Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

ABSTRACTQuantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genusBacteroidesare among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

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Pollution Source-Targeted Water Safety Management: Characterization of Diffuse Human Fecal Pollution Sources with Land Use Information, Strategic Water Sampling, and Quantitative Real-Time PCR

Global Water Pathogen Project ◽

10.14321/waterpathogens.83 ◽

2019 ◽

Author(s):

Orin Shanks ◽

◽

Lindsay A. Peed ◽

Catherine A. Kelty ◽

Mano Sivaganesan ◽

...

Keyword(s):

Land Use ◽

Real Time ◽

Real Time Pcr ◽

Safety Management ◽

Pollution Source ◽

Fecal Pollution ◽

Pollution Sources ◽

Water Sampling ◽

Water Safety

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Detection and Characterization of Circulating Tumor Cells by Quantitative Real-Time PCR

Methods in Molecular Biology - Quantitative Real-Time PCR ◽

10.1007/978-1-4939-9833-3_11 ◽

2019 ◽

pp. 139-151 ◽

Cited By ~ 1

Author(s):

Francesca Salvianti ◽

Filomena Costanza ◽

Gemma Sonnati ◽

Pamela Pinzani

Keyword(s):

Real Time ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Real Time Pcr ◽

Quantitative Real Time Pcr

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Characterization of mtDNA SNP typing using quantitative real-time PCR with special emphasis on heteroplasmy detection and mixture ratio assessment

International Congress Series ◽

10.1016/j.ics.2005.09.021 ◽

2006 ◽

Vol 1288 ◽

pp. 1-3 ◽

Cited By ~ 1

Author(s):

H. Niederstätter ◽

M.D. Coble ◽

T.J. Parsons ◽

W. Parson

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Mixture Ratio ◽

Snp Typing ◽

Mtdna Snp

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Characterization of Three Key MicroRNAs in Rice Root Architecture under Drought Stress using In silico Analysis and Quantitative Real-time PCR

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/1306 ◽

2014 ◽

Vol 11 (2) ◽

pp. 555-565 ◽

Cited By ~ 1

Author(s):

Behnam Bakhshi ◽

Ghasem Hosseini Salekdeh ◽

Mohammad Reza Bihamta ◽

Masoud Tohidfar

Keyword(s):

Drought Stress ◽

Real Time ◽

Real Time Pcr ◽

In Silico ◽

Root Architecture ◽

In Silico Analysis ◽

Rice Root ◽

Quantitative Real Time Pcr ◽

Silico Analysis

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Characterization of fecal concentrations in human and other animal sources by physical, culture-based, and quantitative real-time PCR methods

Water Research ◽

10.1016/j.watres.2013.02.060 ◽

2013 ◽

Vol 47 (18) ◽

pp. 6873-6882 ◽

Cited By ~ 41

Author(s):

Jared S. Ervin ◽

Todd L. Russell ◽

Blythe A. Layton ◽

Kevan M. Yamahara ◽

Dan Wang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Physical Culture ◽

Quantitative Real Time Pcr

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Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR

Journal of Virological Methods ◽

10.1016/j.jviromet.2009.12.001 ◽

2010 ◽

Vol 164 (1-2) ◽

pp. 75-82 ◽

Cited By ~ 15

Author(s):

G. Ananthakrishnan ◽

T. Venkataprasanna ◽

Avijit Roy ◽

R.H. Brlansky

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Virus Isolate ◽

Citrus Tristeza Virus ◽

Quantitative Real Time Pcr ◽

Citrus Tristeza Virus Isolate ◽

Tristeza Virus ◽

Citrus Tristeza

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Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii

Molecular Biology Reports ◽

10.1007/s11033-011-1402-1 ◽

2011 ◽

Vol 39 (5) ◽

pp. 5905-5912 ◽

Cited By ~ 10

Author(s):

Gang Zhang ◽

Mingming Zhao ◽

Chao Song ◽

Anxiong Luo ◽

Jianfa Bai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Anoectochilus Roxburghii

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Quantitative Real-Time PCR Assays for Sensitive Detection of Canada Goose-Specific Fecal Pollution in Water Sources

Applied and Environmental Microbiology ◽

10.1128/aem.00110-10 ◽

2010 ◽

Vol 76 (14) ◽

pp. 4886-4889 ◽

Cited By ~ 27

Author(s):

B. Fremaux ◽

T. Boa ◽

C. K. Yost

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fecal Pollution ◽

Sensitive Detection ◽

Rrna Gene ◽

Canada Goose ◽

Canada Geese ◽

Quantitative Real Time Pcr ◽

Gene Markers ◽

Pcr Assays

ABSTRACT Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

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