both Cryptosporidium oocysts and Giardia cysts in both purified and seeded environmental samples, and is illustrated in Figure 6. The following seeded samples were constructed. a. Environmental sample alone without oocysts or cysts. Two hundred and fifty ul of environmental pellet LA149K (A Los Angeles, CA., concentrated surface water sample which was stored in 2.5% dichromate) was placed in a 1.5 ml microcentrifuge tube. b. Environmental sample seeded with oocysts and cysts. Two hundred and fifty ul of environmental sample LA149K + 50 ul of Cryptosporidium parvum oocysts (total of 500,000 oocysts) + 200 ul of Giardia lamblia cysts (total of 8,000 cysts) were combined in a 1.5 ml microcentrifuge tube. c. Cysts alone. Two hundred ul of Giardia lamblia cysts were placed in a 1.5 ml microcentrifuge tube (total of 8,000 cysts.

PAGE 50

Do dilutions to 1 0(-4) Transfer 50 ul of supernatant to PCR tubes Load PCR tubes in tempcycler and run profile 39 Wash 1 ml of sample and wash the sample x4 to remove preservative Do freeze/thaw procedure on samples Add master mix to samples L Analyze with gel electrophoresis and dot blot Figure 6. Flow Chart for Simultaneous Detection using Split Master Mix PCR.

PAGE 51

40 A tube containing oocysts alone was not prepared as this was not needed due to the difference in cyst and oocyst concentrations used) Samples a and b, were washed xS in 1X PCR buffer. Sample c was washed, x3 in the same buffer (samples a and b were washed an additional 2 times due to the presence of the environmental debris, as the dichromate is harder to remove from a environmental sample) All samples were resuspended to 250 ul in 1X PCR buffer. From samples a, b, and c, the following samples were made in 1.5 ml microcentrifuge tubes; 1. 100 ul of LA149K non-seeded. 2. 100 ul of LA149K non-seeded. 3. 100 ul of LA149K which contained both oocysts and cysts. 4 100 ul of LA149K which contained both oocysts and cysts. 5. 100 ul of Giardia cysts. To each of the tubes, 20 ul of 20% Chelex 100 was added and then the samples were vortexed lightly. The samples were then processed through freeze/thaw x 6. The samples were then boiled an additional 30 min with vortexing every 10 min. All tubes were then centrifuged for 3 min at 12 x 1,000 g, and SO ul of supernatant was transferred to PCR tubes. The following controls were then prepared.

PAGE 52

a. + Cryptosporidium control of lX PCR buffer. 41 10 ul of 106 Al + 40 ul b. + Giardia control = 10 ul of 10"2 G+ (Isolated PCR product from a dual amplification run which was used as a control) + 40 ul of lX PCR buffer. c. control = 50 ul of lX PCR buffer (x2, one for each master mix) The following two master mixes were then prepared. Cryptosporidium master mix Volume (Prepared 250 ul) Reagents 25 ul lOX PCR buffer 25 ul ---------JR 3 Giardia master mix Volume (Prepared 300 ul) 30 ul none 25 ul ----------JR 4 none 30 ul 126.25 ul 17.5 ul 1.25 ul JR 6 -----------------30 ul JR 7 -----------------30 ul dNTPs HPLC H(2)0 100 rnM Mg TAQ polymerase 36 ul 151.5 ul 21 ul 1.5 ul Fifty ul of Cryptosporidium master mix was added to four tubes: the negative control (the negative control for the entire experiment), the Cryptosporidium positive control, one of the tubes containing only the environmental sample, and one of the tubes containing the environmental sample seeded with both oocysts and cysts. Similarly, 50 ul of Giardia master mix

PAGE 53

42 was added to the Giardia positive control, the remaining environmental sample without oocysts and cysts, the remaining environmental sample containing both oocysts and cysts, and the sample containing only cysts. Three drops of autoclaved mineral oil was added to each sample and all of the tubes were capped. The tubes were then loaded into the Model 4 8 0 tempcycler and the amplification profile was started. Results were visualized by electrophoresis. This experimental protocol was evaluated in ten separate experiments. Magnetic-Antibody Capture Detection Protocol The following experimental outline was used to evaluate the use of magnetic-antibody capture for reversal of environmental inhibition to PCR assay, while using the multiplex detection protocol (Figure 7). The Bio Mag secondary antibodies (Goat anti-Mouse IgG, cat. # 8-4335D, used for Giardia cyst capture, and Goat anti-mouse IgM, cat. # 8-4350D, used for Cryptosporidium oocyst capture) and the BioMag Separator, cat. # 8-41028, were obtained from PerSeptive Diagnostics, Inc., Cambridge, MA. The primary antibodies, Mouse IgG anti-Giardia Cryptosporidium oocyst, cyst, were Diagnostics, Inc., Cincinnati, OH. and Mouse IgM anti-obtained from Meridian

PAGE 54

1STWash seeded sa":Jple to 11] remove preservative l Dilute sample in blocking buffer . :------'\1,: "-.._ .. 0 "-.._ Isolate oocysts and cysts by using the BioMag separator L Remove supernatant and discard. Resuspend pellet in PCR buffer. Vortex samples to sheer antibodies Magnet i cally separate antibodies from sample and do dilutions to 1 0( -3) 43 .nli l BioMag anti lgG Wash secondary BioMag antibodies to remove preservative Preload primary anti bodies to secondary antibodies. Incubate on ice and wash Add antibody complex to sample Incubate on ice 7 Add multiplex master mix Load tubes in tempcycler and run PCR \ Do freeze/thaw procedure. Transfer 50 ul of supernatant to PCR tubes lQJ Analyze samples with gel electrophoresis and dot blot Figure 7 Flow Chart for Magnetic-Antibody Capture PCR.

PAGE 55

44 The following seeded samples were set up (in duplicate) in 1.5 ml microcentrifuge tubes. A. 1 ml of environmental sample LA39K + 50 ul of Cryptosporidium parvum oocysts (total of 500,000 oocysts) + 400 ul of Swabby Gerbec Giardia lamblia cysts (total of 16,000 cysts) B. 1 ml of 1X PCR buffer + 50 ul of Cryptosporidium parvum oocysts (total of 500,000 oocysts) + 400 ul of Swabby Gerbec Giardia lamblia cysts (total of 16,000 cysts). Both samples were washed x4 in 1X PCR buffer and resuspended to 1 ml in the same buffer. One of the replicate tubes from both samples A and B were placed in the refrigerator. These two samples were used for the standard multiplex reaction protocol without antibody capture, and served as a comparison group. The remaining A and B sample sets were added to 8 5 ml of 1X PBS with 5% NFDM (Non-fat dried milk) in separate 15 ml plastic conical tubes. These 15 ml tubes were then processed through the following magnetic antibody capture protocol. Prior to addition of the antibody-complex t o the samples, the antibodies were washed. One ml of both secondary BioMag antibodies were placed in separate 1. 5 ml microcentrifuge tubes (the following protocol follows the protocol as described by PerSeptive Diagnostics, Inc., magnetic-antibody cell sorting protocol. Cambridge, MA). The tubes containing the antibodies were placed in the BioMag separator and

PAGE 56

45 magnetically separated at room temperature for 3 min. The supernatant was then carefully removed from both tubes and discarded. The tubes were then removed from the BioMag separator and the antibodies were resuspended to 1 ml in 1X phosphate-buffered saline (PBS) buffer. This procedure was repeated x3, using 1X PBS. The primary antibody mixture (Meridian antibodies) was diluted 20:1 in 1X PBS. To increase specificity of the assay, the primary antibody were preloaded (Conjugation of the secondary antibodies to their primary antibody) to the secondary antibody by adding 2 ml of the 20:1 primary antibody suspension to each 1 ml (approximately 5 x 108 magnetic particles) of secondary antibody suspension. The antibody were then incubated on ice for 20 min (the procedure is done on ice to minimize absorption of the antibody complexes by microbial contaminates, as described by the manufacturer, PerSeptive Diagnostics) The antibody complexes were then washed x3 in 1X PBS using the BioMag separator as previously described. The preloads were resuspended to a final volume of 1 ml each (in 1X PBS), and then 0.5 ml of each preload was added to the 8.5 ml of sample in the 15 ml tubes. The samples were then incubated on ice for 20 min, with continuous rocking at low speed. The oocysts and cysts were magnetically separated from the 15 ml samples, by placing the samples in the BioMag separator for 10 minutes. The supernatant was carefully removed from each tube and discarded. The samples were then resuspended to 1 ml in 1X PCR buffer after the tubes were removed from the separator. The resuspended samples were then

PAGE 57

46 transferred to separate 1. 5 ml microcentrifuge tubes. The samples were rapidly vortexed for 30 seconds (this was done to sheer the antibodies from the oocysts and cysts walls) The antibodies were then separated from the oocysts and cysts by placing the tubes in the BioMag separator and incubating for 10 min at room temperature. The supernatant (which contains the oocysts and cysts) was then carefully removed from the tubes and transferred to separate tubes. The tubes containing the 1. 5 ml microcentrifuge antibody pellets were discarded. The tubes containing the oocysts and cysts were revortexed and magnetically separated again (this was done to assure removal on the magnetic beads) The supernatant from this separation step was centrifuged for 3 min and the supernatant was removed and discarded. Both resulting pellets were then resuspended to 100 ul each in 1X PCR buffer. The duplicate tubes not processed through the magneticantibody-capture protocol were used for the standard protocol. One-hundred ul (after light vortexing for 5 seconds) of each of the samples were transferred to separate 1.5 ml microcentrifuge tubes. These tubes (100 ul each) contained 50,000 oocysts and 1,600 cysts, as compared to the antibody capture tubes which theoretically contained 500,000 oocysts and 16,000 cysts. Ten-fold dilutions were made to 103 (10 ul of sample on 90 ul of 1X PCR buffer) for all of the tubes, both the antibody capture and the non-antibody capture samples. Twenty 20 ul of 20% Chelex 100 were then added to all undiluted and diluted samples. All of the samples were then processed

PAGE 58

47 through freeze/thaw x6, with boiling an additional 30 min while vortexing every 10 min. All of the samples were then centrifuged 12,SOO rpm for 3 minutes and SOul of supernatant was transferred to PCR tubes. The following controls were prepared in PCR tubes. a. + Cryptosporidium control of lX PCR buffer. 10 ul of 10-5 Al + 40 ul b. + Giardia control = 10 ul of G+ -z + 40 ul of lX PCR buffer. c. -control = SO ul of lX PCR buffer. The following master mix was prepared for 22 tubes. lOX PCR buffer 110 ul JR3 JR4 JR6 JR7 d.NTPs 100 mM Mg TAQ polymerase HPLC H(2)0 110 110 110 110 132 77 ul ul ul ul ul ul S.S ul 33S.S ul Fifty ul of master mix was then added to each sample. Three drops of autoclaved mineral oil was added to each tube, and each tube was then capped. The tubes were then loaded in the Model 480 tempcycler and the amplification profile was started. PCR results were then visualized by gel electrophoresis.

PAGE 59

48 4. RESULTS The goal of this project was to develop a sensitive simultaneous PCR assay for detection of Cryptosporidium oocysts and Giardia cysts. In order to do so, it was necessary to evaluate the detection of oocysts and cysts at varying levels. Numbers of oocysts and cysts were calculated in 100 ul of prepared sample, which was used for the DNA extraction method (freeze/thaw) The actual amount used for the PCR reaction was 50 ul, to which the master mix (SO ul) was added. After amplification, 10 ul of the total reaction volume (100 ul) was analyzed by electrophoresis or dot blot. Thus, sensitivity was limited in part due to the limited volumes assayed. Numbers of oocysts and cysts will be reported in the extraction tubes (freeze/thaw tubes) for all experiments in regard to comparison of sensitivity of the procedures.

PAGE 60

49 Standard Cryptosporidium PCR Protocol/Preliminary Inhibition Studies The first set of experiments evaluated three sample pretreatments to determine their ability to decrease inhibition and increase PCR detection of oocysts. Two different profiles were also evaluated to determine their ability to enhance sensitivity. Table 5 (PCR 141) shows the results of an inhibition experiment where the cycle number for the amplification portion of the profile was set at 30 cycles. This experiment was conducted using a Coy Model 60 tempcycler. Tubes 1 through 5 in Table 5, represent a purified dilution set. Cryptosporidium oocysts were only detected by dot blot in the undiluted samples (5, 000 oocysts) and the 10-1 diluted samples (500 oocysts) Tubes 6 through 10 (seeded environmental dilution set) in Table 5, represent no pretreatment and Cryptosporidium oocysts were only detected by dot blot in the undilute sample. Tubes 11 through 15 (seeded environmental dilution set) in Table 5, represent the tRNA pretreatment. No detection of Cryptosporidium oocysts was noted by either electrophoresis or dot blot. Tubes 16 through 20 (seeded environmental dilution set) in Table 5, represent the 20% Chelex 100 pretreatment. Detection of oocysts was noted by both electrophoresis and dot blot in the undiluted sample (5, 000 oocysts) and 10-1 dilution sample (500 oocysts). Tubes

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50 Table 5. Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition. (PCR 141) Tube# Oocyst #s Gel Signal Blot Signal 1. Purified oocysts 2. 10(-1) 3. 10(-2) 4. 10(-3) 5. 10(-4) 6. LA42K seeded 7. 10(-1) 8. 10(-2) 9. 10(-3) 10. 10(-4) 11. LA42K seeded + tRNA 12. 10(-1) 13. 10(-2) 14. 10 (-3) 15. 10 (-4) 16. LA42K seeded + 20% Chelex 17. 10(-1) 18. 10 ( -2) 19. 10(-3) 20. 10(-4) 21. LA42 K seeded + 5 % Chelex 2 2 10(-1) 23. 10(-2) 24. 10(-3) 25. 10 ( -4) 26. 2 pg A1 27. 200 fg A1 28. 20 fg A1 29. Negative Control ++++++ +++++ ++++ +++ ++ Heavy signal Medium signal = M edium light signal = Light signal Very light signal No signal Tube/Sample identification. 5,000 500 50 5 0.5 5,000 500 50 5 0.5 5,000 500 50 5 0.5 5,000 500 50 5 0.5 5,000 500 50 5 0.5 ++++++ ++++ ++++++ ++++ ++ +++++ +++ +++ ++ +++ ++++++ +++++ ++++++ +++++ +++ ++ ++++++ +++ *LA42K = Surface water sample used for s eeded studies Tubes 1 through 5 = A dilution set of purified oocysts. Tubes 6 through 10 = A dilution set of a s eede d env. sample. Tubes 11 through 15 = A dilution set of a seeded environmental Tubes 16 through 20 sample pretreated with tRNA. A dilution set of a seeded environmental sample pretreated with 20% Ch elex 100. Tubes 21 through 25 = A dilution set of a seeded environmental sample pretreated with 5% Ch elex 100, and using a modified Chelex protocol.

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51 21 through 25 (seeded environmental dilution set) in Table 51 represent the enhanced 5% Chelex 100 pretreatment. Detection of oocysts by electrophoresis was noted in the 10-1 dilution sample (500 oocysts) 1 the 10-2 dilution sample (50 oocysts) 1 and the 10-3 dilution sample 5 oocysts. Detection by dot blot was also noted in this same group of samples plus the 10-4 dilution sample (0.5 oocysts). Tubes 26 (2 pg of plasmid Al) and 27 (200 fg of plasmid Al) represent two of the positive controls that were detected by both electrophoresis and dot blot. Tube 28 represents 20 fg of Al and was not detected by either electrophoresis or dot blot. No Cryptosporidium oocysts were detected by either electrophoresis or dot blot in the negative control (Tube 29). Both the use of dot blot analysis and 5% Chelex 100 increased the sensitivity of the assay. This experiment represented initial trials and used a preliminary standard protocol as described in Figure 2 (page 24) After these trials the profile was changed to 39 cycles which further optimized the procedure. Further analysis of the use of Chelex 100 showed that the use of 20% Chelex 100 coupled with boiling for thirty minutes after the freeze/thaw step (a hybrid protocol of the 20% Chelex 100 and modified 5% Chelex 100 protocol) I resulted in the most consistent protocol.

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52 Table 6 (PCR 143), Figure 8 (Gel of PCR 143), and Figure 9 (Blot of PCR 143), represent the standard Cryptosporidium oocysts detection protocol (Figure 2, page 26) in which the inhibition studies were repeated with the amplification profile reset to 39 cycles. This experiment also was conducted using the Coy Model 60 tempcycler. Tubes 1 through 5 were the dilution set of purified oocysts that was analyzed with the standard detection protocol. Detection of Cryptosporidium oocysts was noted in the undilute ( 5, 000 oocysts) and the 10-1 dilution ( 500 oocysts)samples, both by dot blot, and electrophoresis. Tubes 6 through 10 were seeded environmental samples Cryptosporidium oocysts were only detected by dot blot in the undilute sample (5,000 oocysts) and the 10-1 dilution (500 Tubes 11 through 15 (seeded environmental sample dilution set) were samples pretreated with tRNA. Detection of oocysts was noted in tubes 11 (undiluted sample, 5, 000 oocysts) and 12 ( 10-1 dilution, 500 oocysts) with electrophoresis, and dot blot. Tubes 16 through 20 (seeded environmental dilution set) were samples pretreated with 20% Chelex 100. Cryptosporidium oocysts were detected in tubes 16 (Undiluted sample, 5,000 oocysts) and 17 (10-1 dilution, 500 oocysts) by both electrophoresis and dot blot. Tubes 21 through 25 (seeded environmental dilution set) were sample pretreated with an enhanced modified 5% Chelex 100 pretreatment protocol_ Cryptosporidium oocysts were detected in tubes 21 (Undiluted

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53 Table 6. Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition. (PCR 143) Tube # Oocyst #s Gel Signal Blot Signal ---------------------------------------------------1. Purified Oocysts 2. 10 ( -1) 3 10(-2) 4. 10(-3) 5. 10(-4) 6. LA42K seeded 7. 10 ( -1) 8 10(-2) 9 10(-3) 10. 10(-4) 11. LA42K seeded + tRNA 12. 10 ( -1) 13. 10 (-2) 14. 10(-3) 15. 10(-4) 16. LA42K seeded + 20% Chelex 17. 10 (-1) 18. 10 (-2) 19. 10(-3) 20. 10 ( -4 ) 21. LA42K seeded + 5% Chelex 22. 10 ( -1) 23. 10(-2) 24. 10(-3) 25. 10(-4) 26. 2 pg A1 27. 200 fg A1 28. 20 fg A1 29. Negative Control ++++++ = Heavy signal +++++ = Medium signal ++++ +++ + + = Medium light signal = Light signal = Very light signal = No signal Tube/Sample identification. 5,000 ++++++ ++++++ 500 +++ +++ 50 5 0 5 5,000 ++++ 500 +++ 50 5 0 5 5,000 +++++ +++++ 500 ++++++ +++++ 50 5 0 5 5,000 ++++++ ++++++ 500 ++++++ ++++++ 50 5 0 5 5,000 +++++ 500 ++ 50 5 0 5 -+++++ ++++++ +++ ++++ *LA42K = Surface water sample used seeded studies Tubes 1 through 5 = A dilution set of purified oocysts. Tubes 6 through 10 = A dilution set of a seeded env. sample. Tubes 11 through 15 A dilution set of a seeded environmental sample pretreated with tRNA. Tubes 16 through 20 = A dilution set of a seeded environmental sample pretreated with 20% Chelex 100. Tubes 21 through 25 = A dilution set of a seeded environmental sample pretreated with 5% Chelex 100, and using a modified Chelex protocol.

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54 Figure 8. Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition. (PCR 143, gel) Tubes (Dilution sets) 1 through 29, left to right, starting after the Lambda Hind III marker which is located in lane 1. Tube/Sample identification. Lanes 2 through 6 = A dilution set of purified oocysts. Signals seen in lanes 2, and 3 (can not be seen due to poor picture quality) Lanes 7 through 11 = A dilution set of a seeded environmental sample. No signals seen. Lanes 12 through 16 = A dilution set of a seeded environmental sample pretreated with tRNA. Signals seen in lanes 12, and 13. Lanes 17 through 21 =A dilution set of a seeded environmental sample pretreated with Chelex 100. Signals seen in lanes 17, and 18. Lanes 22 through 26 A dilution set of a seeded environmental sample pretreated with Chelex 100, and using a modified Chelex protocol. No signals seen. Lanes 27 through 29 = Positive controls. Signals seen in lanes 27, and 28 (neither can be seen due to poor picture quality) Lane 30 Negative control. No signal seen Results also represented in Table 5, page 48.

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55 Figure 9. Analysis of Sample Pretreatments to Determine Their Effectiveness in Reversing Inhibition. (PCR 143, Blot) Tube/Sample identification. Row 1, columns 1 through 5, tubes 1 through 5 ( A dilution set of purified oocysts, left to right) Row 1, columns 6 through 10, tubes 6 through 10 (A dilution set of a seeded environmental sample, left to right) Row 2, columns 1 through 5, tubes 11 through 15 (A dilution set of a seeded environmental sample pretreated with tRNA, left to right) Row 2, columns 6 through 10, tubes 16 through 20 ( A dilution set of a seeded environmental sample pretreated with Chelex 100, left to right). Row 3, columns 1 through 5, tubes 21 through 25 (A dilution set of a seeded environmental sample pretreated with the enhanced Chelex 100 protocol, left to right). Row 3, columns 6 and 7, positive controls Row 3, column 8, negative control Results also represented in Table 5, page SO.

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56 sample, 5, 000 oocysts) and 22 (10-1 dilution, 500 oocysts) with dot blot, but no detection was noted with electrophoresis. Tubes 26 (2 pg of plasmid Al = amount of plasmid used for amplification) and 27 (200 fg of plasmin Al) were the positive controls and were detected by both electrophoresis and dot blot. Tube 28 represented 20 fg of plasmid Al and was not detected by either electrophoresis or dot blot. Tube 29 represented the negative control for the experiment and no detection electrophoresis or dot blot. was noted by either In seeded environmental samples, profiles run at 30 cycles showed only light positive signals with dot blot analysis. Both Chelex 100 pretreatments (20% and 5%) improved this level of detection. Although in PCR trial 141 (Table 5, page 50), the 5% modified Chelex procedure appeared to be very sensitive, this could not be replicated in the remaining experiments that evaluated the procedure. At 39 cycles, good signals were seen on the gel in purified oocysts samples, but these signals were inhibited below the detection limit of the gel with the addition of the same number of oocysts to environmental samples. Signals were only seen when the samples were analyzed by dot blot. The 20% Chelex protoco l was the most consistent protocol at reversing the observed inhibition. This suggested that the use of 39 cycles in the amplification profile, as well as Chelex 100 pretreatment and dot blot analysis, were useful in increasing sensitivity of the assay when working with environmental samples.

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57 Chelex 100 Cryptosporidium PCR Protocol It appeared from previous studies that both 20% Chelex 100 pretreatment and probing (dot blot) enhanced PCR detection of oocysts in environmental samples. The 20% Chelex 100 pretreatment developed as protocol and used in the next a hybrid protocol the modified 5% previously stated. series of experiments was from the 20% Chelex 100 Chelex 100 protocol, as Table 7 (PCR 196), Figure 10 (PCR 196 gel) and Figure 11 (PCR 196 blot), show the results of experiments using three purified calf Cryptosporidium oocysts samples that were analyzed using the hybrid 20% Chelex 100 Cryptosporidium detection protocol. This experiment demonstrated the sensitivity and reproducibility of the assay when working with purified samples. The three samples used in this set of experiments were received on the following listed dates and were shipped and stored in dichromate: Texas # 2 03-16-93, Texas # 5 06-17-93, Texas-HRS 01-18-94 (The experiments were conducted on 03-24-94). Triplicate samples were taken from two preparations of oocysts (Texas # 2 and Texas # 5). The Texas # 2 preparation contained 1 X 108 oocysts per ml. Both the Texas # 5 and the Texas-HRS preparations contained 1 x 106 oocysts per ml. After dilutions, oocyst concentrations of 90,000, 9,000, and 900 were detected by gel electrophoresis in tubes 1 through 6.

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58 Table 7. Analysis of Three Purified Calf Oocyst Isolates using the Chelex 100 Cryptosporidium Protocol. (PCR 196) -----------------------------------------------Sample and Dilution 1. Texas# 2 10(-2) 2. 10(-3) 3 10(-4) 4. 10(-5) 5. 10(-6) 6. 10 (-7) 7. Texas# 2 10(-2) 8. 10(-3) 9. 10(-4) 10. 10 (-5) 11. 10 (-6) 12. 10(-7) 13. Texas # 2 10(-2) 14. 10 (-3) 15. 10(-4) 16. 10 (-5) 17. 10(-6) 18. 10(-7) 19. Texas # 5 10(-2) 20. 10 (-3) 21. 10(-4) 22. 10(-5) 23. 10(-6) 24. 10(-7) 25. Texas# 5 10(-2) 26. 10 (-3) 27. 10(-4) 28. 10 (-5) 29. 10(-6) 30. 10 (-7) 31. Texas# 5 10(-2) 32. 10(-3) 33. 10(-4) 34. 10 (-5) 35. 10 (-6) 36. 10(-7) 37. Texas-HRS Undilute 38. 10(-1) 39. 10(-2) 40. 10 (-3) 41. 10(-4) 42. 10(-5) 43. 10(-6) 44. 10(-7) 45. Positive control Oocyst #s 90,000 9,000 900 90 9 0.9 90,000 9,000 900 90 9 0.9 90,000 9,000 900 90 9 0.9 900 90 9 0.9 0 0 900 90 9 0.9 0 0 900 90 9 0.9 0 0 90,000 9,000 900 90 9 0.9 0 0 Gel Signal Blot Signal +++++ +++ ++ +++++ +++ ++ +++++ +++ ++ ++++ +++ +++++ +++ +++++ +++ ++++++ ++++++ +++++ +++++ ++++++ ++++++ +++++ ++++ ++++++ ++++++ +++++ +++ ++++++ ++++++ +++++ +++++ + ++++++ ++++++ ++++ +++ ++++++ ++++++ +++++ ++++++ ++++++ +++++ ++++++ ++++++ ++++++ ++++++ +++++ ++ ++ ++++++ ++++++ (Continued on next page)

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Table 3. (Continued) 46. Negative control ++++++ Heavy signal +++++ = Medium signal ++++ Medium light signal +++ Light signal ++ Very light signal + Ultra light signal No signal Tube/sample identification. 59 Tubes 1 through 18 =Three dilution sets of Texas sample# 2 Tubes 19 through 44 = Four dilution sets of Texas sample # 5. Tubes 45 and 46 = Controls.

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60 Figure 10. Analysis of Three Purified Calf Oocyst Isolates using the Chelex 100 Cryptosporidium Protocol. ( PCR 196, gel) Tube/sample identification. Row 1, and row 2, lane 1 = Marker. Lanes 2, 3, 4, 8, 9, 10, 14, 15, and 16 (Row 1), the three sets of Texas #2 dilution sets (Detection of 90,000 through 90 oocysts, times 3). Lanes 20, 21, 26, and 27 (Row 1), the first two Texas #5 dilution sets (Detection of 900 through 90 oocysts, times 2) Lanes 3, 4, 9, 10, 11, and 12 (Row 2), the third Texas #5 dilution set, and the Texas-HRS dilution set (Detection of 900 through 90 oocysts in the third Texas # 5 set, and 90,000 through 90 oocysts in the Texas-HRS set). The signal in the 17th lane represents the positive control. See Table 7 for dilution set layout

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61 Figure 11. Analysis of Three Purified Calf Oocyst Isolates using the Chelex 100 Cryptosporidium Protocol. (PCR 196, blot) Tube/sample identification. Row 1, columns 1 through 12 = First and second Texas # 2 dilution sets, left to right. Detection of 90,000 through 90 oocysts in both sets. Row 2, columns 1 through 6 = Third Texas # 2 dilution set, left to right. Detection of 90,000 through 9 oocysts. Row 2, columns 7 through 12 = First Texas # 5 dilution set, left to right. Detection of 900 through 0.9 oocysts. Row 3, columns 1 through 12 = Second and third Texas # 5 dilution sets, left to right. Detection of 900 through 9 oocysts. Row 4, columns 1 through 8 = Texas-HRS dilution set, left to right. Detection of 90.000 through 0.9 oocysts. Row 4, column 9 = Positive control, signal seen. Row 4, column 10 = Negative control, no signal. *See Table 7 for dilution set layout

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62 Oocysts were. detected in the same samples, plus tube 4 (90 oocysts) by dot blot. The same oocyst concentrations were also detected by electrophoresis and dot blot in the second Texas # 2 dilution set (tubes 7 through 12) In tubes 13 through 18 (Third Texas# 2 dilution set), oocysts were detected at the same level as observed with the first two set by electrophoresis. An additional dilution of oocysts (9 oocysts, in tube # 17) was detected in this set by dot blot. In the three Texas # 5 trials, oocysts were detected at levels down to 90 with gel electrophoresis, and to 9 by dot blot. In one trial 0. 9 oocyst was detected by dot blot analysis. In tubes 19 through 36 (the Texas # 5 dilution sets) oocyst concentrations of 900 and 90 were detected by gel electrophoresis, and 900 through 9 by dot blot. Additionally, dot blot analysis detected a concentration of .9 oocysts (tube 22) in the first Texas # 5 dilution set. In the Texas-HRS dilution set (tubes 37 through 44), oocyst concentrations of 90,000 through 90 were detected by electrophoresis, and 90,000 through 0 9 range by dot blot. Tube 45 was the plasmid A1 positive control and was detected by both electrophoresis and dot blot. Tube 46 was the negative control and no detection was noted with either electrophoresis or dot blot. This experiment demonstrated the consistency and sensitivity of the hybrid 20% Chelex 100 Cryptosporidium PCR detection assay, and served as a basis for development of simultaneous detection.

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63 Simultaneous Detection Protocol Multiplex Protocol In initial trials purified cysts and oocysts were tested for simultaneous detection using the multiplex detection protocol as described in the Material and Methods section, page 33, and illustrated in Figure 5, page 37. Figure 12 (PCR 175 gel), shows the results of a simultaneous detection protocol utilizing a multiplex master mix. In this trial the Cryptosporidium primers were used along with the HSP 70 Giardia primer set. All oocysts and cysts were suspended in 1X PCR buffer and thus represent a purified sample. Lane 2, contained only Cryptosporidium oocysts (50, 000 oocysts) and the PCR product was detected using electrophoresis. Lane 3, contained only Giardia cysts (Swabby Gerbec, 2,000 cysts) and the PCR product was detected using electrophoresis. Lane 4, contained both oocysts (50,000) and cysts (2,000) and both protozoa were detected by electrophoresis. Lane 5, was the negative control (50 ul of 1X PCR buffer) and no detection was noted. Figure 13 (PCR 205 gel) represents the results of a repeat of this experiment. It was noted that when this same type of experimental protocol was applied to a seeded environmental sample, detection of cysts (2,000 per sample) was not possible.

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64 Figure 12. Simultaneous Detection of Purified Cryptosporidium Oocysts and Giardia Cysts using Multiplex PCR. (PCR 175, gel) Lane 1 = Marker (Hae III) Lane 2 = Oocysts only. Lane 3 = Cysts only. Lane 4 = Oocysts and cysts. Lane 5 = Cryptosporidium positive control. Lane 6 = Negative control. The lack of signal in the positive control in this experiment was attributed to a contaminated diluent, as evaluation of a fresh preparation of A1 resulted in the expected signal. The diluent used in this experiment was simultaneously evaluated along with the fresh preparation and again produced no signal.

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Figure 13. Simultaneous Detection of Purified Oocysts and Cysts using Multiplex PCR. (PCR 205, gel) Lane 1 = Marker (RAE III) Lane 2 = Cysts only. Lane 3 = Oocysts only. Lane 4 = Cysts and oocysts. Lane 5 = Giardia positive control. Lane 6 = Cryptosporidium positive control. Lane 7 = Negative control. 65

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66 Cryptosporidium was only detectable in one of four environmental samples at concentrations of 5,000 oocysts per 100 ul of sample. Table 8 (PCR 184), and Figure 14 (PCR 184 Cryptosporidium dot blot), show the results of an experiment which evaluated simultaneous detection of oocysts and cysts seeded into 5 different environmental samples. Table 8, lane 1, contained 5,000 oocysts seeded into 1X PCR buffer and was detected by both electrophoresis and dot blot. Lane 2, contained 2,000 Giardia cysts which were detected by electrophoresis. Cryptosporidium product was also detected in this sample by both electrophoresis and dot blot which suggests that the sample also contained C. oocysts. It is unclear at this time how the contamination took place. Lane 3, contained 5,000 oocysts and 2,000 cysts seeded into 1X PCR buffer (which represents a purified sample) Both oocysts and cysts were detected by electrophoresis, and the oocysts were also detected by dot blot. In the following 5 seeded environmental samples (all surface water samples), LA37K, LA39K, LA149K, LA161K, and LA170K, seed counts were the same as the seed counts in the purified sample (5,000 oocysts and 2,000 cysts per sample). The only detection noted in any of these seeded environmental samples, was detection of Cryptosporidium oocysts by dot blot in the LA149K sample (See Figure 14) Giardia was not blot ted in these series of experiments, as the HSP 70 probes exhibited low sensitivity in previous attempts to use them.

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Table 8. Simultaneous Detection in Seeded Environmental Samples using Multiplex PCR. (PCR 184) 67 Tube # Gel Signal Cryptosporidium Giardia Blot Signal Cryptosporidium 1. Oocysts +++++ 2. Cysts ++++++* 3. Oocysts/Cysts ++++++ 4. LA 37K* 5. LA 39K* 6. LA 149K* 7. LA 161K* 8. LA 170K* 9. Cryp. + cont. ++++++ 10. Giard. + cont. 11. Negative cont. ++++++ Heavy signal. +++++ Medium signal. +++ Light signal. +++++ +++++ ++++++ = Contamination of sample with oocysts. ++++++ ++++++@ ++++++ +++ ++++++ Seeded environmental samples (5,000 oocysts and 2,000 cysts) Tube/sample identification. Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Tube 10 Tube 11 = 5,000 oocysts. 2,000 cysts. = 5,000 oocysts and 2,000 cysts. 5,000 oocysts and 2,000 cysts seeded into environmental sample LA37K (K = Sample stored/preserved in dichromate) 5,000 oocysts and 2,000 cysts seeded into environmental sample LA39K. 5,000 oocysts and 2,000 cysts seeded into environmental sample LA149K = 5,000 oocysts and 2,000 cysts seeded into environmental sample LA161K. 5,000 oocysts and 2,000 cysts seeded into environmental sample LA170K Cryptosporidium positive control. = Giardia positive control. = Negative control.

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f'Cit ''" Figure 14. Simultaneous Detection in Seeded Environmental Samples using Multiplex PCR. (PCR 184, blot) 68 Tubes 1 through 11, one row only, left to right (Columns) starting with tube 1. Detection was noted in columns 1, 2, 3, 6, and 9. Tube/sample identification. Tube 1 Column 1, oocysts only. Tube 2 = Column 2, cysts only. Tube 3 = Column 3, oocysts and cysts. Tube4 = Column 4, Oocysts and cysts seeded into environmental sample LA.3 7K (K = Sample stored/preserved in dichromate) Tube 5 = Column 5, oocysts and cysts seeded into environmental sample LA.39K. Tube 6 = Column 6, oocysts and cysts seeded into environmental sample LA149K. Tube 7 = Column 7, oocysts and cysts seeded into Tube 8 = environmental sample LA161K. Column 8, oocysts and cysts seeded into environmental sample LA170K. Tube 9 Column 9, Cryptosporidium positive control. Tube 10 = Column 10, Giardia positive control. Tube 11 = Column 11, Negative control. *See Table 8 for tabled results of this blot, page 67

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69 Table 9 (PCR 207), and Figure 15 (PCR 207, blot) demonstrated simultaneous detection of oocysts and cysts using a multiplex reaction with a purified seeded sample. The Giardia primer set used in these series of experiments targeted the 171 bp sequence of the giardian gene as described by Baker (Table 4, page 20) Simultaneous detection was noted with both electrophoresis and dot blot. This experiment further demonstrates the ability to simultaneously detect both oocysts and cysts, using an alternate set of Giardia primers. Although multiplex PCR enabled simultaneous detection of both Cryptosporidium oocysts and Giardia cysts (using two different sets of Giardia primers) the assay was still subject to inhibition when used with seeded environmental samples. Experiments designed to increase sensitivity by decreasing primer concentrations were attempted with no noted effect. As optimization of the master mix is important in regard to the efficiency of the assay, it was determined that the next approach to increase sensitivit y would be to attempt simultaneous detection by using a split master mix method. The results of those experiments follow.

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70 Table 9. Simultaneous Detection in a Purified Sample using Multiplex PCR with the Giardian Gene Primer Set. (PCR 217) Tube # Gel Signal Cryptosporidium Giardia 1. Oocysts ++++++ 2 G. lamblia 3. Oocysts/cysts ++++++ 4 G. muris 5 Oocysts/cysts +++ 6 Crypto. + cont. ++++++ 7. HSP 70 control 8 Negative cont. ++++++ = Heavy signal +++ = Light signal ++ = Very light signal Tube/sample identification. Tube 1 = 5,000,000 oocysts. ++ ++++++ ++++++ Tube 2 12,000 Giardia lamblia cysts. Blot Signal Crypto. Giardia ++++++ ++++++ +++ ++++++ ++++++ ++++++ ++++++ Tube 3 5,000,000 oocysts and 12,000 Giardia lamblia cysts. Tube 4 825,000 Giardia muris cysts only. Tube 5 5,000,000 oocysts and 825,000 Giardia muris cysts. Tube 6 Cryptosporidium positive control. Tube 7 HSP 70 primer set positive control, which was used in this experiment as an additional negative control (No signal expected) Tube 8 Negative control

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71 Figure 15. Simultaneous Detection in a Purified Sample using Multiplex PCR with the Giardian Gene Primer Set. (PCR 217, blot) CYPtosporidium dot blot(probed) Row 1, columns 1 through 8, left to right. CYPtOsporidium negative control dot blot(unprobed) Row 2, columns 1 through 8, left to right. Giardia dot blot(probed with the giardian gene probe) Row 3, columns 1 through 8, left to right. Giardia negative control dot blot(unprobed) Row 4, columns 1 through 8, left to right. Tube/sample identification = columns 1 through 8 Tube/column 1 = Oocysts only. Tube/column 2 = Giardia lamblia cysts only. Tube/column 3 = Oocysts and Giardia lamblia cysts. Tube/column 4 = Giardia muris cysts only. Tube/column 5 = Oocysts and Giardia muris cysts. Tube/column 6 = Cryptosporidium positive control. Tube/column 7 = HSP 70 primer set positive control which was used as an additional negative control (No signal expected) Tube 8/column = Negative control. *See Table 9 for tabled results of this blot

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72 Separate Master Mix Protocol As a comparison to the multiplex assay, PCR runs utilizing a separate mastermix protocol were tested. Figure 16 (PCR 180 gel photo) shows the results of experiments that addressed the feasibility of using a split master mix protocol to simultaneously detect both oocysts and cysts in environmental samples. Lanes 2 through 5 were evaluated with the Cryptosporidium master mix only. Lane 2, contained the environmental sample without oocysts or cysts and neither was detected. Lane 3, contained oocysts (5,000) and cysts (1,600) seeded into environmental sample LA149K, and Cryptosporidium PCR product was detected by electrophoresis. Lane 4, contained the Cryptosporidium positive control (A1) which was detected by electrophoresis. Lane 5 contained a negative control (50 ul of 1X PCR buffer) and no detection was noted. Lanes 6 through 10 were evaluated only with the Giardia master mix. Lane 6, contained only environmental sample LA149K with no oocysts or cysts, and no detection was noted. Lane 7, contained both oocysts (5,000) and cysts (1,600) seeded into the environmental sample, and detection of the Giardia product was observed. Lane 8, contained only cysts (4,000) and product was detected using electrophoresis. Lane 9, contained the Giardia positive control and detection was observed with electrophoresis. Lane 10, was a negative control and detection was initially noted however upon probing the gel

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Figure 16. Simultaneous Detection using Split Master Mix PCR. (PCR 180, gel) Samples arranged left to right starting in lane 2. identification. Lane 1 = Marker (Hae III) Samples for CYPtosporidium detection. Lane 2 = Environmental sample LA 149K only. 73 Lane 3 = Environmental sample LA 149K seeded with oocysts and cysts. Lane 4 = Cryptosporidium positive control. Lane 5 = Negative control. Samples for Giardia detection. Lane 6 = Environmental sample LA 149K only. Lane 7 = Environmental sample LA 149K seeded with oocyst s and cysts. Lane 8 = Cysts only. Lane 9 Giardia positive control. Lane 10 Negative control.

PAGE 85

74 signal proved to be a false positive signal. Figure 17 (PCR 180 dot blot), shows the dot blot of the previous experiment (PCR 180) Using the probe made from random prime synthesis (BioPrime DNA Labeling System, GIBCO BRL, Gaithersburg, MD), Giardia was detected at levels of 4,000 alone, 1,600 in environmental samples, and in the positive control. No detection was noted on the negative controls or the blots containing only Cryptosporidium oocysts. Giardia was also detected in column 2, rows 3, 4, 5, and 6, which were blots of samples from a replicate experiment of similar design. The same blot was probed with the oligonucleotide probe (HSP 70 biotinylated probe, Table 4, page 20) and produced extremely light signals in column 2 (Giardia positive control and the environmental sample seeded with cysts), but no signals in column 1 (results not shown) The results of using the split master mix method to evaluate 5 seeded environmental samples are shown in Table 10 (PCR 190). Tubes 1 through 13 were evaluated with the Cryptosporidium master mix, and tubes 14 through 27 were evaluated with the Giardia master mix. All detection was by electrophoresis. Tube 1, contained only purified oocysts (1,000) and a light signal was noted with electrophoresis. Tube 2, contained oocysts (1,000) and cysts (1,600) and the Cryptosporidium 435 bp product band was noted with electrophoresis. All of the remaining seeded environmental samples were seeded with 1,000 oocysts and 1,600 cysts. Tubes

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2 t!E'Y )(M 75 Figure 17. Dot Blot Hybridization with Giardia 163 bp Probe of an Environmental Sample Seeded with Oocysts and Cysts. (PCR 180, blot) Tube/sample identification (Filter on right only) Row 1, column 1 = Environmental sample plus cysts. Row 1, column 2 = Cryptosporidium positive control. Row 2, column 1 = Cysts only. Row 2, column 2 Cryptosporidium negative control. Row 3, column 1 Giardia positive control. Row 3, column 2 Environmental sample only. Row 4, column 1 = Giardia negative control. Row 4J column 2 = Environmental sample plus cysts. Row 5, column 2 Cysts only. Row 6, column 2 Giardia positive control. Row 7, column 2 = Giardia negative control. Row 8, column 2 = 2.5 uL of 100 ng/uL of Giardia DNA. Rows 5 through 8, column 1 = Nothing

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76 Table 10. Simultaneous Detection using Split Master Mix PCR. (PCR 190} Seed numbers Gel signals Sample/dilution Oocysts/Cysts Cryptosporidium Giardia --------------------------------------------1. Oocysts only 1,000/+ 2 Oocysts and cysts 1,000/1,600 + 3. LA 37K 1,000/1,600 4. LA 37K 10(-1} 100/160 5. LA 39K 1,000/1,600 6. LA 39K 10(-1} 100/160 7. LA 149K 1,000/1,600 + 8. LA 149K 10(-1} 100/160 + 9. LA 161K 1,000/1,600 10. LA 161K 10(-1} 100/160 11. LA 170K 1,000/1,600 12. LA 170K 10 ( -1} 100/160 ..: 13. Crypto. + cont. +++++ 14. Cysts only -/1,600 ++++++ 15. Oocysts and cysts 1,000/1,600 ++++++ 16. LA 37K 1,000/1,600 17. LA 37K 10 ( -1} 100/160 18. LA 39K 1,000/1,600 19. LA 39K 10(-1} 100/160 ++++++ 20. LA 149K 1,000/1,600 21. LA 149K 10 ( 1} 100/160 + 22. LA 161K 1,000/1,600 23. LA 161K 10(-1} 100/160 24. LA 170K 1,000/1,600 25. LA 170K 10 ( -1} 100/160 26. Giardia + cont. ++++++ 27. Negative control ---------------------------------------------++++++ Heavy signal +++++ = Medium signal + Ultra light signal No signal All environmental samples listed, were seeded with both oocysts and cysts.

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77 3, and 4 (10"1 dilution of tube 3) contained oocysts and cysts seeded into environmental sample LA37K, and no detection was noted. Tubes 5, and 6 ( 10"1 dilution of tube 5 ) contained both oocysts and cysts seeded into environmental sample LA39K, and no detection was noted. Tubes 7, and 8 (10"1 dilution of tube 7), contained oocysts and cysts seeded into environmental sample LA149K, and oocysts were detected in both samples. Tubes 9, and 10 ( 10-1 dilution of tube 9) contained oocysts and cysts seeded into environmental sample LA161K, and no detection was noted. Tubes 11, and 12 (10-1 dilution of tube 11) contained oocysts and cysts seeded into environmental sample LA170K, and no detection was noted. Tube 13, contained the Cryptosporidiwn positive control, and detection was noted. Tube 14, contained only Giardia cysts (1,600) and detection of cysts was noted. Tube 15, contained both oocysts and cysts seeded into 1X PCR buffer, and detection of cysts was noted. Tubes 16, and 17 (10"1 dilution of tube 16) contained oocysts and cysts seeded into environmental sample LA37K, and no detection was noted. Tubes 18, and 19 (10-1 dilution of tube 18), contained oocysts and cysts seeded into environmental sample LA39K and cysts were only detected in tube 19. Tubes 20, and 21 (10-1 dilution of tube 20), contained oocysts and cysts seeded into environmental sample LA149K, and cysts were only detected in tube 21. Tubes 22, 23 (10-1 dilution of tube 22), 24, and 25 (10"1 dilution of tube 24) contained oocysts and cysts seeded into environmental samples LA161K and LA170K,

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78 and no detection was noted with either sample. Tube 26, contained the Giardia positive control and detection was noted. Tube 2 7, contained the experiments negative control and no detection was noted. Out of four seeded environmental dilution sets, oocysts were detected in only one sample, and cysts were only detected in two. These experimental results demonstrated that while simultaneous detection was also possible using the split master mix approach, the protocol was as susceptible to environmental inhibition as the multiplex protocol was. It was determined at this point to readdress the problem of inhibition. Antibody Capture Detection Protocol The magnetic-antibody capture protocol was tested on seeded environmental samples for simultaneous detection of cysts and oocysts to determine its effect on reversal of inhibition. Table 11 (PCR 200), and Figure 18 (PCR 200 photo) show the results of a multiplex experiment that analyzed the usefulness of magnetic-antibody capture to reverse or limit environmental inhibition of the PCR assay. All undiluted samples contained 1,000 oocysts and 3,200 cysts. All following blots were only for Cryptosporidium detection. As previously stated the HSP 70 probe showed limited sensitivity, and the

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79 Table 11. Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR. (PCR 200) Tube # Gel Signal Crypto. Giardia Blot Signal Cryptosporidium Seeded Env. samples evaluated without antibodies 1. undilute 2. 10(-1) 3 10 (-2) ++ +++++ 4. 10 (-3) +++ Seeded Env. samples evaluated with antibodies 5. undilute ++++++ ++++++ 6. 10 ( -1) +++++ ++++++ 7. 10 ( -2) +++ +++ +++++ 8 10(-3) +++ Purified samples evaluated without antibodies 9. undilute +++++ ++++++ 10. 10(-1) ++++++ ++++++ 11. 10 ( -2) ++ +++++ 12. 10(-3) ++++ Purified samples evaluated with antibodies 13. undilute ++++++ ++++++ 14. 10 ( -1) +++++ ++++++ 15. 10 (-2) ++ +++++ 16. 10 (-3) +++ 17. Crypt. + cont. +++++ 18. Giard. + cont. ++++++ 19. Negat. cont. ++++++ Heavy signal +++++ Medium signal ++++ Medium light signal +++ Light signal ++ Very light signal Tube/sample identification. Samples 1 through 4 = Dilution set of seeded environmental Samples 5 through 8 sample LA 37K using our modified Cryptosporidium protocol. Dilution set of seeded environmental sample LA 3 7K using antibody capture. Samples 9 through 12 = Dilution set of a purified seeded sample using our modified Cryptosporidium protocol. Samples 13 through 16 = Dilution set of a purified seeded Sample 17 Sample 18 Sample 19 sample using antibody capture. Cryptosporidium positive control = Giardia positive control Negative control

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80 Figure 18. Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR. (PCR 200, gel) Tubes/samples arranged left to right, starting with tube # 1 in lane 2. Tube/sample identification. Lane 1 = Marker (Hae III) Tubes 1 through 4, in lanes 2 through 5, left to right. Dilution set of seeded environmental sample LA 37K using the modified Cryptosporidium protocol. Tubes 5 through 8, in lanes 6 through 9, left to right. Dilution set of seeded environmental sample LA 37K using antibody capture. Tubes 9 through 12, in lanes 10 through 13, left to right. Dilution set of a purified seeded sample using the modified Cryptosporidium protocol. Tubes 13 through 16, in lanes 14 through 17, left to right. Dilution set of a purified seeded sample using antibody capture. Tube 17 in lane 18. Tube 18 in lane 19. Tube 19 in lane 20. Cryptosporidium positive control = Giardia positive control = Negative control

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81 giardian gene probe was not available for use at the time of these experiments. Tubes 1 through 4, were dilutions set up to evaluate the multiplex protocol using seeded environmental sample (LA37K) Cryptosporidium oocysts were detected in the 10-2 (10 oocysts) dilution with electrophoresis and dot blot, and in the 10-3 (1 oocyst) dilution with dot blot. LA37K had previously inhibited detection of Cryptosporidium oocysts in an undiluted sample in a simultaneous detection protocol using a multiplex reaction (see Table 8, page 67). In this case inhibition was overcome through dilution of the samples (this detection of target at dilutions but not in the undilute sample, has been observed previously in other experiments) It is thought that when the ratio of target DNA to inhibitor is exceeded, detection becomes possible. Diluting a sample to reverse inhibition is only an option with samples containing large numbers of oocysts or cysts, as log dilutions with samples containing less than ten target organisms would result in dilutions with no targets present. No Giardia detection was noted in any of the samples. Tubes 5 through 8, were seeded environmental samples (LA37K) evaluated with antibody capture. Oocysts were detected in all but the last dilution with electrophoresis (concentrations of 1,000, 100, 10, and 1 oocysts), and in all dilutions with dot blot (concentrations of 1,000 through 0.1 oocyst) Giardia was detected by electrophoresis in the 10-2 dilution sample which contained 32 cysts.

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82 Tubes 9 through 12, was a purified dilution set (contained purified oocysts and cysts seeded into 1X PCR buffer) and were evaluated with the standard multiplex protocol (without antibody capture) Cryptosporidium was detected in all but the last dilution with electrophoresis, and in the complete dilution set with dot blot. No Giardia was detected. When treating this same sample with magnetic-antibody capture, oocysts were detected in all but the last dilution with electrophoresis, and in the complete dilution set with dot blot. No Giardia was detected. Tube 17, contained the Cryptosporidium positive control and was only detected with dot blot. Tube 18, contained the Giardia positive control and was detected with electrophoresis. Tube 19, contained the experiments negative control and no detection was noted. Table 12 (PCR 202) and Figure 19 (PCR 202 gel), show the results of an experiment that addressed the use of magneticantibody capture with an environmental sample (LA161K) that had previously inhibited detection in all seeded experiments. Seeds were 100,000 oocysts and 3,200 cysts in the undiluted samples. Tubes 1 through 5 (dilution set) were evaluated with the standard multiplex protocol and Cryptosporidium was only detected in the 10-2 dilution. No Giardia was detected. Tubes 6 through 10, were seeded environmental samples treated with the magnetic-antibody capture protocol, and Cryptosporidium

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83 Table 12. Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR. (PCR 202) ---------------------------------------------Tube# Gel Signal Cryptosporidium Giardia -----------------------------------------------------1. LA 161K undilute 2. 10(-1) 3. 10(-2) 4. 10(-3) 5. 10(-4) 6. LA 161K undilute 7. 10(-1) 8. 10(-2) 9. 10(-3) 10. 10 (-4) +++ +++++ +++ + 11. Cryptosporidium + control ++++++ 12. Giardia + control 13. Negative control ++++++ +++++ +++ Heavy signal Medium signal Light signal ++++++ + = Ultra light signal Tube/sample identification. Tubes 1 through 5 Dilution set of seeded environmental Tubes 6 through 10 Tube 11 Tube 12 Tube 13 sample LA161K, using the standard simultaneous detection protocol. Dilution set of seeded environmental sample LA161K, using antibody capture. Cryptosporidiurn positive control. Giardia positive control. = Negative control.

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84 Figure 19. Results of a Magnetic-Antibody Detection Experiment using Multiplex PCR. (PCR 202, gel) Tubes/samples arranged left to right starting in lane 2 with tube/sample 1. Tube/sample identification. Lane 1, marker ( Hae III) Lanes 2 through 6, tubes 1 through 5, dilution set of seeded environmental LA161K, using the standard simultaneous detection protocol. Lanes 7 through 11, tubes 6 through 10, dilution set of seeded environmental sample LA161K, using antibody capture. Lane 12, tube 11, Cryptosporidium positive control. Lane 13, tube 12, Giardia positiv e control. Lane tube 13, negative control.

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85 was detected in the first three tubes of the dilution set by electrophoresis. No Giardia was detected in any of the dilutions. Tubes 11 and 12, were the positive controls and both were detected as expected. Tube 13, was the negative control and no detection was noted. The antibody capture procedure was shown to improve detection of oocysts in environmental samples to levels as low as one. However, the procedure was not successful for detection of cysts. The antibody capture procedure was shown to improve detection of oocysts in environmental samples to levels as low as one. However, the procedure was not successful for detection of Giardia cysts.

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86 5. DISCUSSION Early experiments utilizing a standard Cryptosporidium detection protocol demonstrated the need to address environmental inhibition of the PCR assay. The first area addressed was optimization of the master mix as variable results were being observed from run to run while analyzing the same sample. It was noted from these experiments that if the concentration of polymerase was increased from one unit to two, and the magnesium concentration increased from 1.5 mM to 3.5 mM, the reaction produced more consistent results. Various primer concentrations were also analyzed with no noted increase in sensitivity. It became apparent that optimization of reaction reagent concentrations was vital to producing experiments with reproducible results. Figure 20 illustrates a hypothesis that each reagent in the master mix fluctuates in performance around a mean value within a bell curve. For optimal performance of the assay, all mean values must overlap (Represented in Figure 20, panel A, by stacking, i .e. when represented in an xy-graph, all mean values would exist at the same point on an x-axis) so that any fluctuation of reagent performance will still fall within the bell curve of the other reagents. It is this authors belief that when one of the

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Master Mix Reagents: dNTPs, Magnesium, Primer set, PCR buffer, TAq Polymerase, and water. 1----Amplification --l occurs Optimized master mix. All reagent performance means which are based on concentrations, overlap. This results in good reproducible results. A X No Amplification ..J No I npiiiCIIIan !amplification One reagent performance mean is shifted due to non-optimal concentration. This results in variable results, i.e. amplification some but not all of the time B Figure 20. Optimization of Master Mix Reagents based on Reagent Concentrations. 87

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88 reagent concentrations is not optimal (Figure 20, panel B), then the position of the mean and thus the bell curve is shifted to the left or right of the other reagent values which contributes to variable experimental results. Variability can then be explained; when the reagent that is not at its optimal concentration fluctuates to the side of its mean which still exists within the bell curves of the other reagents then amplification occurs, when it fluctuates to the other side (outside the range of the other be.ll curves) then amplification does not occur or potentially not as efficiently. A standard Cryptosporidium PCR protocol developed during this study was found to produce reproducible results (sensitivity was one oocyst when coupled with dot blot analysis) when used to analyze purified samples, and it is believed that the reagent concentrations used are near optimal. The assay however, demonstrated susceptibility to inhibition when used to analyze seeded environmental samples. After the master mix was optimized, the protocol was applied to analysis of seeded environmental samples to evaluate its usefulness in screening environmental samples for the presence or absence of oocysts. Experiments again produced variable results due to inhibition of the assay by unknown constituents in the environmental pellet. The inhibition has not been defined or the inhibitors identified, but it is thought to be a result of many factors. One inhibitor type may

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89 be divalent cations which compete with the magnesium, resulting in inactivation of the polymerase. Nucleic acid binding molecules that interfere with the primer to primer target annealing complex are another possible type of inhibitor. This type of inhibitor could also block the polymerase from binding to its primer/primer target complex. Several other possibilities include the presence of chemicals that alter the reaction buffer characteristics, and chemicals that alter the ability of the dNTPs to complex with the polymerase (blockage of chain elongation) Observations in these experiments with oocysts demonstrated that the amount of inhibition varied with sample type (i.e .. water samples from different locales demonstrated varying degrees of inhibition, from little or no inhibition, to total inhibition) In light of these various factors, or combination of factors and possibilities, various sample pretreatments were analyzed for the ability to block or limit inhibition. Previous work, had evaluated different pretreatments including the use of EDTA, sucrose gradient centrifugation, tRNA, electrophoresis, sample filtration, density columns and Chelex 100 (Johnson et. al. In Press). The use of EDTA (a heavy metal chelator) resulted in no noticeable improvements in sensitivity. Sucrose gradient centrifugation (separation of particulates based on density) reduced sample pellet size but at the same time resulted in large losses of oocysts (> 80%). Percol-sucrose is used to

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90 clarify samples for IFA and microscopic analysis. However, for PCR assays there is not sufficient clarification to remove the inhibitors. tRNA which was used to counter any nucleic acid binding molecules ( tRNA was used as a sacrificial lamb; meaning that if nucleic acid binding molecules were present, the addition of tRNA would bind to them and thus protect the Cryptosporidium DNA once it was released from the oocysts) The effect of the tRNA varied with sample type and therefore was not usable with a broad range of samples. Electrophoresis of the sample was also tried and was based on the possibility that the oocysts would migrate to the positive pole of the electrophoresis apparatus based o n cell charge (net negative internal charge) The results have not been presented in this thesis and the few experiments conducted indicated that oocyst migratio n was random. This random migration could have been the result of the apparatus set up, as the desired apparatus, whic h w ould contain a well to trap the migrating particles and oocysts (if they did migrate in an electrical field) was not available. Sample filtration was also evaluated (using a large pore filter to remove large particulates whil e letting the small particulates such as the oocysts, pass through) .The results indicated that large losses of oocysts occurred due to particle entrapment at the filter/particle interface. Density columns, using a SO% glycerol liquid column were also analyzed. The theory behind the use o f this type of

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91 pretreatment is the same as sucrose gradient centrifugation (separation of particles based on density), except that no centrifugation was used. It was noted that rapid heavy particle fallout occurred, and results indicated that as the heavy particles fell through the column they trapped oocysts, resulting in large losses of oocysts (> 1 log). Chelex 100 (a chelator of divalent cations) was analyzed and it was determined through comparison to the other pretreatments analyzed in this laboratory, to be the most consistent protocol at reversing inhibition. It is believed, that Chelex 100 is superior to other chelators such as EDTA, because it can be removed by centrifugation (therefore, removing the inhibitors from the supernatant containing the DNA) In addition, Chelex 100 could also remove other types of inhibitors based on charge interactions. It should be noted that although this protocol was superior to all others tried, the protocol did not eliminate inhibition, and that the effect of Chelex pretreatment also varied (Chelex 100 pretreatment did not effect inhibition in some samples analyzed) Analysis of the Chelex 100 assay demonstrated the consistency of the assay when analyzing purified samples by both electrophoresis and dot blot. Sensitivity of the assay was approximately 900 to 90 oocysts with electrophoresis detection, and 9 to 0.9 oocysts with dot blot. Purified and young oocysts (< 2 months) from calf feces were used in these studies, and this level of sensitivity should be considered optimal for the assay. In

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92 addition as oocysts age then the numbers of empty oocysts increase. LeChevallier, found that in surface water samples, up to 68% of the oocysts collected were void of sporozoites (LeChevallier et al., 1991). Therefore, PCR would not detect these oocysts (empty oocysts) It also should be noted that the oocysts counts were based dilution extrapolation, and represent the number of oocysts in the 100 ul sample prior to freeze/thaw. When taking this into account, it must be kept in mind that only 50% of the sample is amplified (50 ul of the 100 ul sample) and only 10% of the sample after amplification (10 ul of the 100 ul reaction volume) is evaluated by gel electrophoresis or dot blot. Sensitivity lower than 1 oocyst is possible because of the presence of up to four sporozoites per oocyst. Small differences in sensitivity from dilution set to dilution set, which was observed when using this assay can be explained in the following manner; if a 1 ml environmental sample contains 5 oocysts (which is equivalent to 0 5 oocysts per 100 ul; the volume used for freeze/thaw), and the maximum amount of the sample that can be assayed is 50 ul, then the probability that one oocyst will be in that 50 ul aliquot will vary from one extraction to another. Simultaneous detection of Cryptosporidium oocysts and Giardia cysts is desirable for any new method, as the current IFA method is applicable to both protozoa. For PCR, it was determined that incorporation of a Giardia primer set into our existing modified Cryptosporidium protocol would produce the

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93 desired results through the use of a multiplex reaction, or a reaction that used separate master mixes. The Giardia HSP 70 primer set was the set of primers used for the initial development of this protocol, and the sequences were obtained from Dr. Morteza Abbazadegan. Two master mix approaches were analyzed for their usefulness using our existing Cryptosporidium protocol for simultaneous detection. The first method tried was the use of a multiplex master mix. This type of polymerase chain reaction has been successfully used to detect and study various types of microbes and cells such as v iruses, Bacillus thuringiensis, and Salmonella spp. (Steuerwald et. al., 1994, Bourque et. al., 1993, Way et. al., 1993). The multiplex reaction utilized both the Cryptosporidium primer set and the Giardia primer set simultaneously. The concentration of both primer sets in the master mixes were similar to those used for the Cryptosporidium primer set (200-800 nM of eac h primer set). Simultaneous detection was noted using this protoco l with purified samples, but a noted drop-off in gel signal was observed when compared to detection of either purified oocyst or purified cysts in separate reactions. This drop-off in sensitivity could be attributed to an increase in the amount of DNA within the sample, which would effect the efficiency of the primer set in annealing to its target complex (an increase in more foreign DNA within a sample, would increase the time required for the primer set to find and anneal to the target

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94 sequence) dNTP concentration could also effect sensitivity by being to low (not enough dNTP' s for simultaneous amplification) No simultaneous detection was noted when using this protocol with seeded environmental samples using Chelex 100 pretreatment protocol. An experiment to analyze the effects of varying primer concentrations on sensitivity of the assay produced no remarkable results (lower primer concentrations are believed to increase sensitivity by limiting primer dimer formation, and the formation of primer dimers results in limited availability of the primers for their respective target sequences) The second approach used for simultaneous detection was to use a split master mix method. This protocol utilized two separate master mixes (each master mix containing only one primer set) to analyze a split environmental sample (two equal aliquots of the sample, with one being analyzed by the master mix containing the Cryptosporidium primer set, and the other being analyzed by the master mix containing the Giardia primer set) This protocol enabled simultaneous detection of both oocysts and cysts in both purified seeded samples and seeded environmental samples. A light signal was noted in the negative control in one of these experiments (while using the HSP 70 primer set) that was approximately the same size as the Giardia PCR product. The use of dot blot demonstrated that no probe signal was detected in the negative control. The dot blot results thus indicated that the negative control signal

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95 was not due to contamination, and was probably the result of primer dimers. The use of dot blot analysis avoids reporting of false-positive results. The detection of signals (falsepositives) on gels, followed by a negative results when probing occurred in only two experiments conducted over a two year period (> 80 experiments). The real value of dot blot analysis is verification of positive results and the prevention of false-negative results. Because of the increase in sensitivity seen through dot blot analysis, false-negative results occurring from electrophoresis analysis are often prevented. Further use of the split master mix protocol, demonstrated that simultaneous detection of both protozoa was possible in some but not all environmental samples analyzed. These experiments further illustrated the varying levels of assay inhibition observed when using different environmental water samples for seeded studies. These experiments also demonstrated that inhibition could be countered (in some environmental samples) with dilution of the seeded samples, as seen in earlier work. It was noted that when inhibition was seen in the undiluted sample, detection could be observed upon a 1 to 2 log dilution of the sample. The problem of using dilutions to monitor environmental samples is that when analyzing samples containing small numbers of oocysts and cysts (less than 10 of each), a 1 log dilution may not contain any of the target organism(s). These results further

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96 demonstrated that although the use of Chelex 100 did to some degree counter inhibition, it was by no means as effective as needed. It was determined at this point that further work was needed to address the problem of inhibition, in order to increase the sensitivity of the developed assays. Magneticantibody capture was chosen for analysis as a sample pretreatment that could counter assay inhibition. Magneticantibody capture allows separation of the target cell from the surrounding debris. This method has been used successfully with clinical samples to determine the presence of lymphoma cells in bone marrow specimens (Gribben et. al., 1992), and used to purify transiently transfected HeLa cells from cell cultures (Padmanabhan et. al., 1988). Therefore, its application to environmental samples seeded with oocysts and cysts was analyzed. The methodology not only purifies the sample to be analyzed, but also allows concentration of the sample. For PCR, this means that a large volume can be effectively concentrated to the sample reaction volume of 50 ul. In this set of experiments magnetic-antibody capture allowed a 1 log increase in sample volume to be analyzed. This would explain why an increase in sensitivity was seen in seeded environmental samples versus purified samples analyzed with our modified Chelex 100 Cryptosporidium protocol (analysis of 1 ml versus analysis of 50 ul). This increase in sample volume is by no means the limit of sample volume that

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97 can be analyzed using this protocol (with the current magnetic sorting apparatus, the maximum volume that can be analyzed is 50 ml aliqouts), but is just the sample volume chosen by this author to be analyzed in this set of experiments. The antibody capture method chosen, used indirect capture. This involved using two primary monoclonal antibodies (an IgG for cyst capture, and an IgM for oocyst capture) that attached to oocyst and cyst wall epitotes, respectively. Secondary antibodies designed to bind to the primary antibodies, and tagged with an iron particle, then allow capture of the target cells using a magnetic field. When working with seeded environmental samples it was noted that a blocking reagent (non-fat dried milk) had to be used. Without a blocking reagent, non-specific binding of the antibodies to environmental debris prohibited both purification and concentration of the oocysts and cysts. Analysis of the effect on sensitivity of the assay when another blocking reagent such as bovine serum albumin, or fetal calf serum was used, were not completed. Sensitivity of the assay could also be improved through antibody titers, and analysis of other blocking reagents as described. The antibody titers used in this set of experiments were estimates of the titers needed to ensure capture during the initial evaluation of the assay. Experimentation demonstrated the usefulness of antibody capture as a pretreatment protocol. As an example, seeded

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environmental samples that were pretreated with capture produced superior sensitivity (detection through 1,000 oocysts, Table 11, page 79). The same 98 antibody of 0.1 samples analyzed without antibody capture were at limits of detection of 10 oocysts only (Table 11, page 79) An increase in sensitivity was also seen in purified seeded samples through an increase in signal strength in the undilute samples when analyzed by both gel electrophoresis and dot blot. In addition, magnetic-antibody capture showed multiplex detection of seeded environmental samples when all previous attempts using our modified multiplex protocol had failed. As noted in the photograph of PCR 200 (Figure 18, page 80), the 10-2 dilution of the environmental sample that was analyzed with antibody capture resulted in simultaneous detection of both protozoa, while simultaneous detection was not noted in any of the other samples including the purified samples. This result suggests that concentration of the cysts resulted in detection. In one out of eight experiments (12.5%, Table 11, page 79) using antibody capture with a multiplex reaction, simultaneous detection was noted. No simultaneous detection was noted in samples without the use of antibody capture. Oocysts were detected in six of eight samples (75%) when using antibody capture, and in four of eight without antibody capture (50%). The usefulness of antibody capture was further

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99 demonstrated in an experiment in which a seeded environmental sample (LA161K) which had previously inhibited all experiments (multiplex, separate master mix, and oocyst detection only) using our modified Cryptosporidium protocol, was analyzed using antibody capture (Figure 19, page 84) The standard Chelex 100 protocol using a multiplex PCR reaction only allowed detection of oocysts at the 10-2 dilution level. When magnetic-antibody capture was coupled to our Chelex 100 protocol, detection of oocysts was observed in the undiluted sample through the 10-2 dilution. Giardia cysts were not detected with either protocol. This experiment (Even though simultaneous detection of both oocyst and cyst was not noted), demonstrated that the magnetic-antibody capture protocol when coupled to our modified Chelex 100 Cryptosporidium detection protocol, was superior to any protocol tried to date. One of the obstacles to successful simultaneous detection, was the specificity and sensitivity of the Giardia primer sets. Low specificity can directly effect sensitivity when the experimenter can not distinguish the Giardia band pattern from random bands (resulting from non-specific amplification), during gel electrophoresis analysis. Specificity experiments were conducted on the Giardia HSP 70 primer set and multi banding (and streaking) of various other cell types was observed with electrophoresis analysis of PCR product using the Cryptosporidium PCR profile. This multi banding observed with the other cell types can be attributed

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100 the homology of heat shock genes across cell lines. As an example the homology between the HSP 70 gene between Homo sapiens and E. coli is approximately SO% (Craig et. al., 1993) It is believed that the resulting low specificity observed with this primer set when used on our Cryptosporidium PCR profile, results in observable streaking (i.e ... random amplification of various segments of foreign genomes) This streaking was only observed with samples containing large numbers of cell types (endemic bacteria, protozoa, and fungi) found in undiluted environmental samples, which further confirms the assumption that the streaking is attributed to random amplification. This effect of primer specificity directly effects sensitivity, resulting in target signal masking (HSP 70 gene 163 bp Giardia PCR product) by the observed streaks of PCR product. This streaking or PCR product banding was also noted when analyzing the Giardia lamblia cyst seed stocks. The cyst stock is a gerbil feces concentrate contains not only cysts, but also natural gerbil gut flora. The presence of gut flora was thought to cause this banding, as analyzes of isolated Giardia lamblia DNA resulted in only the expected 163 bp PCR product band. This problem of low sensitivity due to the HSP 70 Giardia primer set, coupled with low probe signals, indicated the need to analyze another Giardia primer set for use in simultaneous detection experiments. The giardian gene primer set was analyzed as an alternate Giardia primer set for detection and produced good

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101 strong signals with both electrophoresis and dot blot analysis when used in a multiplex detection experiment that analyzed a purified sample. No magnetic-antibody capture experiments were analyzed using this primer set, as the primer set was not received until after completion of the initial round of antibody capture experiments. It is anticipated that this primer set will produce results similar to Cryptosporidium results when used with magnetic-antibody capture.

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CONCLUSION The primary obstacle for the successful application of PCR in analysis of environmental samples has been the problem of inhibition of the assay. This research has illustrated the effects on analysis of environmental samples by the inhibitors, and has addressed several means for countering their effect. Of the methods evaluated by this author, magnetic-antibody capture was the most effective method at countering inhibition of the assays. The magnetic-antibody capture protocol can be further optimized, and its potential usefulness as a viable assay has been illustrated in this study. The antibody capture protocol demonstrated an increase in sensitivity ( a minimum of 2 logs) in seeded environmental samples over purified seeded samples analyzed by the Chelex 100 protocol (i.e. detection in undiluted samples) Magneticantibody capture also allowed detection of oocysts in a seeded environmental sample that had inhibited all previous seeded experiments. Further analysis of magnetic-antibody capture in regard to optimization of the assay, should produce a protocol that very sensitive for environmental reaching detection limits of one organism, and rivaling the current IFA procedure.

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103 The simultaneous detection of oocysts and cysts using PCR assay, was shown to be feasible with both the multiplex and separate master mix protocols. This is the first set of protocols developed, as known by the author for the simultaneous detection of enteric protozoa by PCR. Of the two methods (multiplex versus separate master mixes) developed, multiplex offers the most direct and time efficient protocol. Coupled with antibody capture to deal with the problem of environmental inhibition of the assay, multiplex PCR was the most promising protocol. The primary obstacle to development of an assay for simultaneous detection that is as sensitive as needed, has been the low specificity of the primer set (HSP 70 primer set) specificity of as described. As previously stated, low the primer set and the subsequent random amplification of foreign genomes, resulted in low sensitivity to small numbers of cysts in environmental samples. This effect was not noted with the Cryptosporidium primer set which can be attributed to the specificity of the 18s rRNA sequence region from which they were designed. In order to develop a simultaneous detection protocol that is useful to industry, it is this authors opinion that a primer set designed to amplify a segment of the Giardia 18s rRNA gene is needed. An 18s rRNA Giardia primer set would target a highly repetitive genomic sequence, and allow species specificity. As with our Cryptosporidium primer set, good specificity and thus good sensitivity would then be possible. With the right Giardia

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104 primer set (in view of the differences seen between the HSP 70 primer set and the giardian gene primer set), a simultaneous detection protocol that is as sensitive as needed (simultaneous detection of less than 10 oocysts and cysts) could be developed. In conclusion, this work demonstrated that simultaneous detection of both oocysts and cysts in seeded environmental samples using PCR assay is possible. This type of protocol could lead to the development of an assay that is useful to the water industry for outbreak prevention, environmental monitoring, and research. This research also demonstrated that magnetic-antibody capture was the most effective sample pretreatment for reversal of inhibition, of the methods analyzed. Magnetic-antibody capture is a protocol that once optimized, could lead to development of PCR assays that are applicable to many environmental studies and may also be useful for detection procedures employing microscopic procedures such as IFA.

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