[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Detection of cancer micrometastases is required for improvement of cancer therapy. The aim of this study was to establish a rapid and practical genetic assay to detect micrometastasis in gastric cancer and to assess its clinical significance with respect to prognosis.
A novel RNA amplification system with transcription-reverse transcription concerted reaction (TRC) was introduced for quantitative detection of cancer-specific carcinoembryonic antigen messenger RNA. The sensitivity and quantitative aspects of the assay were assessed with the full-length carcinoembryonic antigen messenger RNA, a gastric cancer cell line (MKN-45), and metastatic lymph nodes obtained from patients with gastric cancer. Peritoneal lavage fluid specimens that were collected from gastric cancer surgery were subjected to the assay, and the clinical significance of the results was examined for prediction of recurrence and survival.
The quantification, sensitivity, and reproducibility of the assay with the TRC reaction were equal to those of quantitative reverse transcriptase-polymerase chain reaction with LightCycler. The most important advantages of the assay were its simplicity and rapidity. Molecular diagnosis of peritoneal lavage fluid by the TRC reaction significantly correlated with depth of invasion, peritoneal metastasis, clinical stage, overall survival, and peritoneal recurrence-free survival.
Molecular diagnosis of peritoneal lavage fluid with the TRC reaction could be a useful prognostic indicator for peritoneal recurrence and survival. Because the TRC reaction is more rapid and simpler than reverse transcriptase-polymerase chain reaction as a format for detecting RNA sequences, it may enhance the genetic diagnosis of cancer micrometastasis and may improve cancer therapy.