副溶血性弧菌实时重组酶聚合酶扩增检测技术研究

Real-time recombinase polymerase amplification for detection of Vibrio parahaemolyticus

目的建立快速检测副溶血性弧菌的实时重组酶聚合酶扩增技术(RPA)。方法根据副溶血性弧菌tlh基因保守序列设计筛选引物及探针,建立检测副溶血性弧菌的实时RPA方法。通过检测不同浓度副溶血性弧菌标准株DNA模板评价方法灵敏度,通过检测不同种属弧菌及其他细菌标准株评价方法特异度,通过重复试验评价方法稳定性,通过实样检测评价方法应用效果。结果建立的实时RPA方法在39℃恒温下20min内完成副溶血性弧菌扩增,最低检测限为5pg/反应,与其他致病菌无交叉反应,不同浓度的副溶血性弧菌DNA模板和大肠杆菌DNA模板隔日3次实时RPA检测结果一致,实时RPA对51份鲜活/冰鲜鱼、虾、贝、蟹类样品的检测结果与GB4789.7—2013《食品微生物学检验副溶血性弧菌检验》检测结果一致。结论建立的实时RPA方法可定性检测副溶血性弧菌,且实验操作及结果判读简单,适用于突发公共卫生事件、食品安全监管中的副溶血性弧菌快速检测。

Objective To establish real-time recombinase polymerase amplification(RPA)for the rapid detection of Vibrio parahaemolyticus(VP). Methods An exo probe and primers were designed according to the conserved sequence of thermolabile hemolysin(tlh)gene of VP and then RPA for detection of VP was established. The sensitivity of the assay was evaluated by detecting different concentration of VP;the specificity was evaluated by detecting different bacteria;the stability was evaluated by repeat trials;the application effect was evaluated by detecting food samples which were simultaneously tested with traditional culture method according to GB 4798.7-2013 Detection of VP. Results A real-time RPA was established to complete VP amplification within 20 min at a constant temperature of 39 ℃. The analytical sensitivity of the assay was five pg per reaction and no cross-reactivity with other pathogenic bacteria observed. The RPA detection results with different concentration of VP and E. coli DNA templates at three time points were consistent. The detection results of 51 food samples by RPA were the same as those by traditional culture method. Conclusion The established real-time RPA can qualitatively detect VP,with simple operation and interpretation of results,which is suitable for rapid detection of VP in public health emergencies and food safety supervision.