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 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- 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.