Drug-resistant (DR) tuberculosis (TB) is a global threat to health security and TB control programs. Since conventional drug susceptibility testing (DST) takes several weeks, we have developed a molecular method for the rapid identification of DR strains of Mycobacterium Tuberculosis (M.tb) utilizing DNA bio-chips. DNA bio-chips were prepared by immobilizing oligonucleotides (probes) on highly microporous polycarbonate track-etched membranes (PC-TEM) as novel support. Bio-chip was designed to contain 15 specific probes to detect mutations in three genes ( rpoB, embB, and inhA ). A sensitive and specific chemiluminescence based bio-chip assay was developed based on multiplex PCR followed by hybridization on bio-chip. Fifty culture isolates were used to evaluate the ability of in-house developed bio-chip to detect the mutations. Bio-chip analysis shows that 37.7% of samples show wild type sequences, 53.3% of samples were monoresistance showing resistance to either rifampicin (RMP), isoniazid (INH), or ethambutol (EMB). 4.4% of samples were polydrug resistant showing mutations in both the rpoB gene and embB gene while 4.4% of samples were multidrug-resistant (MDR), harboring mutations in the rpoB and inhA genes. The results were compared with DST and sequencing. Compared to sequencing, bio-chip assay shows a sensitivity of 96.5% and specificity of 100% for RMP resistance. For EMB and INH, the results were in complete agreement with sequencing. This study demonstrates the first-time use of PC-TEMs for developing DNA bio-chip for the detection of mutations associated with drug resistance in M.tb . Developed DNA bio-chip accurately detected different mutations present in culture isolates and thus provides detailed and reliable data for clinical diagnosis.
Keyphrases
- mycobacterium tuberculosis
- high throughput
- drug resistant
- multidrug resistant
- circulating tumor cells
- single molecule
- circulating tumor
- pulmonary tuberculosis
- genome wide
- single cell
- public health
- nucleic acid
- cell free
- wild type
- small molecule
- bioinformatics analysis
- human immunodeficiency virus
- mental health
- genome wide identification
- escherichia coli
- electronic health record
- klebsiella pneumoniae
- high resolution
- emergency department
- living cells
- antiretroviral therapy
- real time pcr
- photodynamic therapy
- climate change
- pseudomonas aeruginosa
- transcription factor
- loop mediated isothermal amplification