Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

ABSTRACT Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r 2 = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.

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Triplex Real-Time PCR without DNA Extraction for the Monitoring of Meningococcal Disease

Diagnostics ◽

10.3390/diagnostics8030058 ◽

2018 ◽

Vol 8 (3) ◽

pp. 58 ◽

Cited By ~ 1

Author(s):

Melissa Whaley ◽

Laurel Jenkins ◽

Fang Hu ◽

Alexander Chen ◽

Seydou Diarra ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Real Time ◽

Dna Extraction ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Limit Of Detection ◽

Detection Range ◽

Clinical Specimens ◽

Large Numbers ◽

Pcr Assays

Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178–5264 CFU/mL by monoplex and 68–2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.

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Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers

Applied and Environmental Microbiology ◽

10.1128/aem.02343-08 ◽

2009 ◽

Vol 75 (10) ◽

pp. 3045-3054 ◽

Cited By ~ 109

Author(s):

Sophie Mieszkin ◽

Jean-Pierre Furet ◽

Gï¿½rard Corthier ◽

Michï¿½le Gourmelon

Keyword(s):

16S Rrna ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Fecal Contamination ◽

Fecal Pollution ◽

Rrna Gene ◽

River Catchment ◽

Pig Farms ◽

Cattle Farms

ABSTRACT The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log10 copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log10 copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log10 copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.

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Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

Applied and Environmental Microbiology ◽

10.1128/aem.07749-11 ◽

2012 ◽

Vol 78 (8) ◽

pp. 2613-2622 ◽

Cited By ~ 57

Author(s):

Jana Junick ◽

Michael Blaut

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Housekeeping Gene ◽

Cell Number ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Bifidobacterium Adolescentis ◽

Fecal Samples ◽

Content Type ◽

Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

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A comparative study of three different PCR assays for detection of Mycoplasma genitalium in urogenital specimens from men and women

Journal of Medical Microbiology ◽

10.1099/jmm.0.47498-0 ◽

2008 ◽

Vol 57 (3) ◽

pp. 304-309 ◽

Cited By ~ 33

Author(s):

Andreas Edberg ◽

Margaretha Jurstrand ◽

Eva Johansson ◽

Elisabeth Wikander ◽

Anna Höög ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Comparative Study ◽

Real Time ◽

Mycoplasma Genitalium ◽

Prevalence Study ◽

Rrna Gene ◽

True Positive ◽

Men And Women ◽

Pcr Assays

The aim of this study was to compare conventional 16S rRNA gene PCR, real-time 16S rRNA gene PCR and real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR as detection methods for M. genitalium infection. The study also determined the prevalence of M. genitalium in male and female patients attending a sexually transmitted infections clinic in a rural area in the west of Sweden. First void urine (FVU) and/or urethral swabs were collected from 381 men, and FVU and/or cervical swabs and/or urethral swabs were collected from 298 women. A total of 213 specimens were used in the PCR comparative study: 98 consecutively sampled specimens from patients enrolled in the prevalence study, 36 consecutively sampled specimens from patients with symptoms of urethritis and 79 specimens from patients positive for M. genitalium by real-time MgPa gene PCR in the prevalence study. A true-positive M. genitalium DNA specimen was defined as either a specimen positive in any two PCR assays or a specimen whose PCR product was verified by DNA sequencing. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %), respectively. In the PCR comparative study, M. genitalium DNA was detected in 61/76 (80.3 %) of true-positive specimens by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Real-time MgPa gene PCR thus had higher sensitivity compared with conventional 16S rRNA gene PCR and had considerably increased sensitivity compared with real-time 16S rRNA gene PCR for detection of M. genitalium DNA. Real-time MgPa gene PCR is well suited for the clinical diagnosis of M. genitalium.

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Comparison of the Host Specificities of Two Bacteroidales Quantitative PCR Assays Used for Tracking Human Fecal Contamination

Applied and Environmental Microbiology ◽

10.1128/aem.00239-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 6258-6260 ◽

Cited By ~ 20

Author(s):

Laurie C. Van De Werfhorst ◽

Bram Sercu ◽

Patricia A. Holden

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

False Positive ◽

Fecal Contamination ◽

Real Time Quantitative Pcr ◽

Pcr Assays ◽

Positive Results

ABSTRACTThe sewage-associated real-time quantitative PCR (qPCR) assays BacHum and HF183 SYBR were compared for specificity against local fecal sources. Both assays were equally sensitive to sewage, but BacHum showed substantially more false-positive results for cat, dog, gull, and raccoon feces.

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