多重环介导等温扩增快速检测沙门菌、副溶血弧菌和单核细胞增生性李斯特菌方法的建立

目的：建立能够同时检测沙门菌、副溶血弧菌（VPH）和单核细胞增生性李斯特菌（LM）的多重环介导等温扩增（mLAMP）方法。方法分别针对沙门菌 bcfD 基因、VPH 的 tlh 基因和 LM的 iap 基因设计 mLAMP 引物，以LAMP 进行单重 LAMP 检测。实时浊度监测扩增结果，优化引物浓度配比，进而建立此3种食源性致病菌的mLAMP 检测体系，以 PCR 检测灵敏性作为比较，进行特异性和灵敏性分析。结果实时浊度监测表明，该 mLAMP体系具有良好特异性，可在45 min 内一次性检测以上3种致病菌，且无非特异扩增产生。这3种菌的检测最低限分别为300 fg／μl、4．2 pg／μl 和4．5 pg／μl，与 PCR 检测灵敏度相当。结论该多重 LAMP 可同时检测食品中的沙门菌、VPH 和 LM，可作为大规模样品的初筛或传统方法的辅助方法，用于快速判定样品中是否含有这3种致病菌。

Development of multiplex loop-mediated isothermal amplification (mLAMP) for detection of Salmonella,Vibrio parahaemolyticus and Listeria monocytogenes

Objective To establish a multiplex loop-mediated isothermal amplification(mLAMP)method for simultaneous detection of Salmonella,Vibrio parahaemolyticus (VPH)and Listeria monocytogenes (LM).Methods Three sets of mLAMP primers were designed to specifically target bcfD of Salmonella and tlh of VPH and iap of LM.The respective single LAMP assay of the three kinds of bacteria was developed,and the ratio of primer concentration was optimized to develop a multiplex LAMP system.The specificity and sensitivity of multiplex LAMP were observed.Results Turbidity monitoring results in real time suggests that the mLAMP was highly specific and amplification could be obtained within 45 min under isothermal conditions.The sensitivity of this mLAMP was found to be 300 fg/μl genomic DNAs for Salmonella and 4.2 pg/μl for VPH and 4.5 pg/μl for LM,which was consistent with conventional PCR.Conclusion The mLAMP described can potentially facilitate simultaneous detection of three kinds of bacteria in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection methods.