Fluorescent MIRA assay for the rapid detection of Clostridium perfringens in food

Abstract

ObjectiveTo establish a rapid and accurate molecular method for detecting Clostridium perfringens （C. perfringens） in food using fluorescent multi-enzyme isothermal rapid amplification （MIRA）.MethodsPrimers and probes based on the conserved sequence of the C. perfringens alpha toxin-coding gene plc were designed separately， and the fluorescence MIRA method was established and optimized. The genomic DNA of 17 non-C.perfringens strains were extracted for specificity verification， and the isolated DNA and bacterial solutions of different dilutions were used for sensitivity evaluation. Matrix interference testing with 4 types of food media was also performed to evaluate the stability of the method， and the actual sample detection results were compared with the standard method to verify its practicality.ResultsThe fluorescent MIRA method for C. perfringens used in this study had high specificity. The results returned negative for other non-C. perfringens strains. MIRA also had high sensitivity， with detection limits of 1.05×101 fg/μL for genomic DNA and 7.5×100 CFU/mL for pure C. perfringens cultures. It also had a strong anti-interference ability； the detection results were not affected by the matrix， and the detection time was short （approximately 20 min）.ConclusionThis study developed a rapid， accurate， sensitive and specific fluorescent MIRA method for the molecular detection of C. perfringens in food.

Keywords

• multi-enzyme isothermal rapid amplification
• clostridium perfringens
• plc gene
• molecular detection
• foodborne pathogens