Isothermal DNA amplification with functionalized graphene and nanoparticle assisted electroanalysis for rapid detection of Johne’s disease

Abstract

Johne’s disease (JD), which is caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a bacterial infection of the intestinal tract of ruminants. JD is a cause of significant economic and animal loss throughout the world. Sensitive, selective, and on-site detection of MAP in clinical samples has always been a problem. This study outlines a loop-mediated isothermal DNA amplification (LAMP) and electrochemical analysis-based point-of-care (POC) detection methodology for MAP. LAMP contributed to the selective amplification of MAP DNA, and electrochemical analysis assisted in the rapid and sensitive analysis of LAMP products. A graphene and tetracycline (TET)-functionalized screen printed carbon electrode was used for the selective detection of magnesium pyrophosphate (Mg2P2O7) produced during the LAMP. The Mg2P2O7 obtained from the LAMP was sandwiched on the electrode between TET and zirconium dioxide nanoparticles (ZrO2). The complexation of Mg2P2O7 triggered an electrochemical change that was monitored using electrochemical techniques. The complexation mechanism and functionalized electrodes were characterized using several microscopic, spectroscopic, and electrochemical techniques. The optimized MAP biosensor was employed to detect a range of MAP DNA concentrations. Using electrochemical impedance spectroscopy, a detection limit of 6.37 aM (20 fg/μL) with a detection range of 6.37 aM (20 fg/μL) to 6.37 pM (20 ng/μL) for MAP DNA was obtained. The application of the biosensor was also assessed by MAP detection in clinical fecal samples. The biosensor could easily detect the presence of MAP in bovine fecal samples and showed good co-relation with other conventional techniques. Therefore, the developed biosensor has the potential to be used for POC detection of JD in animals.