环介导等温扩增技术在食品中痢疾志贺氏菌快速检测

Loop-mediated isothermal amplification-based rapid detection for Shigella dysenteriae in food samples

【目的】探讨、优化基于环介导等温扩增技术(LAMP)快速检测常规食品中感染性痢疾志贺氏菌的方法。【方法】在NCBI数据库中搜索获取志贺氏菌的特异性基因高度保守区,设计3对LAMP反应引物,建立、优化该LAMP可视化检测方法,并评价其特异性、灵敏度,同时与PCR检测方法和传统检测方法对比,进行结果统计分析。【结果】5株志贺氏菌标准菌株样品均检测为阳性,11株非志贺氏菌标准菌株样品均检测为阴性,无交叉反应。最低检验限为1.6×101 CFU/反应(或1.6×101 CFU/m L),且经比较,LAMP检测灵敏度比PCR检测高出1个数量级。通过对161份实际样品和人工污染样品进行检测,LAMP检测与传统方法检测结果具有较高的一致性。【结论】LAMP具有检测过程快速简便、检测结果稳定、可靠的特点,适用于对常规食品中感染性痢疾志贺氏菌的高效、快速检测需求。

[Objective] To study and optimize loop-mediated isothermal amplification method for rapid detection of Shigella that can cause infectious diarrhea in conventional food consumption. [Methods] High conservative and specific target gene sequence of Shigella gene was aligned and obtained from NCBI （National Center for Biotechnology Information） database. Three pairs of corresponding LAMP primers were designed, synthesized and used to establish the LAMP-based visualized detection method. The detection result of practical samples by using LAMP-based detection method was compared with those obtained by conventional methods for statistical analysis. [Results] All 5 strains of Shigella standard strain samples were detected positive and 11 strains of non-Shigella standard samples were detected negative, which showed no cross reaction. The minimum test limit is 16 CFU per test or 3.2~102 CFU/mL, and by comparison, the sensitivity of LAMP detection was 10 times higher than PCR detection. All 161 sets of samples were detected by LAMP, and the results showed high consistency with conventional detection method. [Conclusion] The LAMP-based detection method is featured with advantages in convenience, rapidity and high reliability, which makes it applicable to meet the requirements of rapid detection for Shigella in different types of food samples.