Establishment and application of a TaqMan-based real-time fluorescence quantitative PCR detection method for Clostridium perfringens

Abstract

ObjectiveA TaqMan-based real-time fluorescence quantitative polymerase chain reaction （PCR） method was established to detect Clostridium perfringens （C. perfringens） in tap water samples.MethodsThe highly conserved plc gene located in the pseudonucleus of the bacterium was amplified and specific primers and TaqMan probes were designed. After optimization， a TaqMan-based real-time fluorescence quantitative PCR detection method was established. Combined with the filter membrane method， simulated polluted water samples of standard strains containing plc genes were treated， and the established method was tested.ResultsThe established TaqMan-based method for detecting C. perfringens showed high specificity. Ct >40 were found in 13 foodborne pathogens， 3 Clostridium difficile and 1 Clostridium putrefaciens strain. The detection limit of this method was 1×10 copies/μL， showing high sensitivity. The minimum detection limit for the simulated polluted water sample was 1.0×102 CFU/mL. This method was also used to detect 4 simulated water contamination samples and 90 imported water samples. The results showed that C. perfringens could be detected in 2 simulated 1.0×102 CFU/mL-contaminated water samples， while not in 2 simulated 1.0×10 CFU/mL-contaminated and imported water samples.ConclusionThe TaqMan real-time fluorescence quantitative PCR method established in this study for the detection of C. perfringens has good specificity， high sensitivity， and practical value for detecting C. perfringens in water samples.

Keywords

• clostridium perfringens
• real-time fluorescent quantitative pcr
• taqman probe method
• water sample detection
• foodborne pathogens