检测肠道细菌Feacalibacterium prausnitzii的三对PCR引物的特异性比较

Specificity comparison of three Feacalibacterium prausnitzii-specific PCR primer pairs

【目的】比较3对基于16S rRNA基因、用于检测人肠道中重要细菌Feacalibacterium prausnitzii的引物(FPR-1/FPR-2、FPR-2F/Fprau645R和Fprau223F/Fprau420R)的特异性。【方法】用Clustal X比对每个引物与F.prausnitzii和其他细菌的16S rRNA基因的序列。在Ribosomal Database Project(RDP)数据库中使用Probe Match工具比较每个引物匹配的Faecalibacterium spp.序列数目。利用本课题组建立的中国人粪便菌群的16S rRNA基因全长文库的7255个克隆序列,用Simulated PCR(SPCR)预测每对引物检测到的F.prausnitzii和其他细菌的克隆数;用3对引物分别对代表克隆进行PCR扩增。用3对引物分别对14个健康人的粪便样品进行实时定量PCR。【结果】引物Fprau645R的3端最后一个碱基与非F.prausnitzii序列的错配度高于其它引物,它在RDP中匹配的Faecalibacterium spp.序列数占其匹配的细菌序列数的百分比(97.6%)显著高于其他引物。SPCR预测,3对引物检测到的F.prausnitzii克隆数均为1171左右;在检测到的非Faecalibacterium spp.克隆中,FPR-2F/Fprau645R主要是Subdoligranulum spp.,而FPR-1/FPR-2和Fprau223F/Fprau420R还有Oscillibacter spp.、Ruminococcus spp.和unclassified Ruminococcaceae等。真实PCR与SPCR的结果吻合。实时定量PCR中,FPR-1/FPR-2和Fprau223F/Fprau420R检测到的细菌数量高于FPR-2F/Fprau645R。【结论】3对引物能检测到F.prausnitzii和Subdoligranulum spp.,FPR-2F/Fprau645R的特异性优于FPR-1/FPR-2和Fprau223F/Fprau420R。

[Objective] To compare the specificity of three 16S rRNA gene-based PCR primer pairs（FPR-1/FPR-2,FPR-2F/Fprau645R and Fprau223F/Fprau420R）,which are used to specifically detect and quantify the important human gut bacterium Feacalibacterium prausnitzii.[Methods] Clustal X was used to align the sequences of individual primer and the 16S rRNA gene of F.prausnitzii and other bacteria.The number of Faecalibacterium spp.sequences in Ribosomal Database Project（RDP） matched by each primer was obtained by the Probe Match tool.With the full-length 16S rRNA gene clone library constructed by our laboratory which contained 7255 clones from the gut microbiota of 7 Chinese people,the Simulated PCR（SPCR） program was applied to predict the clone number of F.prausnitzii and other gut bacteria matched by every primer pair;PCR amplification was performed with three primer pairs and representative clones to verify the SPCR prediction.Real-time quantitative PCR was performed with three primer pairs,respectively,for fecal samples from 14 healthy individuals.[Results] The first base at the 3 end of Fprau645R showed the highest mismatch level for non-F.prausnitzii bacteria.The percentage of the number of Faecalibacterium spp.sequences matched by Fprau645R accounting for that of matched bacteria sequences in the RDP database was 97.6%,which was significantly higher than that of other primers.As SPCR predicted,all three primer pairs can detect about 1171 clones from F.prausnitzii;the clones of non-Faecalibacterium spp.detected by FPR-2F/Fprau645R mainly were Subdoligranulum spp.,but the non-Faecalibacterium spp.clones detected by FPR-1/FPR-2 and Fprau223F/Fprau420R were mainly Subdoligranulum spp.,Oscillibacter spp.,Ruminococcus spp.and unclassified Ruminococcaceae etc.The real PCR showed the same results with SPCR.The real-time quantitative PCR showed FPR-1/FPR-2 and Fprau223F/Fprau420R detected more bacteria than FPR-2F/Fprau645R.[Conclusion] The three primer pairs can detect F.prausnitzii and Subdoligranulum spp.,however,the specificity of FPR-2F/Fprau645R is better than FPR-1/FPR-2 and Fprau223F/Fprau420R.