Nanoparticle-based biosensor integrated with multiple cross-displacement amplification for visual and rapid identification of hepatitis B virus and hepatitis C virus

Infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is a major contributor to liver-related morbidity and mortality worldwide. An accurate and rapid point-of-care (POC) diagnostic approach is the gateway for effective treatment and control of these infections. Here, for the first time, we integrated isothermal multiple cross-displacement amplification (MCDA) with a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB) to successfully develop a novel HBV&HCV-MCDA-AuNPs-LFB assay for simultaneous accurate, sensitive, rapid, inexpensive, and visual identification of HBV and HCV agents. The two unique sets of MCDA degenerate primers were successfully designed targeting the <i>S</i> and 5' untranslated region (5'-UTR) genes from the major HBV genotypes (B, C, D, B/C recombinant, and C/D recombinant) and HCV subtypes in China (1b, 2a, 3a, 3b, and 6a), respectively. The optimal conditions for the MCDA reaction were confirmed to be 64°C for 35 min. The MCDA products were decoded visually using the AuNPs-LFB platform, which was devised for analyzing three targets, including HBV-MCDA, HCV-MCDA amplicons, and a chromatography control. The whole detection procedure, including rapid nucleic acid extraction (~10 min), MCDA reaction (35 min), and AuNPs-LFB interpretation (~2 min), can be completed within 50 min. The HBV&HCV-MCDA-AuNPs-LFB assay can detect the target genes (HBV-<i>S</i> and HCV-5'-UTR) with as low as 10 copies of gene-containing plasmid template per test and does not cross-react with other pathogens. Therefore, our preliminary results indicated that the HBV&HCV-MCDA-AuNPs-LFB assay developed in this study can potentially serve as a useful POC diagnostic tool for the identification of HBV and HCV infections.IMPORTANCEHepatitis B virus (HBV) and hepatitis C virus (HCV) infections have been regarded by the World Health Organization as major threats to human health, especially in low- and middle-income regions. Underdiagnosis of HBV/HCV is a particular challenge for achieving the World Health Organization's goal of eliminating HBV and HCV infections by 2030. Here, for the first time, we integrated isothermal multiple cross-displacement amplification (MCDA) with a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB) to successfully develop a novel HBV&HCV-MCDA-AuNPs-LFB assay for simultaneous accurate, sensitive, rapid, inexpensive, and visual identification and differentiation of HBV and HCV agents.