Related Experiment Video
Updated: Dec 26, 2025
System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
Published on: April 5, 2017
Simultaneous Detection of Drug-resistant Mutations in Mycobacterium tuberculosis and Determining their Role through
Somanna Ajjamada Nachappa1, Sumana M Neelambike2, Ahmad Sarikhani1
1Department of Studies in Genetics and Genomics, Genetics and Genomics Lab, University of Mysore, Mysuru, Karnataka, India.
Abstract:
A molecular method for diagnosis of drug-resistant Tuberculosis is Multiplex allele-specific PCR (MAS-PCR), which is more time-efficient, and its accuracy is studied using DNA sequencing. Also, understanding the role of mutations, when translated to protein, in causing resistance helps in better drug designing.
Aim:
To study MAS-PCR in the detection of drug resistance in comparison to DNA sequencing in Mycobacterium tuberculosis, and understand the mechanism of interaction of drugs with mutant proteins.
Methods:
MAS-PCR was used for the detection of drug-resistant mutations and validation was done through DNA sequencing. MAS-PCR targeted four genes, iniA for the drug Ethambutol, rpsL and rrs for Streptomycin, and gyrA for Fluoroquinolone resistance, respectively. Further, the sequence data was analysed and modeled for in silico docking to study the effect on the interaction of the anti-TB drug molecule with the target protein.
Results:
We identified drug-resistant mutations in four out of 95 isolates with one of them carrying a mutation at codon iniA501, two at gyrA94, and one for both iniA501 and gyrA94 using MASPCR. DNA sequencing confirmed drug-resistant mutations in only two isolates, whereas two others had mutation adjacent to the target allele. Drug-protein docking showed Estimated Free Energy of Binding to be higher for Fluoroquinolone binding with GyrA D94V mutant. Both wild and mutant IniA interact with EMB but had no significant effect on binding energy.
Conclusion:
DNA sequencing-based drug resistance detection of TB is more accurate than MASPCR. Understanding the role of mutations in influencing the drug-protein interaction will help in designing effective drug alternatives.
