Establishment of real-time PCR method for detection of clostridium perfringens

OBJECTIVE To establish a rapid,sensitive and specific detection method of Clostridium perfringens with TaqMan PCR.METHODS A pair of primers and probe depending on gene were designed by way of target sequence of Clostridium perfringens,and apply clostridium perfringens of standard bacterium strain for template to appraise clostridium perfringens.The best primer and probe ratio were optimized.Specificity,sensitivity and stability analysis tests were performed by Clostridium perfringens and 24 other strains associated bacteria.RESULTS The best forward primers concentration was 1μM,reverse primer concentration was 1μM,and probe was 0.1μM.Test showed that the probe were highly conservative and specific.The results of all 24 strains bacteria were negative except of strains of Clostridium perfringens.The quantitative detection limit of the method was 9×102 CFU/ml.Repetitive experiments showed that this system had high stability and repeatability.The test results of 3 cases clinical samples were consistent with bacteria cultivation result.CONCLUSION The Real-time PCR method is of high sensitivity and specialty in the detection of Clostridium perfringens,which can be applied to the rapid detection of the bacteria in the balefulness disease.