乳制品中活双歧杆菌检测试剂盒的研制及应用

在EMAReal-Time PCR检测方法的基础上,组装检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。建立了EMAReal-Time PCR检测乳制品中活双歧杆菌的标准曲线。组装出检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。该试剂盒批内及批间变异系数(CV)均小于5%;对乳制品中常见乳酸菌无交叉反应;最低检测限为2×104CFU/mL。该试剂盒在-20℃下,至少可保存6个月,4℃可保存3个月。分别采用研究所获得的试剂盒和平板菌落计数法检测12份市售酸乳中的活双歧杆菌的数量,2种检测方法计数结果差异不显著(P>0.05)。该方法所建立的标准曲线相关系数大于98%。所研制的试剂盒重复性较好、特异性较强、灵敏度较高,可以准确地定量检测乳制品中的活双歧杆菌。

Development and Application of Detection Kit for Viable Bifidobacteria in Dairy Products

Objective To establish a Real-Time and fluorescent quantitative PCR kit for detection of viable bifidobacteria in dairy products. Methods A Real-Time and fluorescent quantitative PCR kit is developed to detect via-ble bifidobacteria in dairy products with the method of EMA Real-Time PCR. Results The standard curve of determi-nation of viable bifidobacteria by EMA Real-Time PCR was established. A fluorescent quantitative PCR kit was devel-oped for detecting viable Bifidobacterium in dairy products. For this kit, the intra-assay and inter-assay coefficient of variation were both less than 5%, and cross-reaction with other lactic acid bacteria in dairy products didn't exist.The detection limit was 2×104 CFU/mL. This kit could be stored over 6 months at-20 ℃ and 3 months at 4℃. A-mong the 12 market yoghurt detected, the results obtained by kit were not significantly different from that obtained by the plate counting (P > 0.05). Conclusion The correlation coefficient of standard curve was more than 0.98. This kit was sensitive, specific and good repeated for detecting viable bifidobacteria in dairy products. This kit could be used to determine viable bifidobacteria in dairy products quantitatively.