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Development of DNA Bio-chip for Detection of Mutations of rpoB, embB and inhA Genes in Drug-Resistant Mycobacterium Tuberculosis

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Abstract

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.

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Funding

This study was funded by BARC.

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Authors and Affiliations

  1. Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India

    Bharti Jain & Savita Kulkarni

  2. Homi Bhabha National Institute, Mumbai, India

    Bharti Jain & Savita Kulkarni

Authors
  1. Bharti Jain
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  2. Savita Kulkarni
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Contributions

Conceptualization: [SK and BJ]; Methodology: [BJ]; Formal analysis and investigation: [BJ]; Writing—original draft preparation: [BJ]; Writing—review and editing: [SK]; Supervision: [SK].

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Correspondence to Savita Kulkarni.

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Jain, B., Kulkarni, S. Development of DNA Bio-chip for Detection of Mutations of rpoB, embB and inhA Genes in Drug-Resistant Mycobacterium Tuberculosis. Ind J Clin Biochem 38, 242–250 (2023). https://doi.org/10.1007/s12291-022-01044-w

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  • DOI: https://doi.org/10.1007/s12291-022-01044-w

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