双歧杆菌属特异性测序引物筛选及优化

【背景】目前双歧杆菌的益生功能被普遍认可,越来越多的研究开始关注肠道中双歧杆菌的生物多样性。然而双歧杆菌是肠道中的低丰度物种,现有技术尚难以深入研究其多样性。【目的】基于双歧杆菌16SrRNA基因序列筛选一对适用于分析粪便样品中低丰度双歧杆菌属多样性的特异性引物。【方法】依据已有引物的相对位置及其与双歧杆菌属16S rRNA基因序列的匹配率,将引物重组优化得到扩增片段800 bp的双歧杆菌属特异性引物;通过PCR扩增和琼脂糖凝胶电泳对引物进行实验筛选和特异性验证;以细菌通用引物(27f/1492r)为参照,通过单分子实时(Single-molecule real-time,SMRT)测序技术对不同引物的3份粪便样品中细菌的DNA扩增子进行测序,在种水平上分析比较不同引物的优劣。【结果】对文献中已有的9对双歧杆菌特异性引物进行重组并优化,其中2对引物的理论特异性较好且扩增产物大于800 bp,它们分别为Bif164-f/Pbi R2和Pbi F1/Pbi R2。PCR扩增和琼脂糖凝胶电泳实验发现,Bif164-f/Pbi R2的扩增条带明亮且无拖尾。此外,利用SMRT测序平台对引物27f/1492r和Bif164-f/Pbi R2的3份粪便样品中细菌的DNA扩增子进行测序并分析。27f/1492r扩增子的分析结果显示,3份样品依次分别含1、3、4个双歧杆菌种且双歧杆菌的平均相对含量为0.34%;而Bif164-f/Pbi R2扩增子的分析结果显示,3份样品依次分别含2、6和8个双歧杆菌种且双歧杆菌的平均相对含量为98.72%。上述结果表明,Bif164-f/Pbi R2可在种水平上特异地检出粪便中低丰度的双歧杆菌,进而实现样品中双歧杆菌的多样性分析。【结论】筛选出一对双歧杆菌特异性引物Bif164-f/PbiR2,可在种水平上分析粪便样品中低丰度双歧杆菌的生物多样性,同时也验证了理论结合实验进行引物筛选这种方法的可行性。

Screening and optimization of Bifidobacterium-specific sequencing primers

Background] The probiotic functions of Bifidobacterium are widely recognized, and a large body of studies paid attention to the biodiversity of Bifidobacterium in the intestine. However, Bifidobacterium is a low abundance genus in the intestine, and it is difficult to study the bifidobacterial diversity by extant technologies in depth. Objective] To screen a pair of specific primers for the analysis of diversity of Bifidobacterium with low abundance in fecal samples. Methods] Initially, to obtain Bifidobacterium-specific primers with amplicons of over 800 bp, the primers were recombined and optimized according to the relative positions of the extant bifidobacterial primers and their matching rates with the Bifidobacterium 16S rRNA gene sequence. Then, the rest primers were screened by PCR amplification and agarose gel electrophoresis, and their specificity was verified. Finally, taking the universal bacterial primer (27f/1492r) as control, the DNA amplicons of bacterial microbiota in three fecal samples amplified by selected primers were sequenced by SMRT (Single-molecule real-time) sequencing technology, and different sequencing primers were compared and analyzed at the species level. Results] Two pairs of primers, Bif164-f/Pbi R2 and Pbi F1/Pbi R2, were selected as the optimal Bifidobacterium primers theoretically with amplicon greater than 800 bp from 9 pairs of Bifidobacterium-specific primers collected from literature. The PCR amplification and agarose gel electrophoresis showed that the amplified bands of Bif164-f/Pbi R2 were bright and no tail. In addition, sequencing and analysis of DNA amplicons from bacterial microbiota in three fecal samples with primers 27f/1492r and Bif164-f/Pbi R2 using SMRT sequencing platform. The analysis of 27f/1492r amplicons showed that three samples contained 1, 3 and 4 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 0.34%. The analysis of Bif164-f/Pbi R2 amplicons showed that three samples contained 2, 6 and 8 bifidobacterial species respectively, and the average relative content of Bifidobacterium was 98.72%. The above results indicate that Bif164-f/Pbi R2 can specifically detect bifidobacteria with low abundance in feces at species level, which realize the diversity analysis of Bifidobacterium in samples. Conclusion] In summary, a pair of Bifidobacterium-specific primers Bif164-f/Pbi R2 were screened in the experiment, which can be used to analyze the diversity of low-abundance Bifidobacterium in fecal samples at species level. It was also confirmed that the combination of theory and experiment is feasible to perform primers screening.