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

Background: The currently used conventional susceptibility testing for drug-resistant 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 1×10 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had 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 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 . It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.