Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10 3 to 10 7 copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.Vibrio parahaemolyticus can cause seafood-borne gastroenteritis in humans. However, not all strains are considered virulent strains. The Kanagawa phenomenon, -type hemolysis in Wagatsuma agar was formerly considered the marker of virulent strains because early studies demonstrated that most clinical strains, but few environmental strains, exhibit this phenomenon (16,29). Thermostable direct hemolysin (TDH) is responsible for the Kanagawa phenomenon. Subsequently, clinical strains lacking the ability to produce TDH but producing a TDH-related hemolysin (TRH) were discovered (5, 6). The strains carrying the tdh gene encoding TDH, the trh gene encoding TRH, or both genes are now considered to be virulent strains (19); the strains carrying these hemolysin genes are strongly associated with clinical cases (11, 30). Therefore, in investigations of the infection by V. parahaemolyticus, the V. parahaemolyticus strains isolated from clinical specimens are usually examined for the tdh and trh genes in most laboratories. The strains carrying the tdh gene, the trh gene, or both genes are then examined for the O:K serotype and, if possible, the DNA fingerprint for epidemiological investigation.Our study group reported five variants of the tdh genes, tdh1 and tdh2 from a Kanagawa phenomenon-positive strain and tdh3, tdh4, and tdh5 genes from Kanagawa phenomenon-negative strains, and these five variants of the tdh gene share Ͼ97% sequence identity (19). T...