RESUMEN

The aim of this study was to evaluate the performance of the transcription-reverse transcription concerted (TRC) method for the detection of Mycobacterium tuberculosis complex (MTC) 16S rRNA in clinical respiratory samples for the diagnosis of pulmonary tuberculosis. TRC is a novel method that enables the rapid and the completely homogeneous real-time monitoring of isothermal sequence RNA amplification without any postamplification procedure. The detection limit of the TRC method for MTC was one organism per 100 mul of sputum. The specificity of the method was confirmed by the absence of positive signals for sputum containing 10(6) M. avium or M. kansasii organisms per 100 microl. A total of 201 respiratory samples from patients diagnosed with or suspected of having tuberculosis were tested. Of the 72 MTC culture-positive samples, the TRC method was positive for 52 (sensitivity, 72.2%), whereas the Roche COBAS AMPLICOR PCR was positive for 58 (sensitivity, 80.6%). Both the TRC method and the COBAS AMPLICOR PCR showed no positive identification for any of the 129 culture-negative samples. The percent agreement between the two methods was 95% (191 of 201 samples). The high sensitivity and specificity together with shorter detection time (within 1 h) of the TRC method allow it to be proposed as a useful method for the rapid detection of MTC in respiratory samples.

Asunto(s)

Técnicas Bacteriológicas/métodos , Mycobacterium tuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Tuberculosis Pulmonar/diagnóstico , Estudios de Evaluación como Asunto , Fluorescencia , Humanos , Sustancias Intercalantes , Mycobacterium tuberculosis/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Esputo/microbiología

RESUMEN

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.