Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples

The potential of incorporating a real-time FOR for amplification and detection of 16S rRNA gene signatures directly from clinical samples was assessed as a tool for diagnostics. Universal FOR primers spanning short variable regions (similar to 500 bp) were optimized for real-time PCR and tested in comparison with a longer fragment (similar to 1400 bp) generated from block-based amplification. Real-time PCR had improved sensitivity of detection (8% increase), decreased amplification time and simplified downstream processing. The real-time PCR primers also offered an improvement in detection of bacteria from samples that demonstrated inhibition with the block-based primers and in the resolution of mixed-sequence traces. In addition to testing primer sensitivity, the effect of amplifying and sequencing two different variable regions of the 16S rRNA gene on organism identification was compared. By amplifying and sequencing a shorter variable region, the number of species that were identified to the species level was reduced by 18%.