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

Chen, Shan1; Liu, Houming1; Li, Tianpin1; Lai, Wenjie1; Liu, Lei1; Xu, Youchun2; Qu, Jiuxin1

2023

10.2147/IDR.S410779

INFECTION AND DRUG RESISTANCE   影响因子和分区

1178-6973

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 & REG;" 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 1x101 copies/& mu;L. 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.

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

SCI

英语

第一 ; 通讯

State Key Laboratory of Infectious Disease Prevention and Control[2019SKLID302] ; Science and Technology Program of Shenzhen, China[JCYJ20170307095303424] ; Special Support Funds of Shenzhen for Introduced High -Level Medical Team, China[SZSM201412005] ; National Key Ramp;D Program of China[2017YFC0909900]

Infectious Diseases ; Pharmacology & Pharmacy

Infectious Diseases ; Pharmacology & Pharmacy

WOS:001022776200001

DOVE MEDICAL PRESS LTD

Web of Science

1.Southern Univ Sci & Technol, Shenzhen Peoples Hosp 3, Affiliated Hosp 2, Natl Clin Res Ctr Infect Dis,Dept Clin Lab, Shenzhen 518112, Guangdong, Peoples R China
2.Tsinghua Univ, Sch Med, Dept Biomed Engn, Beijing 100084, Peoples R China

Chen, Shan,Liu, Houming,Li, Tianpin,et al. Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis[J]. INFECTION AND DRUG RESISTANCE,2023,16.

Chen, Shan.,Liu, Houming.,Li, Tianpin.,Lai, Wenjie.,Liu, Lei.,...&Qu, Jiuxin.(2023).Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis.INFECTION AND DRUG RESISTANCE,16.

Chen, Shan,et al."Using Microfluidic Chip and Allele-Specific PCR to Rapidly Identify Drug Resistance-Associated Mutations of Mycobacterium tuberculosis".INFECTION AND DRUG RESISTANCE 16(2023).