Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis

Shan Chen; Houming Liu; Tianpin Li; Wenjie Lai; Lei Liu; Youchun Xu; Jiuxin Qu

doi:10.2147/IDR.S410779

Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis

Infect Drug Resist. 2023 Jul 3:16:4311-4323. doi: 10.2147/IDR.S410779. eCollection 2023.

Authors

Shan Chen¹, Houming Liu¹, Tianpin Li¹, Wenjie Lai¹, Lei Liu¹, Youchun Xu², Jiuxin Qu¹

Affiliations

¹ 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, Guangdong Province, 518112, People's Republic of China.
² Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing, 100084, People's Republic of China.

Abstract

Background: 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.

Keywords: Mycobacterium tuberculosis; Sanger sequencing; drug resistance; microfluidic analytical techniques; multidrug-resistance; polymerase chain reaction; resistance-conferring gene mutations.