Quantitative Assessment of the Tetracycline Resistance Gene Pool in Cheese Samples by Real-Time TaqMan PCR

ABSTRACT A TaqMan real-time PCR assay was developed to quantify the tetS gene pool present in retail cheeses. This protocol offers a rapid, specific, sensitive, and culture-independent method for assessing antibiotic resistance genes in food samples rich in fats and proteins.

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Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

Journal of Food Protection ◽

10.4315/0362-028x-70.12.2774 ◽

2007 ◽

Vol 70 (12) ◽

pp. 2774-2781 ◽

Cited By ~ 17

Author(s):

I-CHEN YANG ◽

DANIEL YANG-CHIH SHIH ◽

JAN-YI WANG ◽

TZU-MING PAN

Keyword(s):

Bacillus Cereus ◽

Real Time ◽

Real Time Pcr ◽

Food Samples ◽

Most Probable Number ◽

Pcr Assay ◽

Bacillus Cereus Group ◽

Probable Number ◽

Cooked Rice ◽

Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

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Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

Journal of Medical Virology ◽

10.1002/jmv.20365 ◽

2005 ◽

Vol 76 (3) ◽

pp. 350-355 ◽

Cited By ~ 74

Author(s):

Lawrence Corey ◽

Meei-Li Huang ◽

Stacy Selke ◽

Anna Wald

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Real Time ◽

Clinical Samples ◽

Pcr Assay ◽

Taqman Pcr ◽

Simplex Virus

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Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases

Journal of Medical Microbiology ◽

10.1099/jmm.0.46909-0 ◽

2007 ◽

Vol 56 (1) ◽

pp. 52-55 ◽

Cited By ~ 94

Author(s):

Christopher I. Birkett ◽

Hugo A. Ludlam ◽

Neil Woodford ◽

Derek F. J. Brown ◽

Nicholas M. Brown ◽

...

Keyword(s):

Real Time ◽

Taqman Assay ◽

Esbl Production ◽

Pcr Assay ◽

Extended Spectrum ◽

Genes Encoding ◽

Taqman Pcr ◽

Assay Time ◽

Phenotypic Methods ◽

Single Reaction

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

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Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods

Canadian Journal of Microbiology ◽

10.1139/w10-060 ◽

2010 ◽

Vol 56 (9) ◽

pp. 761-770 ◽

Cited By ~ 21

Author(s):

Hua Yang ◽

Oleksandr A. Byelashov ◽

Ifigenia Geornaras ◽

Lawrence D. Goodridge ◽

Kendra K. Nightingale ◽

...

Keyword(s):

Real Time ◽

Resistance Gene ◽

Water Samples ◽

Real Time Pcr ◽

National Park ◽

Tetracycline Resistance ◽

Commensal Bacteria ◽

Tetracycline Resistance Gene ◽

Antibiotic Resistant ◽

Standard Culture

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.

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