Electrochemical detection of single nucleotide polymorphisms (SNPs) in katG and inhA genes associated with isoniazid resistance in Mycobacterium tuberculosis

Abstract

Tuberculosis is amongst the top ten leading causes of death worldwide and despite medical advancements, is still a global concern. One of the main hurdles associated with the eradication of TB is the rise of new drug-resistant strains of Mycobacterium tuberculosis (MTB), making the treatment less or completely ineffective. The cases of multidrugresistant TB (MDR-TB), that is resistant towards both of the first-line anti-TB drugs, rifampicin, and isoniazid, have increased to an alarming value over the years, resulting in a higher mortality rate. That is why, better point-of-care diagnostics are needed that can detect the bacteria as early as possible for the treatment to be effective. The staining and microscopic methods to detect MTB are very time-consuming, laborious, require a BSL facility, and have low sensitivity. PCR-based detection methods also require multiple steps to give results and are complex and expensive. For this purpose, biosensors, for instance, electrochemical biosensors, have become very popular because they offer inexpensive, rapid, real-time, and sensitive detection of the pathogen with minimal sample preparation. In this work, the electrochemical biosensor was fabricated by first modifying the surface of the glassy carbon electrode (GCE) with polypyrrole (PPy) and gold nanoparticles (AuNPs) and then immobilizing the thiolated ssDNA probes to the gold nanoparticles through chemisorption. The surface of ssDNA probe modified GCE was blocked using MCH and synthetic DNA oligonucleotides designed for katG and inhA genes to carry the specific SNP mutations Ser315Thr and c-15t respectfully, were given as targets. The change in current response was analyzed by differential pulse voltammetry at each DNA concentration. The developed biosensor was able to detect the SNPs at an even picomolar (pM) level of target DNA concentration and by plotting the relative change in current values against the concentration, the LOD of the biosensor for the detection of katG and inhA was calculated as 0.86 pM and 0.61pM respectfully. The performance of biosensor was also evaluated on MDR-TB raw sputum samples on which the biosensor was successful in the detection of the respective SNPs in both katG and inhA genes. The biosensor was also found to be highly specific towards the target depending upon the ssDNA immobilized probe. In the case of mutated (carrying the SNP) or noncomplementary target DNA, the hybridization did not occur, as confirmed by the DPV response. This work highlighted an ultrasensitive biosensor that is able to detect SNPs associated with isoniazid resistance and has the potential to be shifted on to a portable chipbased biosensing system.