Study on the LAMP-PCR-hybridization–thermal melt–ELISA method for molecular detection of multidrug resistance in Mycobacterium tuberculosis isolates

Abstract

In this study, we designed a novel colorimetric method to detect multidrug resistance in Mycobacterium tuberculosis isolates. The assay of loop-mediated isothermal amplification (LAMP) is used to amplify target DNA from multidrug-resistant M. tuberculosis isolates, and enzyme-linked immunosorbent assay (ELISA) is used for the colorimetric determination. This method is designed based on point mutation at the hot spot region in target drug-resistant gene using LAMP-polymerase chain reaction (PCR), hybridization, and thermal melting for differentiating homoduplex DNA (drug-susceptible stain) and heteroduplex DNA (resistance mutant). From ELISA colorimetric detection, color change developed in drug-susceptible strains, and colorless result appeared in resistance mutants. A comparison of this LAMP-PCR-hybridization–thermal melt–ELISA (LAMP–TM–ELISA) method with the automated BACTEC MGIT 960 system showed that the sensitivity of this molecular analysis of resistance to isoniazid, rifampin, amikacin, and ciprofloxacin in M. tuberculosis was 92.3%, 95.3%, 93.1%, and 91.4%, respectively. This method for detection of resistance to isoniazid, rifampin, amikacin, and ciprofloxacin in M. tuberculosis showed a specificity of 95.5–98.2% and a test efficiency of 93.2–96.8%. This LAMP–TM–ELISA method will be a useful tool for rapid diagnosis (within 1 working day) and cost-effectiveness (US$15/reaction) to detect resistance to isoniazid, rifampin, amikacin, and ciprofloxacin via katG, inhA and mabA-inhA promoter, rpoB, rrs, gyrA, and gyrB genes in M. tuberculosis isolates.