Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection ofandby dipstick DNA chromatography.

UNLABELLED: Although human tuberculosis (TB) caused byis clinically, pathologically, and radiologically indistinguishable from-caused TB,is innately resistant to pyrazinamide, a key first-line drug effective against. The rapid differentiation of these two biovars is therefore of high clinical and epidemiologic importance. Most current molecular tools in resource-limited settings identify mycobacteria only to thespecies (MTB) level. In this study, we report a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography for the rapid and easy differential detection ofand. The assay was optimized and validated using 143 isolates comprising six MTB reference strains, 50.isolates, 58.isolates, 24 non-tuberculous mycobacterial (NTM) strains, and five other respiratory pathogens. The multiplex LAMP correctly detected MTB and distinguished betweenandsimultaneously with sensitivities of 500 fg and 1 pg DNA, respectively, within 60 min, and the results were visualized by dipstick chromatography within 10 min. The assay was specific in that no major respiratory pathogens tested, including NTM strains, were positive. The multiplex dipstick LAMP assay is therefore a useful and accurate low-cost method for the differential identification ofand, especially in endemic areas where bovine and human TB coexist. The distinction betweenandcan also aid in monitoring the spread ofto humans and allow for correct treatment, which will ultimately contribute to TB control in both humans and animals.

IMPORTANCE: Human tuberculosis caused byandshows similar clinical symptoms; however, the treatment differs becauseis inherently resistant to pyrazinamide, a key first-line drug effective against. Most available molecular tools cannot distinguish the two biovars. This study addresses this gap by introducing a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography that can simultaneously and differentially detectandwithin 60 min. The LAMP method does not require sophisticated high-cost equipment and can be easily implemented in resource-limited settings. Our LAMP facilitates rapid and accurate tuberculosis diagnosis, enabling appropriate therapeutic agents to be selected in areas where bovine and human tuberculosis coexist. It can also screen forinfection in humans and livestock, providing prevalence data in areas where such information is lacking.