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

Researchers developed an in-house loop-mediated isothermal amplification (LAMP) assay called MTB-LAMP to detect Mycobacterium tuberculosis using six primers targeting the 16S rRNA gene. The assay was evaluated on 200 sputum samples from Nepalese patients. MTB-LAMP demonstrated 100% sensitivity in culture-positive samples (96/96 samples), detecting all smear-positive and smear-negative culture-confirmed cases. In culture-negative samples, the assay showed 94.2% specificity (98/104 samples), with positive and negative predictive values of 94.1% and 100%, respectively. The primers were highly specific for the M. tuberculosis complex, showing no cross-reactivity with other mycobacterial species or non-mycobacterial bacteria tested. The detection limit was approximately 20 copies of M. tuberculosis DNA and 10 live bacteria in sputum samples. These results suggest MTB-LAMP could facilitate rapid tuberculosis diagnosis in resource-limited settings by combining the speed of microscopy with the sensitivity of bacterial culture.

Key findings

• MTB-LAMP achieved 100% sensitivity in culture-positive sputum samples, detecting all 96 confirmed tuberculosis cases including both smear-positive and smear-negative specimens
• The assay demonstrated 94.2% specificity in culture-negative samples with 100% negative predictive value, indicating reliable confirmation of negative cases
• MTB-LAMP primers were highly specific for M. tuberculosis complex members with no cross-reactivity with other mycobacteria or common bacterial species tested
• The assay can detect as few as 20 copies of M. tuberculosis DNA and approximately 10 live bacteria in processed sputum samples
• MTB-LAMP offers advantages for tuberculosis diagnosis in developing countries by providing rapid results comparable to culture methods while maintaining high sensitivity