Development of amperometric biosensor based on cloned hemagglutinin gene of H1N1 (swine flu) virus.

The recent emergence of respiratory viruses especially COVID-19 and swine flu has underscored the need for robust and bedside detection methods. Swine flu virus is a very infectious virus of the respiratory system. Timely detection of this virus with high specificity and sensitivity is crucial for reducing morbidity as well as mortality. Cloning of gene segments into a non-infectious agent helps in the development of detection methods, vaccine development, and other studies. In this study, cloning was used to develop a biosensor for H1N1 pdm09 detection. A segment of the hemaglutinin gene was cloned in a vector and characterized with the help of colony touch PCR and blue-white screening. The recombinant plasmid was extracted, and the gene segment was confirmed with the help of HA-specific primers. A 5' amine group-attached hemagglutinin (HA) gene-specific DNA probe was immobilized on the working gold electrode surface to make a quick, specific, reliable, and sensitive detection method for H1N1pdm09 virus in human nasal swab samples. The HA probe was immobilized on the cysteine applied gold electrode of the screen-printed electrode through 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and -hydroxysuccinimide (NHS). Differential pulse voltammetry was performed with the help of methylene blue, which is a redox indicator for the detection of single-stranded cloned HA gene segment. The developed sensor depicted high sensitivity for the H1N1 influenza virus with a detection limit of 0.6 ng ssDNA/6 µl of the cloned HA sample. Specificity was also checked using H3N2 virus, , influenza A and positive H1N1pdm09 samples.