Comparison of real-time PCR with SYBR Green I or 5′-nuclease assaysand dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantificationof selected faecal bacteria

摘要

PCR primers and hybridization probes were designed for the 16S rRNA genesof six bacterial species or groups typically present in human faeces or usedin the dairy industry. The primers and probes were applied for quantificationof the target bacterial genomes added in artificial DNA mixtures or faecalDNA preparations, using dot-blot hybridization and real-time PCR with SYBRGreen I and TaqMan chemistries. Dot-blot hybridization with 33P-labelled oligonucleotide probes was shown to detect a 10?%target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targetedoligonucleotide probes without pre-enrichment of the 16S gene pool by PCRwas thus limited to the detection of the predominant microbial groups. Real-timePCR was performed using a 96-well format and was therefore feasible for straightforwardanalysis of large sample amounts. Both chemistries tested could detect andquantify a subpopulation of 0·01?% from the estimatednumber of total bacterial genomes present in a population sample. The linearrange of amplification varied between three and five orders of magnitude forthe specific target genome while the efficiency of amplification for the individualPCR assays was between 88·3 and 104?%. Use of a thermallyactivated polymerase was required with the SYBR Green I chemistry to obtaina similar sensitivity level to the TaqMan chemistry. In comparisonto dot-blot hybridization, real-time PCR was easier and faster to performand also proved to have a superior sensitivity. The results suggest that real-timePCR has a great potential for analysis of the faecal microflora.