Development of an Insulated Isothermal PCR-based Method for Rapid Detection of Bacillus cereus
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Abstract:
Specific primers and probe were designed for the gyrB gene of Bacillus cereus encoding the gyrase B subunit, and a method based on insulated isothermal polymerase chain reaction (iiPCR) was established for the rapid detection of B. cereus and subsequently validated. The efficacy of this method was compared with that of traditional PCR and real-time fluorescent quantitative PCR (SYBER Green I fluorescent dye and TaqMan probe methods) detection methods, and applied in the detection of B. cereus in different types of retail food. The results revealed that the newly developed method facilitated the rapid detection of B. cereus within 40 min. Moreover, there was no cross reaction with other bacteria, including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Salmonella enteritidis, thereby indicating good specificity. The minimum detection limit of the method was 1.5×102 CFU/mL, which was significantly superior to that of the traditional PCR method, and equivalent to that of quantitative PCR. Of the 42 retail food samples assessed, 19 (45.23%) were found to be contaminated with B. cereus. The results obtained for method validated were completely consistent with those obtained using traditional PCR detection, although test results were available within a considerably shorter timeframe, saving at least 4 h. The method developed in this study provides a rapid, convenient, specific, and sensitive procedure that is suitable for the real-time on-site detection of B. cereus.
