多重实时聚合酶链式反应法快速检测食品中的产志贺毒素大肠埃希氏菌

Rapid detection of Shiga toxin-producing Escherichia coli in food by multiple real-time polymerase chain reaction

中文关键词:  快速  多重Real-Time PCR  产志贺毒素大肠埃希氏菌  冻干

英文关键词:rapid detection  multiplex real-time polymerase chain reaction  Shiga toxin-producing Escherichia coli  freeze drying

中文摘要:

      目的 建立多重实时聚合酶链式反应(polymerase chain reaction, PCR)法快速检测食品中的产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli, STEC)的方法。方法 以产志贺毒素大肠埃希氏菌携带的毒力基因stx1、stx2和黏附基因eae靶基因引物探针建立多重实时PCR体系, 并采用原位冻干技术将PCR反应体系和阳性质控品进行了预先分装冻干, 制成稳定、便捷的即用型反应体系, 随后对方法的灵敏性、特异性和稳定性进行评价。结果 所建立的多重实时PCR法检测eae、stx1、stx2基因的灵敏性为102 CFU/mL。与32种非STEC菌均无交叉反应。整体检测时长可控制在1 h以内, 冻干体系可在常温条件下保存1年以上。结论 本方法快速、简便, 适用于测定食品中的STEC。

英文摘要:

      Objective To establish a multiplex real-time polymerase chain reaction (PCR) method for rapid detection of Shiga toxin-producing Escherichia coli (STEC). Methods A multiplex PCR system was established using target gene primers of virulence genes stx1, stx2 and adhesion gene eae carried by STEC. The PCR reaction system and positive control samples were pre-packed and lyophilized using in situ lyophilization technology to create a stable, convenient, ready-to-use reaction system. Subsequently, the sensitivity, specificity and stability of the method were evaluated. Results The sensitivity of the multiplex fluorescent PCR method established for detecting eae, stx1, and stx2 genes was 102 CFU/mL. There was no cross reaction with 32 non-STEC bacteria. The overall detection time can be controlled within 1 h, and the lyophilized system can be stored at room temperature for more than 1 year. Conclusions The method is rapid, simple and suitable for the determination of STEC in food.