传统发酵豆制品中3种致病菌的三重荧光PCR快速检测方法的建立

为了建立传统发酵豆制品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光PCR快速检测方法。以单增李斯特菌hly A基因、蜡样芽孢杆菌Cereolysin AB基因和金黄色葡萄球菌nuc基因为靶基因设计引物与TaqMan探针,通过优化PCR反应体系,建立了可同时检测单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的三重荧光定量PCR体系,并进行了特异性和敏感性试验。结果显示,该方法灵敏度高,特异性强,重复性好。对26株非目标菌进行检测,结果均为阴性,而定量检测批内和批间的变异系数均小于2%。单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌敏感性试验结果表明,这三种细菌的最低检测浓度分别为3×103cfu/mL、2×104cfu/mL、2×104cfu/mL。应用该方法可在8h内完成对样品中单增李斯特菌、蜡样芽孢杆菌和金黄色葡萄球菌的同步检测。

Detection of three pathogenic bacteria by triple real-time PCR in traditional fermented soybean food

To develop a triple real-time quantitative PCR assay for detection Listeria monocytogenes,Bacillus cereus and Staphyloccus aureus in traditional fermented soybean,primers and Taqman probes targeting the specific-specific gene hly A of L.monocytogenes,the Cereolysin AB gene of B.cereus and nuc gene of Staphyloccus aureus were selected.In addition,the specificity and sensitivity of the method were monitored.The results showed that the method was highly reproducible,specific and sensitive for detection of three pathogenic bacteria.In the specificity test,26 non-target reference and standard strains were negative.All variation coefficients of intra-assay and inter-assay were less than 2% in the quantitative tests.The detection limitations of the method were 3×103cfu/ml,2×104cfu/ml and 2×104cfu/ml of L.monocytogenes,B.cereus and S.aureus for pure cultures,respectively.It took less 8h to detect L.monocytogenes,B.cereus and S.aureus in samples simultaneously.