Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis.
Traditional culture, followed by a panel of biochemical tests for the diagnosis of tuberculosis (TB), is time-consuming, and rapid identification of Mycobacterium tuberculosis is crucial for the early administration of appropriate therapy. In this study, the reverse transcription loop-mediated isothermal amplification combined with enzyme-linked immunosorbent hybridization (RT-LAMP-ELISA-hybridization) assay has been designed for the rapid detection of 16S rRNA in clinical isolates of M. tuberculosis. This assay reproducibly detected a single copy, as opposed to 2000 copies of MTB 16S rRNA detected by conventional gel electrophoresis. Among the 150 specimens of sputum analysed, RT-LAMP-ELISA-hybridization assay had a sensitivity of 94.1% in the culture method, compared to the Amplified M. tuberculosis Direct Test (AMTD), 91.1% and the 88.2% sensitivity of acid-fast staining. Furthermore, RT-LAMP-ELISA-hybridization assay is more cost-effective when compared to the real-time TaqMan RT-PCR and AMTD assays. In conclusion, our results suggest that the RT-LAMP-ELISA-hybridization assay is a highly sensitive, low cost diagnostic tool useful for the rapid and accurate direct diagnosis of sputum specimens, and is suitable for routine clinical use.
Lee
Mei-Feng
MF
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Chen
Yen-Hsu
YH
Peng
Chien-Fang
CF
D001431:Bacteriological Techniques / Q000379:methods; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D011237:Predictive Value of Tests; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013183:Sputum / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S