scholarly journals Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

2007 ◽  
Vol 74 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Orin C. Shanks ◽  
Emina Atikovic ◽  
A. Denene Blackwood ◽  
Jingrang Lu ◽  
Rachel T. Noble ◽  
...  
Keyword(s):  
Genetic Marker ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Broad Distribution ◽  
Assay Performance ◽  
Target Dna ◽  
Bovine Feces

ABSTRACTAccurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing totalBacteroidetes,Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.

scholarly journals Quantitative PCR for Genetic Markers of Human Fecal Pollution

2009 ◽  
Vol 75 (17) ◽  
pp. 5507-5513 ◽  
Author(s):  
Orin C. Shanks ◽  
Catherine A. Kelty ◽  
Mano Sivaganesan ◽  
Manju Varma ◽  
Richard A. Haugland
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Quantitative Methods ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Target Dna ◽  
Pcr Assays ◽  
Wastewater Samples ◽  
Human Specific

ABSTRACT Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 � 106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

scholarly journals Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

2010 ◽  
Vol 76 (5) ◽  
pp. 1359-1366 ◽  
Author(s):  
Orin C. Shanks ◽  
Karen White ◽  
Catherine A. Kelty ◽  
Sam Hayes ◽  
Mano Sivaganesan ◽  
...  
Keyword(s):  
Genetic Markers ◽  
A Priori ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
End Point ◽  
Assay Performance ◽  
Different Populations

ABSTRACT There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

scholarly journals Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor

2011 ◽  
Vol 77 (19) ◽  
pp. 6972-6981 ◽  
Author(s):  
Ryan J. Newton ◽  
Jessica L. VandeWalle ◽  
Mark A. Borchardt ◽  
Marc H. Gorelick ◽  
Sandra L. McLellan
Keyword(s):  
Sequence Analysis ◽  
Genetic Marker ◽  
Microbial Community Composition ◽  
Fold Increase ◽  
Fecal Pollution ◽  
Plate Count ◽  
Rrna Gene ◽  
Fecal Indicators ◽  
Fecal Indicator ◽  
Content Type

ABSTRACTThe complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within theLachnospiraceaefamily, which was closely related to the genusBlautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for humanBacteroidales(based on the HF183 genetic marker), totalBacteroidalesspp., and enterococci and the conventionalEscherichia coliand enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and humanBacteroidalesincreased specificity to detect sewage compared to general indicators, and the relationship to a human pathogen group suggests that the use of these alternative indicators will improve assessments for human health risks in urban waters.

scholarly journals Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

2020 ◽  
Author(s):  
Abigail Hui En Chan ◽  
Kittipong Chaisiri ◽  
Serge Morand ◽  
Naowarat Saralamba ◽  
Urusa Thaenkham
Keyword(s):  
Genetic Marker ◽  
Genetic Markers ◽  
Molecular Systematics ◽  
Ribosomal Rna ◽  
Ribosomal Rna Genes ◽  
16S Ribosomal Rna ◽  
12S Rrna ◽  
Rrna Gene ◽  
12S Rrna Gene ◽  
Rna Genes

Abstract Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

scholarly journals Correlation of Quantitative PCR for a Poultry-SpecificBrevibacteriumMarker Gene with Bacterial and Chemical Indicators of Water Pollution in a Watershed Impacted by Land Application of Poultry Litter

2011 ◽  
Vol 77 (6) ◽  
pp. 2094-2102 ◽  
Author(s):  
Jennifer L. Weidhaas ◽  
Tamzen W. Macbeth ◽  
Roger L. Olsen ◽  
Valerie J. Harwood
Keyword(s):  
Quantitative Pcr ◽  
Environmental Samples ◽  
Marker Gene ◽  
Poultry Litter ◽  
Land Application ◽  
Fecal Pollution ◽  
Health Concern ◽  
Rrna Gene ◽  
Fecal Indicator ◽  
The Impact

ABSTRACTThe impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene ofBrevibacteriumsp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations ofEscherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.

scholarly journals Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

2014 ◽  
Vol 81 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Vikram Kapoor ◽  
Tarja Pitkänen ◽  
Hodon Ryu ◽  
Michael Elk ◽  
David Wendell ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Fecal Pollution ◽  
Detection Sensitivity ◽  
Rrna Gene ◽  
Fecal Bacteria ◽  
Combined Sewer Overflows ◽  
Fecal Indicator ◽  
Content Type ◽  
Detection Frequency ◽  
Human Specific

ABSTRACTThe identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. Furthermore, it has been shown that rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) assays can be more sensitive than rRNA gene-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host-specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8, 87, and 85% of the samples positive for general enterococci,Enterococcus faecalis, andEnterococcus faeciummarkers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specificBacteroidalesmarkers were not detected when using DNA in 12% of the samples, while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specificBacteroidalesmarkers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of nonhuman sources of fecal pollution or the presence of false-positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.

scholarly journals Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces

2013 ◽  
Vol 80 (5) ◽  
pp. 1588-1594 ◽  
Author(s):  
Orin C. Shanks ◽  
Catherine A. Kelty ◽  
Lindsay Peed ◽  
Mano Sivaganesan ◽  
Thomas Mooney ◽  
...  
Keyword(s):  
Water Quality ◽  
Genetic Marker ◽  
Genetic Markers ◽  
Surface Water Quality ◽  
The United States ◽  
Fecal Pollution ◽  
Human Pathogens ◽  
Fecal Indicator ◽  
Content Type ◽  
Age Related

ABSTRACTCalves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers forBacteroidales,Escherichia coli, andEnterococcusspp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.

scholarly journals Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

2020 ◽  
Author(s):  
Abigail Hui En Chan ◽  
Kittipong Chaisiri ◽  
Serge Morand ◽  
Naowarat Saralamba ◽  
Urusa Thaenkham
Keyword(s):  
Genetic Marker ◽  
Genetic Markers ◽  
Molecular Systematics ◽  
Ribosomal Rna ◽  
Ribosomal Rna Genes ◽  
16S Ribosomal Rna ◽  
12S Rrna ◽  
Rrna Gene ◽  
12S Rrna Gene ◽  
Rna Genes

Abstract Background: Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics.Methods: To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results: The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions: This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking

2017 ◽  
Vol 231 ◽  
pp. 1569-1577 ◽  
Author(s):  
Lihua Fan ◽  
Jiangbing Shuai ◽  
Ruoxue Zeng ◽  
Hongfei Mo ◽  
Suhua Wang ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Fecal Pollution ◽  
Pcr Assays

scholarly journals Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes

2020 ◽  
Author(s):  
Abigail Hui En Chan ◽  
Kittipong Chaisiri ◽  
Serge Morand ◽  
Naowarat Saralamba ◽  
Urusa Thaenkham
Keyword(s):  
Genetic Marker ◽  
Genetic Markers ◽  
Molecular Systematics ◽  
Ribosomal Rna ◽  
Ribosomal Rna Genes ◽  
16S Ribosomal Rna ◽  
12S Rrna ◽  
Rrna Gene ◽  
12S Rrna Gene ◽  
Rna Genes

Abstract Background Molecular advances have accelerated our understanding of nematode systematics and taxonomy. However, comparative analyzes between various genetic markers have led to discrepancies in nematode phylogenies. This study aimed to evaluate the suitability of using mitochondrial 12S and 16S ribosomal RNA genes for nematode molecular systematics. Methods To study the suitability of mitochondrial 12S and 16S ribosomal RNA genes as genetic markers for nematode molecular systematics, we compared them with the other commonly used genetic markers, nuclear internal transcribed spacer 1 and 2 regions, nuclear 18S and 28S ribosomal RNA genes, and mitochondrial cytochrome c oxidase subunit 1 gene. After that, phylum-wide primers for mitochondrial 12S and 16S ribosomal RNA genes were designed, and parasitic nematodes of humans and animals from 75 taxa with 21 representative species were inferred through phylogenetic analyzes. Phylogenetic analyzes were carried out using maximum likelihood and Bayesian inference algorithms. Results The phylogenetic relationships of nematodes based on the mitochondrial 12S rRNA gene supported the monophyly of nematodes in clades I, IV, and V, reinforcing the potential of this gene as a genetic marker for nematode systematics. In contrast, the mitochondrial 16S rRNA gene only supported the monophyly of clades I and V, providing evidence that the 12S rRNA gene is more suitable for nematode molecular systematics. In this study, subclades of clade III containing various nematode families were not monophyletic when the 16S or 12S rRNA gene was used as the genetic marker. This is similar to the phylogenetic relationship revealed by previous studies using whole mitochondrial genomes as genetic markers. Conclusions This study supports the use of the 12S rRNA gene as a genetic marker for studying the molecular systematics of nematodes to understand intra-phyla relationships. Phylum-wide primers for nematodes using mitochondrial ribosomal genes were prepared, which may enhance future studies. Furthermore, sufficient genetic variation in the mitochondrial 12S and 16S rRNA genes between species also allowed for accurate taxonomy to species level, revealing the potential of these two genes as genetic markers for DNA barcoding.

Sign in / Sign up

Export Citation Format

Share Document