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Yersinia enterocolitica bacteria (Yersinia enterocolitica, Ye) since the 1980s caused widespread international attention of a foodborne transmission of pathogens, are widely distributed in nature, from the people, animals, soil, water separated, and a variety of food products. Ye is one of the few that can grow under refrigeration temperatures enteric pathogens, global foodborne disease caused by the bacteria outbreak has dozens. The bacteria pathogenic clinical manifestations are mainly two: gastrointestinal-type symptoms and intestinal infections. Mainly rely on the detection of bacteria in laboratory cultures, including cold enrichment method and selective enrichment method. Both methods required training time is long and tedious, and the last dozen biochemical tests identified needed. The result is often only after a week delay in the diagnosis of disease. Abroad to carry out research using molecular biology and immunological methods to detect Ye, including qualitative analysis of PCR technology, probe technique, ELISA and immunomagnetic beads, but these methods are generally used for the rapid screening of pure bacteria, yet not mature to be applied to detect diarrheal stool Ye. Therefore, the study envisaged application of real-time quantitative PCR and the establishment of a rapid and accurate clinical diagnosis. Objective: To establish a rapid and accurate method to detect Yersinia enterocolitica bacteria in diarrheal stool, and used in clinical testing. Materials and methods: 1. Droppings collected inpatient and outpatient diarrhea patients 200 cases 50 cases and normal human feces. 2 application of four culture methods (YSE-CIN, PBSS-SS, PAE-NYE, PAE-DYS) identification of the 250 cases of feces containing Yersinia bacteria. Cohran's Q test analysis of multiple related samples, four methods of detection rate compared using the chi-square test for paired design. 3 Application against pathogenic Ye chromosome heat-labile enterotoxin (YST) gene designed primers and probes for real-time quantitative PCR reaction (probes), detection of pathogenic (7 serotypes) and non-pathogenic sex (five kinds) enterocolitis Yersinia the Yersinia genus within the other species (8 species) and other enteric bacteria (8 species), in order to test its specificity. Pure culture and applied Ye feces Ye-fold dilution, to the sensitivity of the detection primers and probes. Selective enrichment of fecal DNA extraction of 250 cases, and apply this primer and probe detection. Finally, we compare real-time PCR and culture methods result anastomosis degrees. Results: 1. Improved culture method (YSE-CIN) from 200 cases of diarrheal stool detected 18 cases containing pathogenic Ye, the other three traditional methods (PBSS-SS method, PAE-NYE PAE-DYS Act) the number of positive cases, respectively, for 3,5,8. Cohran's Q-test analysis of multiple related samples obtained for Q = 26.6, P = 0.000 <0.05. Paired chi-square test, improved methods of detection rate, respectively, with the other three methods have significant differences. Four methods in normal feces from 50 cases there was no detected Ye. Primers and probes of pathogenic Yersinia bacteria 100% specificity, and other bacteria, no cross-reactivity. The minimum detectable concentration in pure culture and feces, respectively about of 10 ~ 2CFU/ml 10 3CFU / g. 200 cases of diarrheal stool, real-time PCR positive samples were detected in 18 cases, consistent with the improved culture method. Ye in feces after the enrichment concentration is mainly concentrated in the of of approximately 10 ~~ 5-10 8cfu / g. Not detected in 50 normal feces Y.e. Conclusions: 1. Improved culture method a higher detection rate than the other three kinds of training methods, applications in the general hospital. 2 the yst gene primers and probes for pathogenicity Ye is highly specific. Sensitivity tests show that the minimum detectable concentration of the primers and probes can be used in clinical testing. 3 real-time quantitative PCR fast convenient and reliable results, it is more appropriate relative to the other methods used in clinical testing.
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