Abstract

Clostridium perfringens can cause gas gangrene, a serious and often lethal condition. To evaluate real-time fluorescence-based quantitative PCR as a means of rapidly detectingC. perfringens for fast and effective diagnosis of gas gangrene, we used a C. perfringens16s rDNA gene sequence as a template to design specific primers and probes, optimized reaction conditions, and used clones of the amplified PCR product to make a standard curve for quantitative measurement. We showed that the technique was highly specific toC. perfringens, exhibiting no cross-reaction with any of 24 related bacteria tested. The detection sensitivity was 9×10² cfu/ml pure C. perfringens, or 9 cfu/reaction. The procedure was stable, repeatable and fast, taking only 3 h in total. Detection results of 500 wound secretions using this PCR method were consistent with those obtained using conventional culture detection methods, demonstrating for the first time that thisfluorescence-based quantitative PCR system is effective, accurate and applicable on a large sample scale under clinical conditions. We have thus provided a highly specific, sensitive, fast, and accurate fluorescence-based quantitative PCR technique that can quickly diagnose the early stages of gas gangrene. This may be especially valuable in batch testing, such as in the event an earthquake or other emergency.

Key words: Clostridium perfringens, gas gangrene, real-time fluorescence-based quantitative PCR, 16s rDNA, trauma patients.