Establishment and clinical application of a new quantitative method for rapid detection of Clostridium perfringens in wound secretion

Objective To establish a real-time quantitative fluorescent polymerase chain reaction(QR-PCR) method for rapid detection of Clostridium perfringens in wound secretion,so as to provide a new means to diagnose gas gangrene early and quickly.Methods The 16s rDNA gene sequence of Clostridium perfringens downloaded from GenBank was used as a template,and the specific primers and probes were designed according to the conserved region.The PCR amplified products were ligated to plasmid pMD19-T and used as the standard of the quantitative determination,and then the standard curve was generated based on the determination results.The reaction condition and reaction system of the established QR-PCR were optimized,and the specificity,sensitivity,reproducibility and feasibility of the assay were verified.Results The established QR-PCR method showed a high specificity for Clostridium perfringens,and no cross-reaction with 23 other associated bacteria was observed.The sensitivity was 9×102cfu/ml,i.e.9cfu per reaction.The reaction system showed a high stability and the time spent for the whole operation process was only 3 hours.The results of 5 suspected wound secretion detected by the established QR-PCR were identical with that of bacterial culture.Conclusion The established QR-PCR assay is a quick,sensitive and specific method for determination of Cl.perfingen,and it can be used in routine test and batch emergency examination(in war or unexpected events) for Clostridium perfringen in wound secretion.