Rapid simultaneous detection of katG S315T and rpoB S531L mutations for molecular screening of multidrug-resistant tuberculosis using a dual LAMP-lateral flow assay
5 affiliations
- 1Department of Clinical Pathology, Faculty of Medicine, Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand
- 2Department of Pathology, Faculty of Medicine, Srinakharinwirot University, Nakhonnayok, Thailand
- 3Genetic Engineering and Bioinformatics Interdisciplinary Program, Graduate School, Kasetsart University, Bangkok, Thailand
- 4Center of Excellence in Medical and Environmental Innovation Research (CEMEIR), Srinakharinwirot University, Bangkok, Thailand
- 5Innovative Learning Center, Srinakharinwirot University, Bangkok, Thailand
Abstract
The global spread of multidrug-resistant tuberculosis (MDR-TB) remains a major challenge to tuberculosis control programs, particularly in resource-limited settings where access to rapid molecular drug-susceptibility testing is limited. Resistance to isoniazid and rifampicin is most commonly associated with mutations in the katG and rpoB genes, respectively, making these loci valuable molecular markers for early MDR-TB screening. In this study, we developed a dual loop-mediated isothermal amplification assay coupled with lateral flow detection for the simultaneous identification of the katG S315T and rpoB S531L mutations in Mycobacterium tuberculosis. Mutation-specific primer sets were designed using mismatch amplification principles to achieve single-nucleotide specificity. Amplification was performed at 61°C for 60 min, followed by visual detection on a three-line lateral flow strip within 5 min. The assay demonstrated a preliminary detection threshold of approximately 10 CFU/mL equivalents and exhibited no cross-reactivity with wild-type M. tuberculosis H37Rv, non-tuberculous mycobacteria, or non-target resistance-associated mutations, including the inhA −15 promoter mutation and rpoB codon 526 variants. Preliminary clinical feasibility was evaluated using genetically characterized clinical sputum specimens and showed complete concordance with GenoType MTBDRplus and Sanger DNA sequencing results. Because the assay targets only two resistance-associated mutations, a negative result does not exclude isoniazid or rifampicin resistance mediated by alternative mutations.
IMPORTANCE: These proof-of-concept findings demonstrate the initial feasibility of a rapid, low-complexity molecular platform for the simultaneous detection of clinically important resistance-associated mutations in Mycobacterium tuberculosis. The assay represents a promising step toward low-complexity molecular screening for selected multidrug-resistant tuberculosis-associated mutations, bringing us closer to simplified testing in settings where access to conventional molecular drug-resistance testing remains limited.
Related
- Real-world evaluation of the FluoroType MTBDR 2.0 assay for detection of rifampicin and isoniazid resistance-conferring mutations in Mycobacterium tuberculosis complex clinical isolates
- Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
- Development and Validation of a Point-of-Care Triage Scorecard to Enhance Tuberculosis Case Detection During Active Community Screening in Yogyakarta, Indonesia
- Comparative cost analysis of community-based tuberculosis screening: Computer-Aided Detection (CAD) alone versus CAD with point-of-care C-reactive protein testing in South Africa and Lesotho
- Rifabutin Cross-resistance and Associated rpoB Mutations in Rifampicin-resistant Mycobacterium tuberculosis Complex Isolates
BibTeX
@article{ckumdee2026rapid,
title = {{Rapid simultaneous detection of katG S315T and rpoB S531L mutations for molecular screening of multidrug-resistant tuberculosis using a dual LAMP-lateral flow assay}},
author = {Jutturong Ckumdee and Somchai Santiwatanakul and Nakarin Vutipow and Thongchai Kaewphinit},
journal = {Microbiology Spectrum},
year = {2026},
month = oct,
doi = {10.1128/spectrum.02195-26},
url = {https://doi.org/10.1128/spectrum.02195-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Rapid simultaneous detection of katG S315T and rpoB S531L mutations for molecular screening of multidrug-resistant tuberculosis using a dual LAMP-lateral flow assay
AU - Jutturong Ckumdee
AU - Somchai Santiwatanakul
AU - Nakarin Vutipow
AU - Thongchai Kaewphinit
PY - 2026
DA - 2026/10/08
JO - Microbiology Spectrum
DO - 10.1128/spectrum.02195-26
UR - https://doi.org/10.1128/spectrum.02195-26
AB - The global spread of multidrug-resistant tuberculosis (MDR-TB) remains a major challenge to tuberculosis control programs, particularly in resource-limited settings where access to rapid molecular drug-susceptibility testing is limited. Resistance to isoniazid and rifampicin is most commonly associated with mutations in the katG and rpoB genes, respectively, making these loci valuable molecular markers for early MDR-TB screening. In this study, we developed a dual loop-mediated isothermal amplification assay coupled with lateral flow detection for the simultaneous identification of the katG S315T and rpoB S531L mutations in Mycobacterium tuberculosis. Mutation-specific primer sets were designed using mismatch amplification principles to achieve single-nucleotide specificity. Amplification was performed at 61°C for 60 min, followed by visual detection on a three-line lateral flow strip within 5 min. The assay demonstrated a preliminary detection threshold of approximately 10 CFU/mL equivalents and exhibited no cross-reactivity with wild-type M. tuberculosis H37Rv, non-tuberculous mycobacteria, or non-target resistance-associated mutations, including the inhA −15 promoter mutation and rpoB codon 526 variants. Preliminary clinical feasibility was evaluated using genetically characterized clinical sputum specimens and showed complete concordance with GenoType MTBDRplus and Sanger DNA sequencing results. Because the assay targets only two resistance-associated mutations, a negative result does not exclude isoniazid or rifampicin resistance mediated by alternative mutations. IMPORTANCE: These proof-of-concept findings demonstrate the initial feasibility of a rapid, low-complexity molecular platform for the simultaneous detection of clinically important resistance-associated mutations in Mycobacterium tuberculosis. The assay represents a promising step toward low-complexity molecular screening for selected multidrug-resistant tuberculosis-associated mutations, bringing us closer to simplified testing in settings where access to conventional molecular drug-resistance testing remains limited.
SN - 2165-0497
ER -