Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods

Abstract

The aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus -tag CH49 -3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5' nuclease PCR protocol. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result.The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus -tag CH49 -3099 -positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia enterocolitica

“…The real-time RPA sensitivity experiments can detect a minimum concentration of 10 −4 ng/μL, which is almost identical to RT-PCR [ 4 ] and LAMP [ 15 ] sensitivity. It is 10 times more sensitive compared to RPA-SYBR Green I [ 32 ] and normal PCR.…”

“…Existing rapid detection methods (RT-PCR and LAMP) for Y. enterocolitica require at least 50 min to complete [ 4 , 15 ]. The detection time of DNA in this study was less than 20 min, and the detection results can be viewed in real time, which greatly improves the detection efficiency of Y. enterocolitica .…”

“…Yersinia enterocolitica , a member of the Enterobacteriaceae family, is a kind of zoonotic pathogen that exists widely all over the world [ 1 , 2 ]. It can infect humans or animals through contaminated food or water, causing self-limiting disease, lymphadenitis and terminal ileal inflammation, sepsis and even death in severe cases [ 3 , 4 ].…”