ABSTRACT
We developed a novel method named the transcription-reverse transcription concerted (TRC) method and an instrument that allowed rapid and completely homogeneous real-time monitoring of RNA isothermal sequence amplification without any post-amplification analysis in our previous study [Ishiguro et al., Anal. Biochem., 314, 77-86 (2003)]. In this study, we newly established rapid and sensitive TRC systems for the detection of the mRNAs transcribed from two major virulence genes of Vibrio parahaemolyticus: the tdh gene encoding the thermostable direct hemolysin (tdh) and the trh gene encoding the thermostable direct hemolysin-related hemolysin. Examination of the standard RNAs prepared in vitro showed that a positive result, increase in the fluorescence intensity to the cut-off value within 25 min, was obtained for as few as 100 copies of RNA. The TRC method specific to the trh mRNA detected the mRNAs transcribed from the trh1 and trh2 genes, two representative trh variants sharing 84% sequence identity. The detection time gave a linear relationship to the logarithm of starting RNA copies ranging from 10(3) to 10(7) copies, showing that quantitative analysis is possible. The detection time for 10(3) copies of the standard RNAs ranged from 11 to 15 min. Examination of the total RNAs extracted from the standard strains of V. parahaemolyticus demonstrated that the new TRC systems are sufficiently sensitive to detect as few as 100 CFUs of the strains carrying the target genes. Total RNA preparations extracted from 24 strains of V. parahaemolyticus, 52 strains belonging to 31 other species of the genus Vibrio and 11 strains belonging to 8 species of non-Vibrio genera were examined. The results of the detection of tdh- and trh-specific mRNAs by the two TRC systems and those of the respective genes by the DNA colony hybridization method agreed. We conclude that the new TRC systems are rapid, highly sensitive, easy to manipulate, and are suitable for routine examination of virulent strains of V. parahaemolyticus in microbiological laboratories.
