Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

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

Water Research ◽

10.1016/j.watres.2013.03.061 ◽

2013 ◽

Vol 47 (18) ◽

pp. 6921-6928 ◽

Cited By ~ 32

Author(s):

Meredith R. Raith ◽

Catherine A. Kelty ◽

John F. Griffith ◽

Alexander Schriewer ◽

Stefan Wuertz ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fecal Pollution ◽

Pollution Sources ◽

Quantitative Real Time Pcr

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Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam

Journal of Water and Health ◽

10.2166/wh.2016.173 ◽

2016 ◽

Vol 15 (1) ◽

pp. 155-162 ◽

Cited By ~ 5

Author(s):

Pierangeli G. Vital ◽

Nguyen Thi Van Ha ◽

Le Thi Hong Tuyet ◽

Kenneth W. Widmer

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Surface Waters ◽

Real Time Pcr ◽

Membrane Filtration ◽

Quantitative Real Time Pcr ◽

Wet Season ◽

Culture Methods ◽

Fecal Indicator ◽

Filtration Method

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

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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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Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

Journal of Food Protection ◽

10.4315/0362-028x-69.4.891 ◽

2006 ◽

Vol 69 (4) ◽

pp. 891-896 ◽

Cited By ~ 14

Author(s):

FEDERICA BELLAGAMBA ◽

SERGIO COMINCINI ◽

LUCA FERRETTI ◽

FRANCO VALFRÈ ◽

VITTORIO M. MORETTI

Keyword(s):

Real Time ◽

Prion Protein ◽

Dna Sequences ◽

Real Time Pcr ◽

Animal Feed ◽

Quantitative Estimation ◽

Taqman Assay ◽

Quantitative Real Time Pcr ◽

The Real ◽

Species Specific

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

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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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COMPARISON OF A CONVENTIONAL PCR AND QUANTITATIVE REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION OF HUANGLONGBING DISEASE ASSOCIATED WITH ‘CANDIDATUS LIBERIBACTER ASIATICUS’ IN CITRUS VARIETIES

The Journal of Animal and Plant Sciences - February ◽

10.36899/japs.2021.5.0346 ◽

2021 ◽

Vol 31 (5) ◽

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Qpcr Assay ◽

Candidatus Liberibacter Asiaticus ◽

Quantitative Real Time Pcr ◽

Conventional Pcr ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Varieties

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Reverse Transcription-Quantitative real-time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

Journal of Clinical Microbiology ◽

10.1128/jcm.00779-21 ◽

2021 ◽

Author(s):

Bryanna Lexus Freitas ◽

Lynn Leach ◽

Vishnu Chaturvedi ◽

Sudha Chaturvedi

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Real Time Pcr ◽

Environmental Samples ◽

Ribosomal Gene ◽

Qpcr Assay ◽

Molecular Diagnostic ◽

Quantitative Real Time Pcr ◽

Candida Auris ◽

Healthcare Environments

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to detect live C. auris in healthcare environments rapidly. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT-qPCR reaction. Validation studies yielded positive Ct values from sponge matrix samples spiked with 10 2 to 10 5 CFU of live C. auris while dead (bleach-killed) C. auris (10 5 /ml) or other live Candida species (10 5 /ml) had no cycle threshold (Ct) values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

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