Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

Abstract

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Rapid Identification of Mycobacterium tuberculosis in BACTEC MGIT960 Cultures by In-House Loop-Medicated Isothermal Amplification

“…Presumptive identification of isolates recovered on solid media, such as L-J slant, can be performed based on colony morphology, but this cannot be applied with MGIT-positive cultures. In our previous study, TB-LAMP was used for direct detection of M. tuberculosis in sputum specimens (19); however, the direct detection of M. tuberculosis in sputum includes bacilli that died during anti-TB therapy. The detection of live bacilli is essential for the accurate diagnosis of active TB following proper treatment.…”

“…A set of six primers recognizing eight distinct regions on the 16S ribosomal RNA gene of M. tuberculosis was used (19) Biolabs, Ipswich, Mass., USA), and 1 ml fluorescent detection reagent (FDR; Eiken Chemical Co., Tokyo, Japan) with 7.0 ml DNA lysates. Amplification was performed at 659 C for 60 min in a small heat block, followed by incubation at 809 C for 2 min to terminate the reaction.…”

“…LAMP generates a large amount of DNA with high specificity, allowing amplification to be detected by the naked eye as fluorescence or turbidity (13)(14)(15)(16)(17). LAMP has been used to detect a number of infectious organisms, including M. tuberculosis (18,19), and has been proposed as a useful tool for the rapid and accurate diagnosis of M. tuberculosis in resource-limited laboratories (20,21). In this study, we used an in-house LAMP assay to rapidly identify MTBC from MGIT culture in Thailand.…”

Detection of Mycobacterium tuberculosis complex in sputum specimens using a loop-mediated isothermal amplification assay in Korea

“…Although several clinical evaluations of LAMP assays have been published (Boehme et al, 2007;George et al, 2011;Mitarai et al, 2011;Pandey et al, 2008), to the best of our knowledge, this is the first study to clinically evaluate the use of the LAMP method to detect the hspX gene of MTC and is also the first report of the LAMP method for diagnosing TB in Korea. Sputum specimens obtained from patients at the Korea University Medical Center (KUMC) who were suspected of having TB were subjected to culture confirmation, smear microscopy and the LAMP method.…”

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

“…10 The system shows a high amplification efficiency and has been used to diagnose several other diseases. [10][11][12][13][14][15][16][17] The objective of our study was to evaluate the applicability of the LAMP system, in the veterinary field, to detect MTC directly from suspected TB lesions of cattle and wildlife being slaughtered for food.…”