title: 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.
abstract: 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 M. tuberculosis complex (MTC), 156 M. tuberculosis 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 M. tuberculosis in sputum specimens. The sensitivity and specificity of LAMP assay were evaluated for the detection of M. tuberculosis. Phylogenetic analysis of the clpP sequences revealed that the Mycobacterium strains were split into two major clusters: i) MTC; ii) NTM strains and M. leprae. 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 M. tuberculosis from sputum. Our result indicated that Rv2461c coding gene was an efficient and promising alternative nucleic acid amplification test target for the detection of M. tuberculosis.
authors: Department of Respiratory Medicine, The First Affiliated Hospital of Chongqing Medical University Chongqing, P. R. China.
authors: Dairong
authors: D
authors: Li
authors: Key Laboratory of Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University Chongqing, P. R. China.
authors: Jianing
authors: J
authors: Zhao
authors: Chongqing Public Health Medical Center Chongqing, P. R. China.
authors: Xiaoping
authors: X
authors: Nie
authors: Department of Respiratory Medicine, The First Affiliated Hospital of Chongqing Medical University Chongqing, P. R. China.
authors: Tao
authors: T
authors: Wan
authors: Key Laboratory of Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University Chongqing, P. R. China.
authors: Wenchun
authors: W
authors: Xu
authors: Department of Pathology, Chongqing Medical University Chongqing, P. R. China.
authors: Yong
authors: Y
authors: Zhao
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis*
chemical_list: D001426:Bacterial Proteins; C570836:Rv2468c protein, Mycobacterium tuberculosis
keywords: Mycobacterium tuberculosis; Rv2461c gene; clpP genes; diagnostic marker; gene amplification; loop-mediated isothermal amplification