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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.

SUBMITTER: Li D

PROVIDER: S-EPMC4313969 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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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.

Li Dairong D Zhao Jianing J Nie Xiaoping X Wan Tao T Xu Wenchun W Zhao Yong Y

International journal of clinical and experimental pathology 20141201 12

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-mycoba ...[more]

PMID: 25674236

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Project description:BackgroundTuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease. Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries.MethodsIn the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process. To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB.ResultsOur SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA.ConclusionTo our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Dataset Information

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.

Publications

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.

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