Development of a real time PCR taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

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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Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens

Journal of Applied Microbiology ◽

10.1111/j.1365-2672.2010.04930.x ◽

2011 ◽

Vol 110 (3) ◽

pp. 769-777 ◽

Cited By ~ 22

Author(s):

X.S. Qu ◽

L.A. Wanner ◽

B.J. Christ

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Common Scab ◽

Simultaneous Detection ◽

Taqman Assay

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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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