| Yersinia enterocolitica is a food-borne pathogen that causes various enteric syndromes. One of the features of the disease process is the ability of the bacteria to translocate through the intestinal epithelium and proliferate within the Peyer's Patches, causing a wide variety of clinical and immunological manifestations. Y. enterocolitica is widely distributed in nature and has a broad animal reservoir including swine, dogs, rodents, rabbits and birds among which the swine are the reservoir that most threaten infection in humans. And as the bacterium has the ability to multiply in foods at low temperatures, approaching 0℃, as well as in vacuum-packed and modified-atmosphere packages, it is of significant concern from.a food safety and public health perspective.We chose 271 pathogenic and 27 non-pathogenic Y. enterocolitica strains isolated from diarrhea patients, animals, food and the environment in China. They included 205 strains of serotype 0:9,72 of serotype 0:3,10 of serotype 0:8, five of serotype 0:5, three of serotype 0:6,30 and three of undetermined serotype, together with 12 reference strains from Europe, the United States and Japan.The ail from the 283 pathogenic strains showed three sequence patterns:A1, A2 and A3. In the primary coding region of the foxA ORF (Genebank:X60447 nt 433-1,866, nt 28 to 1,461 in the ORF) from the pathogenic strains, the sequences formed three groups and were further divided into eight sequence patterns. The results showed 13 sequence patterns for the 27 non-pathogenic strains with tens to hundreds more polymorphic sites and no apparent regularity. Analysis of polymorphisms in ail and foxA of pathogenic Y. enterocolitica strains from different times and regions showed ail to be an important virulence gene for pathogenic Y. enterocolitica and to have a highly conserved sequence, while the gene encoding the ferrioxamine receptor, foxA, is also conserved in pathogenic strains, where two primary sequence patterns were found. The results showed ail and foxA both could be the target genes for detection of pathogenic Y. enterocolitica.We developed a TaqMan probe-based real-time PCR method for the rapid detection of Y. enterocolitica based on the research for ail and foxA genes. Two primer sets, ail-1(ail-1-F and ail-1-R) and ail-2(ail-2-F and ail-2-R), were used for ail gene because of the deference for pattern Al and A2, and they were suitable for the same probe. One primer set and one probe were used for foxA gene detection. The target genes were cloned into the vector pMD18-T to make the standard curve. The sensitivity and specificity of the PCR method was tested with a diverse range of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The detection limit of the optimized reaction system to ail and foxA plasmids is 1.0×102 copies/μl. The result showed the probe for ail could detect the pathogenic Y. enterocolitica, while the probe for foxA could detect all the Y. enterocolitica. In addition, the method was tested on separate specimens, in all,112 out of 150 samples were positive for ail-1 and ail-2 gene, and 118 positive for foxA gene. Compared to traditional culture method and conventional PCR, real-time PCR assay provides greater specificity and requires less time and labor. |
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