Abstract: A new method for selectively quantitative detection of trh-positive viable cells of Vibrio parahaemolyticus in oysters was developed using ethidium bromide monoazide (EMA) staining in combination with real-time PCR (RT-PCR, real-time polymerase chain reaction). The results showed the optimum light exposure time to achieve DNA crosslinking by EMA in dead cells and to photolyze the free EMA in solution was 20 min. The use of 2.0μg/mL EMA or less did not inhibit the RT-PCR amplification of DNA derived from viable cells of Vibrio parahaemolyticus. The minimum amount of EMA to completely inhibit the RT-PCR amplification of DNA derived from heat-killed cells was 1.0μg/mL. In artificial contaminated oyster samples without enrichment process, there was a strict negative correlation between the log cell number and the Ct values in the range of 2.0 × 103 to 2.0 × 107 CFU. The detection limit of the real-time PCR assay was 2 × 103 CFU in both pure cultures and artificial contaminated oyster samples, which indicated the sensitivity of RT-PCR was 400 CFU/g in artificial contaminated oyster sample. The freezing/thawing experiments showed thawing in water bath at temperature below 55 ℃ had little effect on viable cells of Vibrio parahaemolyticus. Therefore, this method avoided the defection in traditional PCR, which could not distinguish viable bacteria cells from dead ones. It may offer a fast, sensitive method to identify and quantify pathogenic viable cells effectively.

Key words: EMA, RT-PCR (real-time polymerase chain reaction), seafood, Vibrio parahaemolyticus, viable cells