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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(mL AMP)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mL AMP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mL AMP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mL AMP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。
Objective To establish a multiplex cyclic isothermal amplification (mL AMP) method capable of simultaneously detecting Salmonella, Vibrio parahaemolyticus (VPH) and Listeria monocytogenes (LM). Methods Amplicon AMPAMP primers were designed for the bcfD gene of Salmonella, the tlh gene of VPH and the iap gene of LM respectively. Real-time turbidity monitoring amplification results, optimizing the primer concentration ratio, and then establish these three kinds of food-borne pathogens mL AMP detection system to detect the sensitivity of PCR as a comparison for specificity and sensitivity analysis. Results The real-time turbidity monitoring showed that the mL AMP system had good specificity and could detect the above three kinds of pathogenic bacteria in one time within 45 min, with no specific amplification. The minimum detection limits for these three strains were 300 fg / μl, 4.2 pg / μl and 4.5 pg / μl, respectively, comparable to PCR assays. Conclusions The multiplexed LAMP can detect Salmonella, VPH and LM in food at the same time. It can be used as a screening method for large-scale samples or as an adjunct to traditional methods to rapidly determine whether the samples contain the three pathogenic bacteria.