Establishment of EMA-PCR detection method of Vibrio parahaemolyticus

Abstract:

PCR technology has been widely used in the detection of Vibrio parahaemolyticus. However, traditional PCR appeared higher false-positive result because of the lack of differentiation between DNA from viable and dead microorganisms. Therefore, Ethidium Monoazide Bromide (EMA) was added in the process of PCR to establish a rapid and accurate detection method of V. parahaemolyticus. The dnaJ gene was used as the target gene for PCR detection of V. parahaemolyticus by utilizing its pure isolates and genomic DNA as the template, and the sensitivity was 2.5×104 CFU/mL and 6×102 fg/μL respectively. The use of 5 mg/L or less EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The PCR amplification of DNA derived from 1×108 CFU/mL V. parahaemolyticus dead cells can be inhibited by 2 mg/L EMA. The results show that EMA-PCR can be used to minimize the false-positive results by inhibiting the PCR amplification of V. parahaemolyticus dead cells from a mixed bacterial population.