Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR

In this study we developed a specific and sensitive quantitative PCR (qPCR) method combined with a propidium monoazide (PMA) sample treatment to quantify tdh-positive viable cells of V. parahaemolyticus in raw seafood (PMA-qPCR). The high selectivity of primers and probes were demonstrated by using purified DNA from 57 strains belonging to 18 species. Using these primers and probes for qPCR and in artificial contamination samples, a good correlation was obtained between Ct values and log CFU/reaction in the range of 12–1.2 × 106 CFU/reaction both from qPCR and PMA-qPCR with R2 values of 0.9973 and 0.9919, respectively. The optimization of PMA concentration showed that 8 μg/mL was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction in compromised cells. However, turbidity and cell concentration experiments showed that PMA treatment was not effective in samples where turbidities were ≥ 10 NTU and OD600nm values were ≥ 0.8. PMA-qPCR was compared with culture isolation and traditional qPCR in environmental samples (including oyster, scallop, shrimp, and crab). The PMA-qPCR resulted in lower numbers of log CFU g− 1 than qPCR, with values having better agreement with numbers determined by culture isolation. In conclusion, this method is an effective tool for producing reliable quantitative data on viable V. parahaemolyticus in raw seafood.

► Detection sensitivity is 12 CFU/reaction in artificial contamination oyster samples for both qPCR and PMA-qPCR.
► The optimum concentration of PMA is 8 μg mL− 1.
► The efficiency of PMA treatment is decreased greatly and even resulted in the inefficiency of PMA treatment with higher turbidity (> 10 NTU) and cell density of samples (OD600 nm ≥ 0.8).
► For seafood samples, the values obtained by PMA-qPCR are in better agreement with the numbers determined by culture isolation.