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.

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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...

A transcription-reverse transcription (RT) concerted reaction (TRCR) method was developed for rapid and specific detection of EV71 from clinical specimens. This method was validated with EV71 strains from all of the known genotypes (genotypes A, B1 to B5, and C1 to C5), with detection limits of 10 to 10 3 copies, and was useful for identification of EV71 from throat swabs of patients with hand, foot, and mouth disease (HFMD). Enterovirus 71 (EV71) is a major etiological agent for hand, foot, and mouth disease (HFMD) and herpangina, along with coxsackievirus A (CVA) types such as CVA10 and CVA16 (1, 10). In contrast to CVA infection (3), EV71 infection is also associated with severe neurological diseases, such as brain-stem encephalitis and poliomyelitis-like paralysis (4, 9, 14) with a high incidence (case severity rate of Ͻ0.3%) (6). Effective vaccines are not available at present, and only supportive care can be provided for the treatment of severe brain-stem encephalitis (reviewed in reference 15). Because of the rapid progression of symptoms in fatal cases of EV71 infection (Յ12 h after hospitalization) (2), rapid diagnosis of EV71 infection is essential for severely EV71-infected patients to provide proper care among patients with or without HFMD caused by other EV infections.In this study, we aimed to develop a diagnostic test for EV71 by using a transcription-reverse transcription (RT) concerted reaction (TRCR) method (7, 13). The TRCR method is an isothermal RNA amplification method as well as an RT-loop-mediated isothermal amplification (LAMP) method, but it utilizes T7 RNA polymerase and avian myeloblastosis virus (AMV) reverse transcriptase instead of DNA polymerase (7, 13). The major advantages of the TRCR method are (i) rapid detection (20 min of reaction time), (ii) high specificity (by intercalation activating fluorescence [INAF] probes) (8), and (iii) high sensitivity (as low as 10 copies). For its rapid measurement of RNA, the TRCR method has been used as a diagnostic tool for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare in some hospitals

Successful preemptive therapy for cytomegalovirus (CMV) infection after hematopoietic stem cell transplantation (HSCT) depends on the availability of a rapid and sensitive assay to guide early treatment. Currently, the antigenemia assay and the quantitative real-time PCR (qPCR) assay are widely used for this purpose, but they have distinctive concerns. This study aimed to develop and evaluate a novel CMV diagnostic test based on transcription-reverse transcription concerted reaction (TRC), an RNA-detecting technology. The CMV-TRC assay detected CMV β2.7 transcripts within 10 min over a five-log range. Among a total of 219 samples obtained from 24 allogeneic HSCT recipients, samples detected as positive by the CMV-TRC assay showed a relatively strong correlation with those detected as positive by the qPCR assay and the antigenemia assay. The CMV-TRC assay showed higher sensitivity (77.7%) than the antigenemia assay (68.1%) and detected the first and recurrent episodes of active CMV infection in HSCT patients significantly earlier than the antigenemia assay (P < 0.001). Although the CMV-TRC assay (87.8%) showed low sensitivity compared to the qPCR assay (96.3%), the performance of the CMV-TRC assay was equivalent to that of the qPCR assay in detecting the appearance of active CMV infection episodes (P < 0.092) or rather superior in detecting the clearance of episodes (P < 0.001). The CMV-TRC assay with several advantages may be useful for guiding preemptive anti-CMV therapy in HSCT recipients.