不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.

Analysis of Different Lactobacillus rhamnosus by Random Amplified Polymorphic DNA

Objective] We developed a molecular method to mark and differentiate Lactobacillus rhamnosus strains and to analyze genetic diversity among isolates from different sources. Methods] Ten strains of Lactobacillus spp. were isolated from 56 feces samples of elderly people above 90 years of age in regions of Hetian, Xinjiang and Bama, Guangxi, China. We used API 50CHL test chip for strain identification. These isolates belonged to Lactobacillus rhamnosus with 98.6%~99.9% satisfactory. Ten L. rhamnosus isolates and one reference strain ATCC7496 were analyzed by random amplified polymorphic DNA (RAPD) technique to differentiate L. rhamnosus strains at molecular level. We screened 5 random primers named P14, OPG28, OPG25, P7 and P4 and developed optimum RAPD amplifying system. Results] The clear and stable DNA fingerprints of each strain were obtained; the amplicon size ranged from 100 to 2000 bp, and the band patterns showed polymorphism among different L. rhamnosus strains. Genetic similarity coefficients based on RAPD patterns varied from 0.581 to 0.935, which suggested genetic diversity and different genetic relationship among these strains. Eleven strains of Lr could be clustered into 5 groups (group A to E) at the level of 80% similarity. Seven L. rhamnosus strains isolated from Hetian, Xinjiang, were grouped in group B and C, 3 isolates from Bama, Guangxi, were grouped in group D and E. Conclusion] RAPD method is feasible for molecular mark and differentiation of L. rhamnosus strains and high level of genetic diversity and different relationship are found among L. rhamnosus isolates.