Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food.

Applied and Environmental Microbiology
S Thisted LambertzM Lindblad

Abstract

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail ...Continue Reading

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Citations

Jan 29, 2013·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·R StephanM Fredriksson-Ahomaa
Nov 16, 2010·Epidemiology and Infection·A BackhansS Thisted Lambertz
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