Detection Of Polymerase Chain Reaction Products Research Articles

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR)

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10 2 cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore™ 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.

Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction.

In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens.

Calculation and Verification of the Ages of Retroprocessed Pseudogenes

To verify the existence of processed pseudogenes in different primates and their correlation with the estimated age of divergence, selected regions of processed pseudogenes of α-enolase, calmodulin II (CALMII), and argininosuccinate synthetase (AS) were amplified by the polymerase chain reaction (PCR) using DNA of blood samples. Published primate divergence times from the accepted paleontological records and the age of the pseudogenes based on molecular clock calculations were compared to data obtained by detection of PCR products exhibiting the expected amplicon size of the pseudogene region. For the α-enolase and the CALMII pseudogenes Ψ2, and Ψ3, calculated divergence times were 11, 19, and 36 Myr, respectively. For the AS pseudogenes Ψ1, Ψ3, and Ψ7, the divergence times were calculated to be 21, 25, and 16 Myr, respectively. Primer design and the annealing temperature are critical factors in the detection of pseudogenes in different species and impact greatly on the interpretation of the PCR analysis. The estimated divergence times of the selected pseudogenes utilizing calculations based on the molecular clock theory correlated well with experimental PCR detection of the selected pseudogenes represented in this study.

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10 3 cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10 3 cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.

Sex Determination by PCR in Single Bovine Blastomeres Biopsied by Extrusion Method at the 8-cell Stage.

We attempted to determine the sex by polymerase chain reaction (PCR) in single bovine blastomeres at the 8-cell stage. PCR was performed on male-specific primers attached to a bovine embryonic sex determination kit, an XY Selector. Embryos at the 8-cell stage were isolated by the EDTA method, and one (1/8 embryo), two (2/8 embryo), and four (4/8 embryo) blastomeres were subjected to PCR. The detection rates for male-specific PCR product were 25.8, 25.8 and 45.2% for 1/8, 2/8 and 4/8 embryos, respectively. In some embryos, despite detection of the male-specific product in 4/8 embryos, the male-specific product was not detected in 1/8, 2/8 or both 1/8 and 2/8 embryos derived from the same embryo (3.2, 3.2 and 16.1%, respectively). Collecting single blastomeres by the extrusion method affected neither the rate of development to blastocysts nor the number of cells in blastocysts. PCR was performed in 1/8 embryos collected by the extrusion method, and the male-specific PCR product was detected. Nevertheless, in 27.5% of embryos, despite detection of male-specific PCR product in 7/8 embryos, the male-specific product was not detected in the 1/8 embryo from the same embryo. These findings indicated that collecting single blastomeres at the 8-cell stage allows the selection of the male embryo by PCR, and also the extrusion method is useful for biopsy of embryo at the 8-cell stage.

Detection and quantification of Salmonella in pure cultures using 5′-nuclease polymerase chain reaction

Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan ™ PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5′-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276–7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5′-nuclease-based kit for the quantification of Salmonella.

Quantitative measurement of Stachybotrys chartarum conidia using real time detection of PCR products with the TaqManTMfluorogenic probe system

The occurrence of Stachybotrys chartarum in indoor environments has been associated with a number of human health concerns, including fatal pulmonary haemosiderosis in infants. Currently used culture-based and microscopic methods of fungal species identification are poorly suited to providing quick and accurate estimates of airborne human exposures to the toxin containing conidia of this organism. In this study, real-time polymerase chain reaction (PCR) product analysis using the TaqManU fluorogenic probe system and an Applied Biosystems PrismS model 7700 sequence detection instrument (model 7700) was applied to the specific detection of S. chartarum ribosomal DNA (rDNA) sequences. Based upon this assay and a recently reported comparative cycle threshold method for quantifying target DNA sequences using data from the model 7700, a simple method for the direct quantification of S. chartarum conidia was developed. In analyses of samples containing several different strains and from two to over 2×105cells, this method consistently provided quantitative estimates of S. chartarum conidia that were within a one-fold range (50–200%) of those determined on the basis of direct microscopic counts in a haemoÍcytometer. The method showed a similar level of agreement with direct counting in the quantification of S. chartarum conidia in air samples collected from several contaminated homes.

Clonal loss of heterozygosity in microdissected Hodgkin and Reed-Sternberg cells.

The typical Hodgkin and ReedSternberg (HRS) cells are thought to represent the malignant cellular elements of Hodgkin’s disease (HD). The detection of immunoglobulin gene rearrangements in HRS cells indicates that they are clonally derived from B cells (1–8), but immunogenotyping, as such, does not provide any information on specific gene alterations possibly involved in the molecular pathology of HD. In many tumors, highly informative polymorphic DNA markers may identify loci harboring clonal loss of heterozygosity (LOH) and thus help to trace tumor suppressor genes (9,10). Although in HD, cytogenetic data suggest that nonrandom chromosomal deletions may occur at several loci, including 1q42, 4q26, and others, very little (if anything) is known about clonal LOH in HRS cells at the molecular level (11– 15). We, therefore, set out to study microdissected HRS cells from classical types of HD at candidate loci for LOH with a highly sensitive microsatellite polymerase chain reaction (PCR) assay. In seven patients with classical HD, HRS cells and surrounding cells including bystander lymphocytes were laser microdissected from formalin-fixed, paraffin-embedded tissue sections (Table 1). From a patient with nodularsclerosing HD (patient 6), samples were taken at presentation (6a) and at relapse (6b). In two patients (patient 6 and patient 7, a patient with mixed cellularitytype HD), frozen sections of lymphoma tissue yielded high-molecular-weight control DNA from microdissected cell populations. Buccal smears provided constitutional high-molecular-weight DNA in patients 5 and 6 (16,17). Patients gave their informed consent to include their material in this study. For molecular analyses, tetranucleotide repeat microsatellites were selected through the Genome Data Base at loci with a high frequency of chromosomal deletions in HD, which were predicted by the cytogenetic literature (11–15), and located at 1q42 (D1S517), 4q26 (D4S2301), 9p23 (D9S254), and 11q23 (D11S1294). Their PCR primer sequences are available in the Genome Data Base (http://www.gdb.org). A highly sensitive seminested PCR assay included a first round that used the primers indicated above. In a second PCR, one of the primers in each pair was replaced by an internal forward primer (Fint) or an internal reverse primer (Rin t ) : 1q42 (D1S517Rin t ) 5 8 CATGTGTCCATCAATGGTAG-38; 4q26 (D4S2301Fint) 58-GATGAGTGCTTAGACCATAGTA-38 ; 9p23 (D9S254Rint) 58-GTCTCCAATGCATGANCTT-38; and 11q23 (D11S1294Rint) 58-CTGGTTTGCTTTCCCTTTCTT-38 (software 4 “Primers! For the WWW”; http://www.williamstone.com) (Fig. 1, A). Allelic dropout during amplification of polymorphic microsatellite fragments may randomly affect either allele mimicking LOH. To exclude this pitfall, pooled samples of 10 purified HRS cells from a given patient were amplified (18,19). Ten picograms of DNA (corresponding to the DNA content of a single diploid cell) was consistently detected by our PCR assay. We also used a seminested PCR (1,3,20–22) to examine HRS cells from patients 3, 4, 6, and 7 for clonally rearranged immunoglobulin heavy-chain genes created through joining of variable, diversity, and joining immunoglobulin gene regions (V-D-J joining). The primers were a framework III immunoglobulin gene VH primer 58ACACGGC(C/T)(G/C)T(G/A)TATTACTGT-38 and a consensus JH primer (21) 58-ACCTGAGGAGACGGTGACC-38. The seminested primer was from VLJH sequences (58-GTGACCAGGT(N)CCTTGGCCCCA-38) (22) (Fig. 1, B). DNA extracted from buccal smears and from bystander lymphocytes showed constitutional individualspecific microsatellite patterns in all patients (Fig. 1, A). Of 259 samples of HRS cells microdissected from formalin-fixed tissue, 59 (23%) yielded a detectable microsatellite PCR product. Mock picks from tissue sections cleared of cellular material were consistently negative by PCR analysis. All patients except patient 5 reproducibly showed clonal LOH in HRS cells at one to three different microsatellite loci (Table 1). LOH was seen at all loci, but the locus most frequently altered was D4S2301 (4q26; four of five informative patients). High-molecular-weight DNA from fresh HRS cells and corresponding degraded DNA from formalin-fixed tissue yielded identical individual-specific microsatellite band patterns (Fig. 1, A). None of the HRS cell samples showed extra microsatellite bands (23). Patients 3, 4, 6, and 7 showed clonal immunoglobulin heavy-chain gene configurations in HRS cells and polyclonal patterns in bystander lymphoid cells (Fig. 1, B). In patient 6, a common rearranged immunoglobulin heavy-chain gene band in HRS cells, observed at presentation (6a) and at relapse (6b), indicated that the initial monoclonal population of HRS cells had relapsed. Our immunogenotype data from formalin-fixed HRS cells are in keeping with published data that HRS cells are mostly monoclonal B-cell populations (1–8,20,24). The detection of LOH has several implications in HD. The patients’ buccal mucosa and bystander lymphocytes showed constitutional microsatellite patterns indicating that LOH in HRS cells was an acquired specific genetic feature of HD. To the best of our knowledge, this is the first report on microsatellite PCR detection of clonal LOH in microdissected HRS cells. In contrast to our approach, classical cytogenetics permit the study of mitotic cells only and usually do not permit the identifi-

Clonal T-cell receptor gamma-chain gene rearrangement by PCR-based GeneScan analysis in advanced cutaneous T-cell lymphoma: a critical evaluation.

Detection of clonal T-cell receptor gamma (TCRgamma)-chain gene rearrangement is a promising approach to distinguish between cutaneous T-cell lymphomas (CTCLs) and reactive T-cell infiltrates. Despite the improved sensitivity by using the polymerase chain reaction (PCR) rather than Southern blot analysis, monoclonality could be demonstrated in only 53-90 per cent of CTCL biopsies in recent studies. In the present study, formalin-fixed, paraffin-embedded specimens of 21 selected patients with clear-cut advanced-stage CTCL were analysed using a semi-nested TCRgamma PCR with newly developed consensus primer pairs. Detection of PCR products was done by GeneScan analysis (GSA); this technique is advantageous due to its sensitivity and accuracy in the detection and size determination of PCR products and it is easier to interpret than direct read-outs from TGGE or DGGE gels. In serial dilution experiments, TCRgamma-PCR-GSA allowed the detection of clonal, rearranged T-cells with a high in vitro sensitivity against a polyclonal background (1-6 per cent). Despite the selection of clear-cut, advanced-stage CTCL cases, however, dominant clonal TCRgamma-chain gene rearrangement was found in only 16 of the 21 patients analysed, indicating an overall clinical sensitivity of 76 per cent. Specificity was evaluated using biopsy specimens from 21 control patients suffering from long-standing psoriasis (n=13) and eczema (n=8). Surprisingly, GeneScan profiles showing apparently single dominant peaks were detected in 14 per cent of these skin lesions, but these profiles turned out to be pseudo-monoclonal by repeated determinations. In conclusion, TCRgamma-PCR-GSA does not suffice reliably to exclude malignancy, due to its limited clinical sensitivity, but with precautions taken to detect pseudo-monoclonality and to secure specificity, TCRgamma-PCR-GSA is a valuable instrument in the diagnosis of CTCL.

Molecular genetic diagnosis of de novo and recurrent bladder cancer.

Bladder cancer, the second most common urological tumor, is usually diagnosed by endoscopy and biopsy of the lower urinary tract. However, this procedure is expensive, can cause discomfort to the patient and is a source of infection. Commercially available diagnostic systems measure protein byproducts of bladder carcinoma in voided urine; their sensitivity is only between 60-80%. Polymerase chain reaction (PCR)-based microsatellite analysis of the urine sediment (MAUS) is a noninvasive, inexpensive and easily performed analytical method which was introduced in the de novo diagnosis and follow-up of bladder cancer. By utilizing the PCR with 20 polymorphic microsatellite markers on different chromosomes and separating PCR products by electrophoresis on 7% denaturing polyacrylamide-formamide-urea slab gels, a 91% diagnostic sensitivity could be achieved. In order to minimize costs and analysis time, the separation and detection of PCR products was carried out by capillary array electrophoresis and two-color fluorescent primer labeling/laser beam detection in another study. The accuracy of both methods was the same. In either detection system, MAUS is an accurate and promising tool in the noninvasive diagnosis of bladder cancer.

Detection of PCR products in solution using surface plasmon resonance.

Polymerase chain reaction (PCR) products were detected using a flow injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7.

Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis

Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and beta-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at approximately 90 degrees C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85+/-1.15 degrees C. In addition, multiplex assays using both MBR/JH and beta-globin primers yielded easily distinguishable fluorescence melting peaks at approximately 90 degrees C and 81.2 degrees C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method for the detection of MBR/JH translocations. The feasibility of specific PCR product detection without electrophoresis or utilization of expensive fluorescently labeled probes makes this method attractive for routine molecular diagnostics.

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 103copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100μl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.

Highly sensitive chemiluminescent method for the detection of cell contamination

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.

Highly sensitive chemiluminescent method for the detection of cell contamination.

Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination.

Colourimetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae

The development of a nested polymerase chain reaction (PCR) assay is described to detect low concentrations of monodon baculovirus (MBV) DNA from total Penaeus monodon postlarval DNA. A modified DNA extraction procedure was also developed to circumvent problems associated with co-purification of PCR inhibitors in total DNA extracted from whole postlarvae. This method involved mechanical disruption of frozen prawn material immediately followed by phenol extraction at high temperature. An assessment of the sensitivity of the assay demonstrated detection down to eight viral genome equivalents. The PCR was shown to be specific for MBV DNA by not amplifying prawn DNA or DNA preparations of Baculovirus penaei (BP), white spot baculovirus (WSBV), bennettae baculovirus and insect Autographica californica nuclear polyhedrosis virus (NPV). A colourimetric method of PCR product detection was used to simplify final analysis.

Detection of Aspergillus fumigatus PCR products by a microtitre plate based DNA hybridisation assay.

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this...

Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts

The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32 P− labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNγ. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNγ mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.

Development of a Sensitive Colorimetric-PCR Assay for Detection of Viruses in Woody Plants.

Diagnostic methods employing the polymerase chain reaction (PCR) provide the most sensitive means currently available for detecting viruses in woody plants. A new technique has been tested that does not rely on gel electrophoresis or molecular hybridization to detect virus-specific PCR products. This colorimetric method for detection of PCR products from woody plants was demonstrated to be at least as sensitive as gel analysis. When combined with immunocapture of virions from plant sap, colorimetric detection provides a means to apply PCR technology to a large number of samples. Here, we report on the use of this technique for detection and quantitation of a walnut isolate of cherry leafroll virus (CLRV-W), citrus tristeza virus (CTV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) in woody and herbaceous plants. For purified virus preparations, detection limits ranged from 100 pg/ml to 100 ag/ml. We also describe the colorimetric PCR detection of CTV in pooled samples.

Detection of Cryptosporidium parvum oocysts in municipal water samples by the polymerase chain reaction

The enteric protozoan, Cryptosporidium parvum, the causative agent for cryptosporidiosis, has been isolated from drinking water, fecal samples from humans and animals, and environmental samples such as sediment and soil. The currently available water sampling methods for detection of this parasite are labor-intensive and the efficiency of oocyst recovery is poor. A recent improved method utilizing membrane filtration and dissolution followed by polymerase chain reaction (PCR) amplification, and confirmatory nested PCR was evaluated for the sensitive and specific detection of C. parvum oocysts. Detection of PCR products by the ELISA-based Digene SHARP Signal™ System Assay was assessed for sensitivity. Seventy-two municipal water samples ranging in volume from 230 to 1,000 l from southwestern Ontario, Canada were spiked with varying concentrations of formalin-killed C. parvum oocysts for use in this study. Oocyst recovery on the filters was determined by the Merifluor immunofluoresence assay for Cryptosporidium/Giardia. Oocyst detection using the PCR assay showed an 84.7% correlation with immunofluoresence assay (IFA) results. During optimization studies, the correlation between PCR and IFA reached 98%. The sensitivity of a primary PCR assay ranged from 1 to 10 oocysts per reaction, which was equivalent to 10 2 to 10 3 oocysts per 100 l municipal water. The PCR assay also showed potential for application to untreated water samples and naturally contaminated municipal water from a recent Cryptosporidium outbreak. Further application of nested PCR may improve overall sensitivity and specificity for detecting C. parvum in municipal water samples since combined primary and nested PCR results showed 97.2% correlation with IFA. The Digene SHARP Signal™ System assay offers a sensitive and specific alternative for detection of C. parvum amplification products.