Protein-coding housekeeping gene Rv2461c can be used as an amplification target in loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis in sputum samples

摘要

The study is to explore the potential of the conserved Rv2461c gene as a biomarker for Tuberculosis (TB) diagnosis. The conservation of the hypothetical genes was evaluated in this study using multiple sequence alignment and phylogenetic analysis. The conservation of Rv2461c coding gene was evaluated by polymerase chain reaction using six reference strains of emM. tuberculosis complex/em (MTC), 156 emM. tuberculosis/em clinical isolates, 25 species of non-tuberculosis mycobacteria (NTM), and 10 non-mycobacterial species. A total of 126 clinical sputum specimens were collected from patients with respiratory symptoms, including 79 specimens from suspected TB patients, and 47 specimens from patients with respiratory diseases other than TB. Genomic DNAs were extracted and subject to polymerase chain reaction for nucleic acid amplification test. In addition, we successfully developed loop-mediated isothermal amplification (LAMP) technology for rapid detection of emM. tuberculosis/em in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of emM. tuberculosis/em. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and emM. leprae/em. During the evaluation of the conservation of Rv2461c coding gene, all MTC strains yielded positive results, and no false-positive results were observed in NTM or other bacterial species. LAMP analysis showed high sensitivity and specificity (84.8% and 95.7%, respectively) for the detection of emM. tuberculosis/em from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of emM. tuberculosis/em.