pHEAA/pMA-DNA Hydrogel Sensing Platform for Detecting Drug-Resistant Gene Variants in <i>Mycobacterium tuberculosis</i>

Mutations in the drug-resistant gene of<i>Mycobacterium tuberculosis</i>can make it challenging to use drugs in clinical practice. Traditional genetic testing for resistance requires cell culture and susceptibility testing, which take 1-2 weeks. In this study, a DNA-sensitive hydrogel (pHEAA/pMA-DNA) has been developed with nucleic acid binding ability, water retention capacity, and high-temperature resistance, allowing it to work normally at 105 °C. Molecular dynamics simulations have been used to obtain the necessary physicochemical parameters. The DNA-sensitive hydrogel acts as a novel biosensor for detecting rifampicin- and isoniazid-resistant gene mutations in <i>Mycobacterium tuberculosis</i>. The microarray sensor's detection range is between 10⁹ copies/ml and 10¹ copies/mL, and its stability coefficient of variation (CV) is 2.424%. The study demonstrates that there is no mutual interference in the gel lattice. In addition, experiments on actual nucleic acid samples reveal accurate detection of bacterial strains and drug-resistant gene mutations. The regression curve conforms to the kinetic characteristics of nucleic acid amplification, exhibiting a sigmoidal shape. The Four-Parameter Logistic Regression (4PL) equation was employed for fitting, achieving an excellent coefficient of determination (R² = 0.99791 > 0.99). The method enables parallel detection of microarray biosensors in multidrug-resistant <i>Mycobacterium tuberculosis</i>. The sensors show high efficiency in detecting resistance mutation sites of <i>Mycobacterium tuberculosis</i> to rifampicin and isoniazid, paving the way for researchers to design different probes in in vitro detection fields.