【摘要】 [目的]初步探讨常规PCR、荧光PCR、PCR产物测序及脉冲场凝胶电泳(PFGE)等核酸检测技术在常见细菌性食源疾病病原菌检测中的应用。[方法]用常规PCR、荧光PCR直接检测经选择性增菌培养的沙门菌和志贺菌,测定PCR扩增产物序列以确定病原菌种类,用限制性内切酶XbaI和SpeI对分离菌株进行PFGE分析。[结果]常规PCR、荧光PCR方法在两份腹泻病人样本中均检出沙门菌,结果与培养法一致。二者未在皮蛋中检出沙门菌,而培养法在市售皮蛋中检出沙门菌。检测时间分别为PCR16h、荧光PCR14h、PCR产物直接测序19h,较培养法缩短4d。来源不同的3株分离菌XbaI和SpeI酶切图谱完全一致(相似度为100%)。[结论]应用常规PCR、荧光PCR可准确、及时检出腹泻病人样本中的沙门菌和志贺菌,有望用于食物中毒的快速检测。但在检测食品和环境样本时需适当延长增菌培养时间。在从农田到餐桌的食品供应链中,PFGE可对疾病暴发进行早期识别并协助对病原菌及其传播途径的溯源,有助于食物中毒的预防。更多还原

【Abstract】 [Objective]To explore the application of PCR、real-time PCR,PCR products sequencing and pulsed-field gel electyophoresis(PFGE)in detection of pathogenic bacteria for food-borne disease.[Methods]PCR and real-time PCR technique were applied to detect Salmonella and Shigella from the selective enrichment cultivation,and the sequence analysis were also conducted for the PCR products.Isolated strains were digested by restriction endonucleases Xba I and Spe I,and were analyzed by PFGE.[Results]Two samples from diarrheic patients were Salmonella positive by PCR and real-time PCR detection,which was in accordance with that of the traditional method.PCR and real-time PCR technique failed to detect Salmonella from preserved egg,while traditional method detected Salmonella.The detection time of PCR,real-time PCR and PCR products sequencing were 16 hours,14hours and 19 hours,respectively,saved 4 days by comparing with traditional method.Three isolates from different origin demonstrated the same PFGE pattern with endonucleases Xba I and Spe I.digestion[Conclusion]Real-time PCR or PCR products sequencing could detect Salmonella and Shigella from samples of diarrheic patients after 10-hours’ selective enrichment cultivation,which provides a rapid,effective and accurate method for identifying pathogenic bacteria.However,when detecting food and environmental samples,the time of cultivation should be suitably prolonged.In food chain from farm to table,PFGE can provide early identification of disease outbreak and trace to pathogenic bacteria and route of transmission.更多还原