Study on PMA-ddPCR Method for Quantitative Detection of Live Vibrio parahaemolyticus in Aquatic Products
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Abstract:
A rapid quantitative PCR detection method for live Vibrio parahaemolyticus in aquatic products was studied. As propidium monoazide (PMA) can inhibit the death of bacteria DNA amplification under at certain lighting condition, and the droplet digital PCR technology can extend detection accuracy to single molecule target genes and achieve absolute quantification, a new method called PMA-ddPCR (combined PMA and ddPCR ) was applied to detect live Vibrio parahaemolyticus. Specific primers and probes targeting the tlh gene of Vibrio parahaemolyticus were designed, and the PCR reaction system was optimized. The PMA concentration and exposure conditions of PMA-ddPCR for target gene were also tested. The results showed that the working concentration of PMA was 16 μg/mL and the exposure time was 8 min. The expansion of dead bacteria could be completely inhibited and the activity of live bacteria was not affected under this condition. The sensitivity of PMA-ddPCR method was higher than that of PMA-qPCR method by comparing the detection limits of PMA-ddPCR and PMA-qPCR (2×101 cfu/mL and 2×102 cfu/mL, respectively). The live Vibrio parahaemolyticus in artificially contaminated marine products (sea shrimp and small scallop) were detected by PMA-ddPCR quantitative method, and the lowest detectable Vibrio parahaemolyticus in sea shrimps was 1.9×101 cfu/g, and the lowest detectable of Vibrio parahaemolyticus in small sail was 0.89×101 cfu/g. This study provided the foundation for the application of PCR technology to the quantitative detection of low-level pollution of live Vibrio parahaemolyticus in aquatic products.
