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

张栋; 冯丽莉; 薛玉玲; 王华; 荀一萍; 李兴佳; 朱宏; 王世杰

doi:10.13386/j.issn1002-0306.2016.18.005

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

摘要

摘要: 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。

Abstract: 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( phe S) of L.paracasei N1115. Propidium monoazide( PMA) was used to suppress dead bacteria DNA amplification,and then the q PCR method was used to detect bacteria powder in the number of live bacteria N1115. Results: Specific and verified primers of L.paracasei N1115 were: C15 F,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- q PCR could detect the number of living L.paracasei N1115 rapidly and accurately.Conclusion: A PMA- q PCR assay had been developed for rapid and accurate detection of viable L.paracasei N1115.