Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography
ABSTRACT Although human tuberculosis (TB) caused by Mycobacterium bovis is clinically, pathologically, and radiologically indistinguishable from Mycobacterium tuberculosis-caused TB, M. bovis is innately resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. The rapid di...
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American Society for Microbiology
2025-06-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02421-24 |
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| author | Mwangala L. Akapelwa Thoko F. Kapalamula Lavel C. Moonga Precious Bwalya Eddie S. Solo Joseph Y. Chizimu Jeewan Thapa Kyoko Hayashida Bernard M. Hang'ombe Musso Munyeme Aki Tamaru Takayuki Wada Shiomi Yoshida Takuya Kodera Mistuo Kawase Stephen V. Gordon Keiko Yamada Chie Nakajima Yasuhiko Suzuki |
| author_facet | Mwangala L. Akapelwa Thoko F. Kapalamula Lavel C. Moonga Precious Bwalya Eddie S. Solo Joseph Y. Chizimu Jeewan Thapa Kyoko Hayashida Bernard M. Hang'ombe Musso Munyeme Aki Tamaru Takayuki Wada Shiomi Yoshida Takuya Kodera Mistuo Kawase Stephen V. Gordon Keiko Yamada Chie Nakajima Yasuhiko Suzuki |
| author_sort | Mwangala L. Akapelwa |
| collection | DOAJ |
| description | ABSTRACT Although human tuberculosis (TB) caused by Mycobacterium bovis is clinically, pathologically, and radiologically indistinguishable from Mycobacterium tuberculosis-caused TB, M. bovis is innately resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. 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 the M. tuberculosis species (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 of M. bovis and M. tuberculosis. The assay was optimized and validated using 143 isolates comprising six MTB reference strains, 50 M. bovis isolates, 58 M. tuberculosis isolates, 24 non-tuberculous mycobacterial (NTM) strains, and five other respiratory pathogens. The multiplex LAMP correctly detected MTB and distinguished between M. tuberculosis and M. bovis simultaneously 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 of M. bovis and M. tuberculosis, especially in endemic areas where bovine and human TB coexist. The distinction between M. bovis and M. tuberculosis can also aid in monitoring the spread of M. bovis to humans and allow for correct treatment, which will ultimately contribute to TB control in both humans and animals.IMPORTANCEHuman tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. 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 detect M. bovis and M. tuberculosis within 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 for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking. |
| format | Article |
| id | doaj-art-6a52dc3acb684a129a59c98fd0f2b11c |
| institution | OA Journals |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | American Society for Microbiology |
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| series | Microbiology Spectrum |
| spelling | doaj-art-6a52dc3acb684a129a59c98fd0f2b11c2025-08-20T02:05:24ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-06-0113610.1128/spectrum.02421-24Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatographyMwangala L. Akapelwa0Thoko F. Kapalamula1Lavel C. Moonga2Precious Bwalya3Eddie S. Solo4Joseph Y. Chizimu5Jeewan Thapa6Kyoko Hayashida7Bernard M. Hang'ombe8Musso Munyeme9Aki Tamaru10Takayuki Wada11Shiomi Yoshida12Takuya Kodera13Mistuo Kawase14Stephen V. Gordon15Keiko Yamada16Chie Nakajima17Yasuhiko Suzuki18Division of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanDivision of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanSchool of Veterinary Medicine, University of Zambia, Lusaka, Lusaka Province, ZambiaDepartment of Pathology and Microbiology, University Teaching Hospital, Lusaka, ZambiaDepartment of Pathology and Microbiology, University Teaching Hospital, Lusaka, ZambiaZambia National Public Health Institute, Ministry of Health, Lusaka, Lusaka Province, ZambiaDivision of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanDivision of collaboration and education, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanSchool of Veterinary Medicine, University of Zambia, Lusaka, Lusaka Province, ZambiaSchool of Veterinary Medicine, University of Zambia, Lusaka, Lusaka Province, ZambiaDepartment of Microbiology, Osaka Institute of Public Health, Osaka, Osaka Prefecture, JapanGraduate School of Human Life and Ecology, Osaka Metropolitan University, Osaka, Osaka Prefecture, JapanClinical Research Center, National Hospital Organization Kinki-Chuo Chest Medical Center, Sakai, Osaka Prefecture, JapanTohoku Bio-Array Co., LTD, Sendai, JapanTohoku Bio-Array Co., LTD, Sendai, JapanHokkaido University, Institute for Vaccine Research and Development, Sapporo, Hokkaido Prefecture, JapanDivision of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanDivision of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanDivision of Bioresources, Hokkaido University International Institute for Zoonosis Control, Sapporo, JapanABSTRACT Although human tuberculosis (TB) caused by Mycobacterium bovis is clinically, pathologically, and radiologically indistinguishable from Mycobacterium tuberculosis-caused TB, M. bovis is innately resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. 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 the M. tuberculosis species (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 of M. bovis and M. tuberculosis. The assay was optimized and validated using 143 isolates comprising six MTB reference strains, 50 M. bovis isolates, 58 M. tuberculosis isolates, 24 non-tuberculous mycobacterial (NTM) strains, and five other respiratory pathogens. The multiplex LAMP correctly detected MTB and distinguished between M. tuberculosis and M. bovis simultaneously 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 of M. bovis and M. tuberculosis, especially in endemic areas where bovine and human TB coexist. The distinction between M. bovis and M. tuberculosis can also aid in monitoring the spread of M. bovis to humans and allow for correct treatment, which will ultimately contribute to TB control in both humans and animals.IMPORTANCEHuman tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis. 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 detect M. bovis and M. tuberculosis within 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 for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking.https://journals.asm.org/doi/10.1128/spectrum.02421-24M. bovisM. tuberculosissimultaneous detectionmultiplex LAMPdipstick chromatographybiovar differentiation |
| spellingShingle | Mwangala L. Akapelwa Thoko F. Kapalamula Lavel C. Moonga Precious Bwalya Eddie S. Solo Joseph Y. Chizimu Jeewan Thapa Kyoko Hayashida Bernard M. Hang'ombe Musso Munyeme Aki Tamaru Takayuki Wada Shiomi Yoshida Takuya Kodera Mistuo Kawase Stephen V. Gordon Keiko Yamada Chie Nakajima Yasuhiko Suzuki Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography Microbiology Spectrum M. bovis M. tuberculosis simultaneous detection multiplex LAMP dipstick chromatography biovar differentiation |
| title | Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography |
| title_full | Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography |
| title_fullStr | Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography |
| title_full_unstemmed | Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography |
| title_short | Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography |
| title_sort | development of a multiplex loop mediated isothermal amplification lamp method for differential detection of mycobacterium bovis and mycobacterium tuberculosis by dipstick dna chromatography |
| topic | M. bovis M. tuberculosis simultaneous detection multiplex LAMP dipstick chromatography biovar differentiation |
| url | https://journals.asm.org/doi/10.1128/spectrum.02421-24 |
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