属特异性T-RFLP技术在双歧杆菌群落分析中的应用

Genus-specific T-RFLP technique for differentiating Bifidobacteria community in probiotics

【目的】以双歧杆菌标准菌株为材料,构建双歧杆菌属特异性末端限制性片段长度多态性分析(T-RFLP)技术,用于微生物群落中双歧杆菌的特异性分析。【方法】采用16S rRNA基因的双歧杆菌属特异性引物,5′-端用HEX荧光标记,结合通用引物1510r进行双歧杆菌特异性PCR扩增,软件模拟酶切后选取Hae III和Alu I进行限制性酶切,对酶切消化产物的荧光标记末端测序得到T-RFLP峰谱图。同时将该技术与实验室已建立的乳酸杆菌属特异性T-RFLP技术相结合,建立多相T-RFLP技术应用于对市面上益生菌产品的时效性检测。【结果】建立的方法能够快速准确地对不同种的双歧杆菌及合生元产品中的益生菌进行定性或半定量分析。【结论】据此,成功搭建T-RFLP技术用于微生态环境中双歧杆菌的检测,并成功将多相T-RFLP技术用于市售益生菌产品的时效性检测。

[Objective] A Bifidobacteria genus-specific T-RFLP technique was developed to differentiate Bifidobacterium strains in microbial communities. [Methods] A Bifidobacteria genus-specific primer(g-bifid-F), based on the bacterial 16 S rRNA gene sequence, was 5′-labeled with hexachlorofluorescein(HEX), followed by PCR with a 16 S universal primer(1510r). The PCR amplicon was then digested by restriction enzyme Hae III and Alu I before terminal sequencing to achieve T-RFLP profiles. Moreover, combined with a Lactobacillus genus-specific T-RFLP technique in our lab's previous study, this technique was used to detect bacterial composition and the compositional dynamic of a commercial probiotic product——Biostime. [Results] This technique was more specific than the traditional T-RFLP using universal primers, which could be used to detect or semi-quantitate different species of Bifidobacteria. [Conclusion] A Bifidobacteria genus-specific T-RFLP technique was well-established, and a combined system of these two T-RFLP techniques was successfully applied to commercial probiotic product detection.