双正交优化多重PCR检测食源性致病菌的研究

为了建立快速检测食源性致病菌的多重PCR检测方法,根据沙门氏菌fimY基因、单增李斯特氏菌hlyA基因和小肠结肠炎耶尔森氏菌ail基因设计3对特异性引物,利用双正交法分别对多重PCR反应体系中的3对引物比进行L9(33)正交优化和Taq酶、dNTPs、镁离子、混合引物的添加量进行L16(44)正交优化,并对Buffer的反应浓度进行优化。结果扩增出了3条特异性目的条带,建立的多重PCR方法具有很好的特异性。人工污染食品,9h富集培养增菌后的检出限可达100cfu/g。因此,该检测方法具有较强的实际应用价值,为检测多种食源性致病菌提供了有效的方法参考。

Double orthogonal design to optimize multiplex PCR reaction system for detection of food-borne bacterial pathogens

Aiming to establish a rapid multiplex PCR method for the simultaneous detection of food-borne bacterial pathogens, three pairs of specific primers were designed according to Salmonella fimY gene, Listeria monocytogenes hlyA gene and Yersinia enterocolitica ail gene. Orthogonal experimental design was used to optimize multiple PCR amplification system of three factors (three primer proportion) at three levels and four factors (Taq DNA polymerase, Mg2+, dNTPs and primers) at four levels, and the concentration of the Buffer was also optimized. The multiplex PCR assay could amplify three specific target channels and the specificity of this detection method was confirmed. The sample was enrichmented culture 9 hours after artificially polluting food, and the detection limit of the multiplex PCR assay for meat was up to 100 cfu/g. The multiplex PCR method established has higher practical application value and provide effective reference approach for the detection of food-borne pathogenic bacteria.