A sequence-specific, nanoparticle-based biosensor platform for rapid and visual identification of serum hepatitis B...

Chronic hepatitis B virus (HBV) infection is a major cause of liver-related morbidity and mortality worldwide. Serum HBV pregenomic RNA (pgRNA) is a surrogate marker for the transcriptional activity of covalently closed circular DNA. Here, we successfully designed a novel point-of-care (POC) diagnostic platform that allows specific, sensitive, rapid, and visual identification of HBV pgRNA. The platform integrates probe-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) with either restriction endonuclease-mediated real-time fluorescence (REF) or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB), termed HBV-RT-LAMP. A unique set of probe-based LAMP primers targeting HBV-pgRNA was successfully designed. The optimal conditions for HBV-RT-LAMP were determined to be 64°C and 30 min. AuNPs-LFB and a pocket fluorescence detector (REF assay) were used for readout of the products. Our assay detected the target gene at concentrations as low as 50 copies/mL of HBV RNA standard and did not produce cross-reactions with HBV DNA (treated with DNase I) or other pathogens. The entire detection process, including HBV RNA extraction (45 min), LAMP (30 min), and the interpretation of results (AuNPs-LFB, less than 2 min), could be performed within 80 min, with no need for expensive devices. Therefore, the HBV-RT-LAMP diagnostic system developed in this study can potentially serve as a useful POC diagnostic tool for the evaluation of chronic HBV infection status and antiviral drug efficacy.IMPORTANCEChronic hepatitis B (CHB) is still a serious global concern that can result in severe liver-related diseases, including liver cirrhosis and hepatocellular carcinoma. Serum hepatitis B virus (HBV)-pregenomic RNA (pgRNA) has been proposed as a surrogate intrahepatic covalently closed circular DNA marker in CHB patients. Here, for the first time, a novel point-of-care diagnostic platform, termed HBV-reverse transcription loop-mediated isothermal amplification (RT-LAMP), which integrates probe-based RT-LAMP with either restriction endonuclease-mediated real-time fluorescence (REF) detection or a gold nanoparticle-based lateral flow biosensor (AuNPs-LFB), was developed and applied for accurate, sensitive, specific, rapid, and visual identification of HBV-pgRNA.