产气荚膜梭菌多重TaqMan荧光定量PCR检测方法的建立OA北大核心CSTPCD

To establish a method for rapid detection and typing of five toxin genes cpa,cpb,etx,itx and cpe of Clostridium perfringens(C.perfringens).Specific primers and TaqMan probes were designed based to the cpa,cpb,etx,itx and cpe toxin gene sequences of C.perfringens published in NCBI database.By con-structing standard positive plasmids,optimizing reaction conditions,constructing standard curves,and performing specificity,sensitivity,repeatability and clinical sample detection tests,a multiplex TaqMan fluorescence quantitative PCR(qPCR)detection method was established that can simultaneously detect five toxin genes.The multiplex TaqMan qPCR assay was established with a good linearity of its amplification curve with R2＞0.99 and 90％＜E＜110％,with a high specificity that this method was no cross-reactivity with different pathogens,with a high sensitivity that the lower limit of detection for all its five positive plasmids was 102 copies/μL,and with a good repeatability that the coeffi-cients of variation were all less than 2％.Thirty samples were tested by using this method,and the re-sults showed a total of 30 positive samples,including 11 strains of C.perfringens type A,1 strain of C.perfringens type C and 18 strains of C.perfringens type D.The multiplex TaqMan qPCR detection method for C.perfringens established in this experiment provides a convenient,rapid and efficient technical support for the diagnosis,prevention and control of C.perfringens disease.