PMA-qPCR方法快速检测Lactobacillus paracasei N1115活菌的初步研究

Study on PMA- qPCR assay for rapid and accurate detection of viable Lactobacillus paracasei N1115

目的:建立快速准确的副干酪乳杆菌N1115(Lactobacillus Paracasei N1115)活菌的荧光定量检测方法并应用于菌粉的活菌计数。方法:根据副干酪乳杆菌N1115的苯丙氨酰-tRNA合成酶α亚基基因(phe S)设计特异性引物;使用叠氮溴化丙锭(Propidium monoazide PMA)抑制死菌DNA扩增,然后通过q PCR的方法检测菌粉中的N1115的活菌数。结果:经验证得到一对N1115特异引物:C15F、C15R;副干酪乳杆菌N1115在90℃水浴条件下热损伤时间为8 min;PMA对于N1115死菌DNA的扩增有明显的抑制效果;PMA-q PCR能够快速准确的测得菌粉中N1115活菌数。结论:建立了一种快速、准确的副干酪乳杆菌N1115活菌的荧光定量检测方法。

Objective ：To develop a fast and accurate live bacteria fluorescent quantitative detection method applied in the viable count of Lactobacillus paracasei in bacterial powder. Methods： Specific primers were designed according to Phenyl alanine-tRNA synthetase alpha subunit gene （pheS）of L.paracasei N1115. Propidium monoazide（PMA） was used to suppress dead bacteria DNA amplification, and then the qPCR method was used to detect bacteria powder in the number of live bacteria N1115.Results：Specific and verified primers of L.paracasei Nlll5 were：C15F,C15R.The thermal damage time of L.paracasei N1115 was 8 min in the 90℃ water bath.PMA could obviously control the amplification of dead L.paracasei N1115 .The way of PMA-qPCR could detect the number of living L.paracasei N1115 rapidly and accurately.Conclusion：A PMA-qPCR assay had been developed for rapid and accurate detection of viable L.paracasei N1115.