食源性致病菌快速检测方法的建立与应用

Rapid Detection Method of Food-borne Pathogenic Bacteria of Establishment and Application by Multiplex PCR

目的:建立一种快速检测空肠弯曲杆菌、副溶血弧菌和小肠结肠炎耶尔森菌的多重PCR方法,并进行市场采样检测。方法:根据空肠弯曲杆菌的hipO基因、副溶血弧菌的tlh基因和小肠结肠炎耶尔森菌的Ail基因的特异性抗原基因序列,设计3对特异性引物,优化各种工作条件;测定特异性和灵敏度,建立一种多重PCR检测方法,并对泰安农贸市场采集的409份样本进行检测。结果:建立的多重PCR方法可以快速、简便地检测3种目的菌;对6种食源性致病菌的检测结果表明,该方法特异性很好,灵敏度高。空肠弯曲杆菌的检出极限为3.0×101cfu/mL,副溶血弧菌的检出极限为6.2×101cfu/mL,小肠结肠炎耶尔森菌的检出极限为1.5×101cfu/mL。样本中空肠弯曲杆菌、副溶血弧菌和小肠结肠炎耶尔森菌的阳性率分别为20.8%,1.5%和13.2%。结论:采用该方法可在5h内得到检测结果,具有操作简便,检测周期短,敏感度和特异性高等优点,为进一步开发空肠弯曲杆菌、副溶血弧菌和小肠结肠炎耶尔森菌快速检测试剂盒及其产业化奠定了基础。

Objective： To develope a rapid method for simultaneously detection of Campylobacter jejuni, Vibrio parahaemolyticus and Yersinia enterocolitica by multiplexes PCR. And sampling inspection in Market. Methods： According to Characteristic genes sequences of the hipO gene of Campylobacter jejuni, the tlh gene of Vibrio parahaemolyticus and the Ail gene of Yersinia enterocolitica, three pairs of specific primers were screened, the specifcity and sensitivity of this system were evaluated. Many factors of multiplex PCR were optimized. And 409 samples were collected from poultry meat market in Tai’an for detection. Results： The multiplex PCR assay could be simple and rapid to detect those three pathogens, and the detection limits were 3.0×101 cfu/mL for Campylobacter jejuni, 6.2×101 cfu/mL for Vibrio parahaemolyticus and 1.5×101 cfu/mL for Yersinia enterocolitica. Campylobacter jejuni was detected in 20.8% of the samples, whereas Vibrio parahaemolyticus and Yersinia enterocolitica were identified in 1.5% and 13.2%, respectively. Conclusion： The whole test can be completed in 5 h. The results of the experiments demonstrated that the multiplex PCR assay was rapid, simple, sensitive and specific, Which establish important foundation for simultaneous detection for these three bacteria in polluted sample and study on instant detection kit.