A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens

Abstract

The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.

Molecular Targets Related Drug Resistance Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains

“…New approaches have also been reported, like FASTPlaque-response bacteriophage assay (Minion and Pai, 2010 ), microscopic observation drug susceptibility assay (MODS) (Peter et al, 2016 ), use of dyes to monitor bacterial growth (Coban et al, 2016 ), sequencing and hybridization, reverse hybridization, direct sequencing (Deggim-Messmer et al, 2016 ), TB Biochip platform (Xue et al, 2016 ) and other molecular approaches. Each method was proposed to provide faster as well as more reliable results for early detection of drug-resistance.…”

Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

“…The model was used to predict hybridization kinetics of eighteen 20-mer probes targeting vancomycin resistant genes. This gene was chosen as a model because emergence of antibiotic resistance associated with pathogenic bacteria is an alarming global issue [14,15] and numerous hybridization-based biochip platforms are being developed to diagnose multi-drug resistant bacteria [16,17,18,19,20]. Effect of target concentration, temperature, recirculation flow rate and rate constants on the hybridization kinetics, with target concentration, rate constants, and temperature being the most important parameters affecting the rate of hybridization were investigated.…”