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.