| Yesinia enterocolitica(abbreviated as Y.e) is a food-borne pathogen that can cause illness to both people and animals and it has also been extensively attractive to scientists because of isolation and detection from people, animals, soil, water and many kinds of foods. Y.e can healthily survive at freezing temperature belonging to the limited intestinal pathogen and tens of food-borne pandemics have been caused by it across the world. Clinical manifestation of yersiniosis may generally be summarized as gastroenteritis and infection outside intestine. At present, clinical detection for Y.e mainly depends on culture methods, including cold enrichment and selective enrichment, both of which are time-consuming and laborious. Suspected colonies are needed to confirm by lots of chemical tests in the end. Therefore, the result will come out in more than 1 week, which may delay the diagnosis. Due to the disadvantages above, foreign scientists have made good use of methods of molecular biology and immunology to detect Y.e, including common PCR, probe, ELISA, immunomagnetic beads and so on. However, all of these methods are confined to pure cultures of Y.e for rapid screen, which are not mature to detect Y.e from diarrhea stool samples. Hence, in this study, we want to carry out real-time PCR for rapid detection and analysis of Y.e from diarrhea stools in the hope of as an alterative to culture methods. Objective: To establish a quick and accurate real-time PCR method to detect pathogenic Y.e from diarrhea stools as an alternative to traditional culture methods. Materials and Methods: 1. 200 stools were collected from inpatients and outpatients with the syndrome of diarrhea. Additionally, 50 stools were collected from healthy people2. All stool samples were detected by 4 culture methods, including YSE-CIN,PBSS-SS,PAE- NYE,PAE-DYS . Relevant samples were analyzed by Cohran's Q test and then positive detection rate of YSE-CIN will be separately compared with other 3 methods by McNemer test.3. The real-time PCR, using the primer-probe particularly designed for the yst gene of Y.e, was applied to detect pathogenic(7 serotypes) and nonpathogenic(5 serotypes) Y.e, other species of Yersinia(8 types) and other enteric bacteria(8 types) for specificity. Sensitivity test for the primer-probe was performed through serial dilution of pure cultures of Y.e and stool samples with Y.e4. DNA was extracted from 250 stool samples after selective enrichment for real-time PCR and then the results would be compared with culture methods.Results:1. Of 200 diarrhea stools, the number of positive samples for YSE-CIN,PBSS-SS,PAE- NYE,PAE-DYS was 18,3,5,8, respectively. Relevant samples were analyzed by Cohran's Q test with notable significance. By McNemer test, the positive detection rate of YSE-CIN was significantly discrepant with other 3 traditional methods. Of the 50 healthy stools, no Y.e was found by 4 culture methods.2. Real-time PCR with the particular primer-probe was found to be 100 percent specific to virulent Y.e and the lowest detection limit in pure cultures and stool samples was about 100CFU /ml and 1000CFU/g, respectively.3. Of the 200 diarrhea stools, the same 18 positive samples were successfully detected by real-time PCR while no positive sample was found from 50 healthy stools. The concentration of Y.e in stool samples mainly focused on 10~5-10~8CFU/g after enrichment.Conclusion:1. The improved culture method(YSE-CIN) with higher positive detection rate than other 3 methods can be utilized in common hospital.2. The primer-probe was highly specific to yst gene of Y.e and its lowest detectionlimit was also suitable for clinical tests.3. In addition to be convenient and timesaving, real-time PCR with reliable results ismuch more suitable to detect Y.e from stool samples compared to other methods. |
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