副溶血弧菌重组酶聚合酶扩增快速检测方法的建立及初步应用

Establishment and preliminary application of a rapid detection method for Vibrio parahaemolyticus

为建立副溶血弧菌快速检测方法,本研究以副溶血弧菌不耐热溶血素(tlh)基因作为靶序列设计特异性引物,经反应条件优化建立了检测副溶血弧菌的重组酶聚合酶扩增(RPA)方法。优化试验结果显示该方法在37℃和35 min条件下检测效果最佳;特异性试验结果显示,该方法除对副溶血弧菌的检测结果为阳性外,对其余8种常见的弧菌检测结果均为阴性,特异性较强;敏感性试验结果显示,该RPA方法对副溶血弧菌的检出限为104cfu/mL,敏感性较高。利用建立的RPA检测方法、培养法及普通PCR方法同时对采集的48份海鲜样品进行检测,结果显示:该RPA方法能够对临床样品快速检测,并且检测结果与国家标准培养法及普通PCR方法的符合率为100%,能够满足临床样品的检测需求。本研究建立了一种检测副溶血弧菌的方法,该方法具有反应快速、特异性强、敏感性高等特点,为副溶血弧菌的快速检测奠定基础。

To establish a rapid detection method for Vibrio parahaemolyticus, this study designed specific primers based on the heat-labile hemolysin(tlh) gene sequence of Vibrio parahaemolyticus, and established the RPA method for the detection of Vibrio parahaemolyticus by optimizing the reaction conditions. The optimized test results show that the method has the best detection effect under the conditions of 37 ℃ and 35 min;the specificity test results showed that, except for the positive test result of Vibrio parahaemolyticus, the other 8 common Vibrio species were all negative, with strong specificity;the results of sensitivity test showed that the detection limit of the RPA method for Vibrio parahaemolyticus was 104 cfu/mL, indicating a high sensitivity. Fortyeight seafood samples were detected by the established RPA detection method, culture method and common PCR method at the same time. The results showed that the RPA method could quickly detect the actual samples, and the coincidence rate of the results was 100% with the national standard culture method and common PCR, which could meet the testing requirements of actualsamples. This study established a method for detecting Vibrio parahaemolyticus, which has the characteristics of rapid response,high sensitivity, and strong specificity, which lays the foundation for the rapid detection of Vibrio parahaemolyticus. It would lay a foundation for the rapid detection of Vibrio parahaemolyticus.