属特异性T-RFLP技术用于乳酸杆菌的群落分析  

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

作　　者：张思璐[1] 刘云霄[1] 张浩琪[1] CHIN James 吴希阳[1] 

机构地区：[1]暨南大学食品科学与工程系,广东广州510632 [2]Immunology and Microbiology,Elizabeth Macarthur Agriculture Institute,NSW Department of Primary Industries,Camden,NSW 2570,Australia

出　　处：《微生物学通报》2012年第8期1179-1189,共11页Microbiology China

基　　金：暨南大学科研培育与创新基金项目(国际及港澳台合作项目;No.21611614)

摘　　要：【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。[Objective] To develop a Lactobacillus genus-specific T-RFLP technique fordifferentiating 14 Lactobacillus strains. [Methods] A Lactobacillus genus-specific primer （LAB-rev） was firstly designed based on the sequence variation of bacterial 16S-23S space re- gion, 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 us- ing 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 in- vestigating lactobacillus communities, evaluating the probiotic function of functional food, dairy drinks and medicine on intestinal micro-ecological environment.

关 键 词：乳酸杆菌 T-RFLP技术 属特异性引物 

分 类 号：TS201.3[轻工技术与工程—食品科学]