产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。

Establishment of Real-Time PCR Method for Detection of Clostridium perfringens

Objective：To establish a rapid,sensitive and specific real-time PCR method for detection of Clostrid-ium perfringens.Methods：A pair of primers and probe were designed depending on gene byway of target se-quence of C.perfringens,and the DNA of C.perfringens of standard strain was applied for template.The best primer and probe ratio were optimized.Specificity,sensitivity and stability analysis tests were performed by C.perfringens and 12 other associated bacteria strains.Results：The best forward primers concentration was 0.45 μmol / L,reverse primer concentration was 0.15 μmol / L,and probe was 0.3 μmol / L.The test showed that the probe were highly conservative and specific.The results of all 12 bacteria strains were negative except of strains of C.perfringens.The quantitative detection limit of the method was less than 10 CFU / reaction.Conclusion：The real-time PCR method is of high sensitivity and specialty in the detection of C.perfringens,which can be applied to the rapid detection of the bacteria in the war disease.