属特异性T-RFLP技术用于乳酸杆菌的群落分析

【目的】设计乳酸杆菌属特异性T-RFLP技术(末端限制性片段长度多态性分析)对14株乳酸杆菌进行分型。【方法】采用源于16S-23S rRNA基因间隔区序列的乳酸杆菌属特异性引物LAB-rev,乳酸杆菌的属特异性引物,6-FAM荧光标记后结合16S上游通用引物7f用于乳酸杆菌的PCR扩增。【结果】选取HaeⅢ和HhaⅠ进行限制性酶切,最后对酶切后的产物末端测序得到T-RFLP峰谱图,该图谱能够快速准确地对不同种的乳酸杆菌进行定性、定量的分析。【结论】实验成功搭建T-RFLP技术用于微生态环境中乳酸杆菌检测的平台,对于在功能性食品、乳酸饮料和药物对肠道微生态的影响及菌种鉴定等领域有重大意义。

Development of a genus-specific T-RFLP technique for differentiating Lactobacillus community

Objective] To develop a Lactobacillus genus-specific T-RFLP technique for differentiating 14 Lactobacillus strains. Methods] A Lactobacillus genus-specific primer (LAB-rev) was firstly designed based on the sequence variation of bacterial 16S-23S space region, which was then labeled with 6-Carboxyfluorescein (6-FAM), followed by PCR with a 16S universal primer (7f). The PCR amplicon was then digested by restriction enzyme Hae III and HhaI before terminal sequencing by AB3730 Gene Scan to achieve T-RFLP profiles. Results] Results showed that this technique is more specific than the traditional T-RFLP using universal primers, which can be used to detect and quantitate Lactobacillus of 14 different species in this study. Conclusion] This technique has a great potential of application in investigating lactobacillus communities, evaluating the probiotic function of functional food, dairy drinks and medicine on intestinal micro-ecological environment.