Molecular Detection of Multidrug Resistance and Characterizations of Mutations in Mycobacterium Tuberculosis Using Polycarbonate Track-Etched Membrane Based DNA Bio-Chip.

Abstract

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes(PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR inM.tbculture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80bprifampicin resistance determining region of rpoB gene, katGgene andmabA-inhAregulatory region. RMP-resistance-associated gene mutation pointsrpoB 516, 526, 531and533, and the INH-resistance-associated gene mutation pointskatG315andinhA-15were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST)were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono-resistant, 6 INH mono-resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection ofrpoB, katGand inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics.