Impact of Capsid and Genomic Integrity Tests on Norovirus Extraction Recovery Rates.

Human norovirus (HuNoV) is the leading pathogen responsible for food-borne illnesses. However, both infectious and non-infectious HuNoV can be detected by RT-qPCR. This study evaluated the efficiency of different capsid integrity treatments coupled with RT-qPCR or a long-range viral RNA (long RT-qPCR) detection to reduce the recovery rates of heat inactivated noroviruses and fragmented RNA. The three capsid treatments evaluated (RNase, the intercalating agent PMAxx and PtCl4) reduced the recovery of heat inactivated HuNoV and murine norovirus (MNV) spiked on lettuce, when combined with the ISO 15216-1:2017 extraction protocols. However, PtCl4 also reduced non-heat-treated noroviruses recovery as estimated by RT-qPCR. The PMAxx and RNase treatments had a similar effect on MNV only. The most efficient approaches, the RNase and PMAxx treatments, reduced the heat-inactivated HuNoV recovery rates estimated using RT-qPCR by 2 and >3 log, respectively. The long RT-qPCR detection approach also reduced the recovery rates of heat inactivated HuNoV and MNV by 1.0 and 0.5 log, respectively. Since the long-range viral RNA amplification could be applied to verify or confirm RT-qPCR results, it also provides some advantages by reducing the risk of false positive HuNoV results.

Molecular identification of Cryptosporidium species in Canadian post-weaned calves and adult dairy cattle.

Cryptosporidium is a zoonotic protozoan parasite that is distributed globally and impacts both human and animal health. There are over 40 species of Cryptosporidium described to date, of which four (C. parvum, C. bovis, C. ryanae and C. andersoni) are routinely reported in cattle. The goal of this study was to identify the Cryptosporidium species infecting dairy cattle from across Canada using cow fecal samples and post-weaned calf rectal swabs obtained through the Canadian National Dairy Study. A total of 353 cattle samples (117 pooled rectal fecal swabs from post-weaned calves and 236 cow fecal samples) from 175 herds across the 10 Canadian provinces were analysed by targeting Cryptosporidium's small subunit ribosomal RNA (SSU rRNA or 18S) gene. Herd prevalence of Cryptosporidium was 27.4% nationally, ranging from 0% in Saskatchewan (SK) to 62% in Prince Edward Island (PE). The national prevalence of Cryptosporidium cattle infections was 15.4% in pooled rectal fecal swab samples from post-weaned calves and 16.1% in adult cows. Sanger sequence analysis of the SSU rRNA gene target revealed that C. bovis, C. andersoni and C. ryanae occurred in both adults and post-weaned calves, with C. bovis as the predominant species detected in pooled fecal swab samples of post-weaned calves (9/18, 50%) and C. andersoni as the predominant species in cows (25/38, 66%). Cryptosporidium parvum was not observed in any of the pooled rectal swab samples from post-weaned calves but was observed in one mixed infection of C. bovis/C. parvum in an adult cow. The fifth species identified in this study was C. muris and was present in two adult cows. Low concentrations of oocyst equivalents, as measured by quantitative real-time PCR (qPCR) of the SSU rRNA gene copy number, were observed in a subset of cattle samples. Cryptosporidium andersoni concentrations varied from province to province, with the widest range and highest counts in cows from PE. In conclusion, oocysts from Cryptosporidium species observed in this study are shed into the environment, contributing to the environmental load. However, the Cryptosporidium species in the post-weaned calves and cows found in this study pose a lower risk to the dairy cattle than if they were infected with C. parvum. Similarly, these Cryptosporidium species pose only a small risk to public health as the three species are infrequently reported in humans globally and have not been reported in Canadians to date.

Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations.

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.

Draft Hybrid Genome Assembly of a Canadian Cyclospora cayetanensis Isolate.

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented in 313 contigs.

CryptoGenotyper: A new bioinformatics tool for rapid Cryptosporidium identification.

Cryptosporidium is a protozoan parasite that is transmitted to both humans and animals through zoonotic or anthroponotic means. When a host is infected with this parasite, it causes a gastrointestinal disease known as cryptosporidiosis. To understand the transmission dynamics of Cryptosporidium, the small subunit (SSU or 18S) rRNA and gp60 genes are commonly studied through PCR analysis and conventional Sanger sequencing. However, analyzing sequence chromatograms manually is both time consuming and prone to human error, especially in the presence of poorly resolved, heterozygous peaks and the absence of a validated database. For this study, we developed a Cryptosporidium genotyping tool, called CryptoGenotyper, which has the capability to read raw Sanger sequencing data for the two common Cryptosporidium gene targets (SSU rRNA and gp60) and classify the sequence data into standard nomenclature. The CryptoGenotyper has the capacity to perform quality control and properly classify sequences using a high quality, manually curated reference database, saving users' time and removing bias during data analysis. The incorporated heterozygous base calling algorithms for the SSU rRNA gene target resolves double peaks, therefore recovering data previously classified as inconclusive. The CryptoGenotyper successfully genotyped 99.3% (428/431) and 95.1% (154/162) of SSU rRNA chromatograms containing single and mixed sequences, respectively, and correctly subtyped 95.6% (947/991) of gp60 chromatograms without manual intervention. This new, user-friendly tool can provide both fast and reproducible analyses of Sanger sequencing data for the two most common Cryptosporidium gene targets.

Molecular characterization of Cryptosporidium isolates from humans in Ontario, Canada.

BACKGROUND: Cryptosporidiosis is a gastrointestinal disease with global distribution. It has been a reportable disease in Canada since 2000; however, routine molecular surveillance is not conducted. Therefore, sources of contamination are unknown. The aim of this project was to identify species and subtypes of Cryptosporidium in clinical cases from Ontario, the largest province in Canada, representing one third of the Canadian population, in order to understand transmission patterns. METHODS: A total of 169 frozen, banked, unpreserved stool specimens that were microscopy positive for Cryptosporidium over the period 2008-2017 were characterized using molecular tools. A subset of the 169 specimens were replicate samples from individual cases. DNA was extracted directly from the stool and nested PCR followed by Sanger sequencing was conducted targeting the small subunit ribosomal RNA (SSU) and glycoprotein 60 (gp60) genes. RESULTS: Molecular typing data and limited demographic data were obtained for 129 cases of cryptosporidiosis. Of these cases, 91 (70.5 %) were due to Cryptosporidium parvum and 24 (18.6%) were due to Cryptosporidium hominis. Mixed infections of C. parvum and C. hominis occurred in four (3.1%) cases. Five other species observed were Cryptosporidium ubiquitum (n = 5), Cryptosporidium felis (n = 2), Cryptosporidium meleagridis (n = 1), Cryptosporidium cuniculus (n = 1) and Cryptosporidium muris (n = 1). Subtyping the gp60 gene revealed 5 allelic families and 17 subtypes of C. hominis and 3 allelic families and 17 subtypes of C. parvum. The most frequent subtype of C. hominis was IbA10G2 (22.3%) and of C. parvum was IIaA15G2R1 (62.4%). CONCLUSIONS: The majority of isolates in this study were C. parvum, supporting the notion that zoonotic transmission is the main route of cryptosporidiosis transmission in Ontario. Nonetheless, the observation of C. hominis in about a quarter of cases suggests that anthroponotic transmission is also an important contributor to cryptosporidiosis pathogenesis in Ontario.

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation.

BACKGROUND: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia. METHODS: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. RESULTS: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia. CONCLUSIONS: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.

Prevalence and genetic characterization of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut, Canada.

There are few epidemiologic studies on the role of dogs in zoonotic parasitic transmission in the Circumpolar North. The objectives of this study were to: (a) estimate the faecal prevalence of Giardia spp. and Cryptosporidium spp. in dogs; (b) investigate potential associations between the type of dog population and the faecal presence of Giardia spp. and Cryptosporidium spp.; and (c) describe the molecular characteristics of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut. We conducted two cross-sectional studies in July and September 2016. In July, the team collected daily faecal samples for 3 days from each of 20 sled dogs. In September, the team collected three faecal samples from each of 59 sled dogs, 111 samples from shelter dogs and 104 from community dogs. We analysed faecal samples for the presence of Giardia spp. and Cryptosporidium spp. using rapid immunoassay and flotation techniques. Polymerase chain reaction (PCR) and sequencing of target genes were performed on positive faecal samples. Overall, the faecal prevalence of at least one of the target parasites, when one faecal sample was chosen at random for all dogs, was 8.16% (CI: 5.52-11.92), and for Giardia spp. and Cryptosporidium spp., prevalence was 4.42% (CI: 2.58-7.49) and 6.12% (CI: 3.88-9.53), respectively. The odds of faecal Giardia spp. in sled dogs were significantly higher than those in shelter and community dogs (OR 10.19 [CI: 1.16-89.35]). Sequence analysis revealed that 6 faecal samples were Giardia intestinalis, zoonotic assemblage B (n = 2) and species-specific assemblages D (n = 3) and E (n = 1), and five faecal samples were Cryptosporidium canis. Giardia intestinalis is zoonotic; however, Cryptosporidium canis is rare in humans and, when present, usually occurs in immunosuppressed individuals. Dogs may be a potential source of zoonotic Giardia intestinalis assemblage B infections in residents in Iqaluit, Nunavut, Canada; however, the direction of transmission is unclear.

Weather, environmental conditions, and waterborne Giardia and Cryptosporidium in Iqaluit, Nunavut.

Indigenous communities in the Arctic often face unique drinking water quality challenges related to inadequate infrastructure and environmental contamination; however, limited research exists on waterborne parasites in these communities. This study examined Giardia and Cryptosporidium in untreated surface water used for drinking in Iqaluit, Canada. Water samples (n = 55) were collected weekly from June to September 2016 and tested for the presence of Giardia and Cryptosporidium using microscopy and polymerase chain reaction (PCR). Exact logistic regressions were used to examine associations between parasite presence and environmental exposure variables. Using microscopy, 20.0% of samples tested positive for Giardia (n = 11) and 1.8% of samples tested positive for Cryptosporidium (n = 1). Low water temperatures (1.1 to 6.7 °C) and low air temperatures (-0.1 to 4.5 °C) were significantly associated with an increased odds of parasite presence (p = 0.047, p = 0.041, respectively). These results suggest that surface water contamination with Giardia and Cryptosporidium may be lower in Iqaluit than in other Canadian regions; however, further research should examine the molecular characterization of waterborne parasites to evaluate the potential human health implications in Northern Canada.

Draft Genome Assembly of a Potentially Zoonotic Cryptosporidium parvum Isolate, UKP1.

Cryptosporidium parvum is a zoonotic protozoan parasite that causes food and waterborne gastrointestinal disease and whose major animal reservoirs are cattle and small ruminants. We report here on a draft whole-genome sequence of a zoonotic isolate of C. parvum isolated from a person with cryptosporidiosis.

Campylobacter in recreational lake water in southern Quebec, Canada: presence, concentration, and association with precipitation and ruminant farm proximity.

Campylobacter is an important cause of gastrointestinal illness and exposure to recreational water is one potential source of infection. The objective of this study was to investigate the presence and concentrations of Campylobacter, and determine the influence of agricultural activities and precipitation on their presence, at lake beaches used for water recreation in southern Quebec, Canada. A total of 413 water samples were collected from June to August, from 22 beaches, between 2011 and 2013. The overall proportion of positive water samples was estimated to be 33.9% (95% CI: 27.7, 40.1) for C. jejuni and 49.7% (95% CI: 41.8, 57.6) for Campylobacter spp. The concentrations of both thermotolerant Campylobacter spp. and C. jejuni ranged from 20 to 900 bacteria/L of water. Logistic regressions showed that the presence of C. jejuni and Campylobacter spp. was significantly associated with the year and season. Other significant predictors of C. jejuni, but not Campylobacter spp., included the presence of precipitation the day before sampling and the presence of ruminant farms within a 5 km radius of the beach. The present study provides insights into the risk of Campylobacter presence in recreational lake water for better understanding public health risks.

Evaluation of real-time PCR assays and standard curve optimisation for enhanced accuracy in quantification of Campylobacter environmental water isolates.

Campylobacter is a major public health and economic burden in developed and developing countries. This study evaluated published real-time PCR (qPCR) assays for detection of Campylobacter to enable selection of the best assays for quantification of C. spp. and C. jejuni in environmental water samples. A total of 9 assays were compared: three for thermotolerant C. spp. targeting the 16S rRNA and six for C. jejuni targeting different genes. These assays were tested in the wet-lab for specificity and sensitivity against a collection of 60, genetically diverse, Campylobacter isolates from environmental water. All three qPCR assays targeting C. spp. were positive when tested against the 60 isolates, whereas, assays targeting C. jejuni differed among each other in terms of specificity and sensitivity. Three C. jejuni-specific assays that demonstrated good specificity and sensitivity when tested in the wet-lab showed concordant results with in silico-predicted results obtained against a set of 211 C. jejuni and C. coli genome sequences. Two of the assays targeting C. spp. and C. jejuni were selected to compare DNA concentration estimation, using spectrophotometry and digital PCR (dPCR), in order to calibrate standard curves (SC) for greater accuracy of qPCR-based quantification. Average differences of 0.56±0.12 and 0.51±0.11 log fold copies were observed between the spectrophotometry-based SC preparation and the dPCR preparation for C. spp. and C. jejuni, respectively, demonstrating an over-estimation of Campylobacter concentration when spectrophotometry was used to calibrate the DNA SCs. Our work showed differences in quantification of aquatic environmental isolates of Campylobacter between qPCR assays and method-specific bias in SC preparation. This study provided an objective analysis of qPCR assays targeting Campylobacter in the literature and provides a framework for evaluating novel assays.

Quantification of viable Giardia cysts and Cryptosporidium oocysts in wastewater using propidium monoazide quantitative real-time PCR.

Real-time PCR (qPCR) is a rapid tool to quantify pathogens in the aquatic environment; however, it quantifies all pathogens, including both viable and nonviable. Propidium monoazide (PMA) is a membrane-impairment dye that penetrates only membrane-damaged cells. Once inside the cell, PMA is covalently cross-linked to DNA through light photoactivation, and PCR amplification is strongly inhibited. The goal of this study was to evaluate PMA-qPCR assays for rapid quantification of viable and heat-treated Giardia cysts and Cryptosporidium oocysts in wastewater. We observed a reduction in detection of heat-treated Giardia duodenalis cysts of 83.2, 89.9, 98.2, or 97% with PMA-qPCR assays amplifying a 75 base-pair (bp) ß-giardin target, 77-bp triosephosphate isomerase (tpi), 133-bp glutamate dehydrogenase (GDH), and 143-bp ß-giardin gene target, respectively. Thus, the exclusion of dead cysts was more effective when qPCR assays that produced larger amplicons were used. The PMA treatment of Cryptosporidium oocysts plus/minus heat treatment abolished the fluorescent signal for dead oocysts with a PMA-qPCR assay amplifying a Cryptosporidium parvum (150-bp) oocyst wall protein (COWP) gene. The PMA-qPCR 143-bp ß-giardin assay for Giardia and the PMA-qPCR 150-bp COWP assay for Cryptosporidium accurately quantified live oo(cysts), and failed to detect dead oo(cysts), when phosphate-buffered saline and tertiary effluent wastewater were spiked with concentrations of 10(3) or 10(2) dead oo(cysts), respectively. Therefore, these assays are suitable for the detection of viable parasites that are typically present in tertiary wastewater effluents at concentrations of <10(3) oo(cysts)/l and can provide rapid risk assessments of environmental water.

Quantification of E. coli O157 and STEC in feces of farm animals using direct multiplex real time PCR (qPCR) and a modified most probable number assay comprised of immunomagnetic bead separation and qPCR detection.

To better understand Escherichia coli O157:H7 on-farm transmission dynamics requires sensitive methods for quantification of a broad range of concentrations of target organisms. For this purpose, a multiplex real time PCR (qPCR) assay was developed for quantification of O157 E. coli from 1g fecal samples of cattle and other animal species, targeting the Shiga toxin genes (stx1 and stx2) and the O157 somatic antigen gene, per. The multiplex qPCR assay provided specific detection across a broad range of bacterial concentrations with a lower limit of detection (LOD) of 10(1) genome copies which is equivalent to 10(1) bacteria. However, the LOD, when direct qPCR was applied to quantification of the targets in the feces of dairy cattle, was 10(3) genome copies per gram of feces. Enumeration below the threshold for direct qPCR was performed using a modified most probable number (mMPN) method whereby E. coli O157 in enriched samples was isolated using immunomagnetic bead separation (IMS) and detected using qPCR, thus reducing the time and logistic constraints of biochemical/serological/gel analysis. Application of the mMPN (IMS/qPCR) assay to samples that were negative when tested using direct qPCR alone permitted quantification of low levels of E. coli O157 below levels detectable with direct qPCR. The direct qPCR and mMPN (IMS/qPCR) assays were applied to fecal samples from dairy, beef, swine and poultry feces. This approach can be employed to gain a better understanding of the patterns of infection in animals for analysis of on-farm transmission dynamics, for evaluating the effects of on-farm control strategies and for risk assessment in public health.

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef.

The purpose of this study was to apply our rapid, integrated double enrichment 5' nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.

A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses.

A rapid test for microbial quantification in carcass and environmental swabs that does not require enrichment and provides results in less than 4 h is described here. Steps in the assay include the rapid concentration of bacteria on sponge swabs by vacuum filtration followed by real-time PCR detection. The assay has been applied for the detection of coliforms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on carcass swabs and environmental samples in a slaughterhouse-processing line. Comparison of this rapid method with standard culture techniques for coliform counts on beef and pork carcass swabs revealed higher numbers of bacteria (2- to 50-fold) by the rapid test compared with the plate counts. This was due to the detection of all bacteria (live, dead, and non-culturable forms) in the rapid assay. To allow detection of only viable bacteria, concentrated samples were treated with ethidium monoazide (EMA) prior to DNA extraction and real-time PCR detection, thereby preventing the amplification of DNA from bacteria with damaged cell walls and allowing only the DNA from bacteria with intact membranes to be detected. EMA treatment resulted in a significant reduction (P < 0.001) in the number of coliforms detected compared to real-time PCR without EMA treatment. In beef swabs, the counts obtained in EMA real-time PCR were not significantly different (P < 0.08) from the culture counts and the correlation coefficient between the two assays was 0.7385. A lower correlation coefficient (0.402) was obtained with pork swabs. The assay described herein has the potential to be applied on a routine basis to slaughterhouse lines for the detection of indicator organisms or specific pathogens.

Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens.

Real-time PCR, using dual-labeled fluorescent probes targeting the beta-giardin gene, was used to detect Giardia lamblia in human stool specimens and to discriminate between isolates from the two major genetic assemblages of G. lamblia infective to humans, assemblages A and B.

Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage.

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.

Application of quantitative real-time PCR with dual-labeled hydrolysis probes to microbial water quality monitoring.

Issues of water quality are a global problem with potentially devastating results in communities if microbial levels are not monitored and controlled effectively.This is especially true with the potential threat of bioterrorist contamination of water supplies.This study presents a method for quantifying microbial water pathogens by 5' nuclease real-time polymerase chain reaction analysis, thus decreasing the assay time (under 2 h for completion of thermal cycling and analysis) and increasing the sensitivity and precision of detection as compared with traditionally used assays. We have quantified Escherichia coli, toxigenic E. coli O157:H7, the microcystin-producing cyanobacterium Microcystis aeruginosa, and the protozoan parasite Giardia lamblia. Our measurements have detected as few as three cells per sample for the bacterial targets and one cell per sample for G. lamblia. The quantification of total E. coli and microcystin-producing cyanobacteria has been extended to the analysis of environmental samples.