Performance of a molecular beacon-based qPCR method for detection of fluoroquinolone resistance in Mycobacterium tuberculosis: A study confirmed by sequencing

Abstract

Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to the control and treatment of tuberculosis (TB). Rapid diagnosis of resistant strains via utilization of molecular techniques is of critical importance on the proper management of the patients. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR) method for the detection of mutation-based fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis isolates. Primers and a molecular-beacon probe were designed to target quinolone-resistance-determining-region (QRDR) of the gyrA gene of M. tuberculosis. Amplification conditions and concentrations of primers/probe were optimized, and the effectiveness of the optimized qPCR method was tested on 50 MDR M. tuberculosis strains. To confirm the qPCR results, all strains were also screened for mutations in the gyrA gene using Sanger sequencing. The optimized qPCR method had analytical sensitivity of 96.5 cfu/ml. The method detected FQ resistance in 7 (14 %) of the 50 MDR-strains via either no or significantly decreased fluorescence signal due to mutations associated with FQ resistance. The sequencing of these seven strains detected three resistance-associated mutations (A90V, D94A and D94G). Of the 43 FQ-susceptible strains, 10 strains with wild-type gene sequences yielded strong fluorescent signals above 450 RFU, while the remaining 33 strains harboring a non-resistance-associated mutation (S95T) showed decreased fluorescence signals <350 RFU. The sensitivity and specificity of the qPCR method for detection of the mutations related to resistance was ≥99.9 % and 98 %, respectively. Consequently, the utility of the optimized qPCR method for the identification of mutation-based FQ resistance is promising.