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Shan Chen,1 Houming Liu,1 Tianpin Li,1 Wenjie Lai,1 Lei Liu,1 Youchun Xu,2 Jiuxin Qu1 1Department of Clinical Laboratory, Shenzhen Third People’s Hospital, National Clinical Research Center for Infectious Diseases, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, Guangdong Province, 518112, People’s Republic of China; 2Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084, People’s Republic of ChinaCorrespondence: Jiuxin Qu, Department of Clinical Laboratory, Shenzhen Third People’s Hospital, National Clinical Research Center for Infectious Diseases, The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen, People’s Republic of China, Email Qujiuxin@163.com Youchun Xu, Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, People’s Republic of China, Email CYX040198@163.comBackground: The currently used conventional susceptibility testing for drug-resistant Mycobacterium tuberculosis (M.TB) is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP).Methods: A total of 300 clinical samples were collected, and DNA extraction was performed using the “isoChip®” Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples.Results: Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1× 101 copies/μL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored rpoB gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had katG gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had embB gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method.Conclusion: The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in M. tuberculosis. It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.Graphical Abstract: Keywords: Mycobacterium tuberculosis, multidrug-resistance, drug resistance, resistance-conferring gene mutations, polymerase chain reaction, microfluidic analytical techniques, Sanger sequencing |
