Real-time quantitative PCR for rapid detection of Clostridium perfringens

Objective To detect Clostridium perfringens with real-time quantitative PCR technique to provide a new tool for diagnosing gas gangrene early.Methods The gene sequences of Clostridium perfringens downloaded from the genebank were aligned using the biologic software and the specific primers and probe were designed in the conserved region of the 16s rDNA gene from Clostridium perfringens.The primers,probe and the reactive condition were optimized to improve the sensitivity,specificity,repetitiveness and feasibility of the assay.Results Only Clostridium perfringens possessing 16s rDNA gene generated fluorescent signals,and no cross-reaction was observed with 24 other associated bacteria.The reactive system had a high stability.The sensitivity achieved was 9×102cfu/ml,i.e.9 cfu/per reaction.It took only three hours to do the real-time quantitative PCR.The results of 300 cases of suspected wound secretion detected by the real-time quantitative PCR were the same with bacteria cultivation.Conclusion The real-time quantitative PCR assay is a quick,sensitive and specific tool for usual monitoring and emergent examination of Clostridium perfringen.