Establishment and application of nanoparticle-based biosensor combined with loop-mediated isothermal amplification for sensitive and visual detection of hepatitis B virus

Objective To establish a novel molecular diagnostic assay for the rapid and visual detection of hepatitis B virus (HBV) based on loop-mediated isothermal amplification (LAMP) combined with gold nanoparticle-based lateral flow biosensor (AuNPs-LFB) and evaluate its clinical application.

Methods The HBV-LAMP degenerate primers against the S gene from the major HBV genotypes (B, C, D, B/C recombinant, and C/D recombinant) in China were designed to establish a HBV-LAMP-AuNPs-LFB assay. The assay conditions, including both HBV-LAMP reaction temperature and time, were optimized. The sensitivity and specificity of the HBV-LAMP-AuNPs-LFB assay were evaluated, and its feasibility in clinical application was compared with fluorescent quantitative PCR.

Results A set of HBV-LAMP degenerate primers was successfully designed and used for the establishment of HBV-LAMP-AuNPs-LFB assay. The assay conditions were optimized at 68 °C for 30 minutes. The specificity of HBV-LAMP-AuNPs-LFB assay was 100.00%, and no cross reactions with other pathogens were observed. The HBV-LAMP-AuNPs-LFB assay can detect the target genes (HBV-S) even the gene-containing plasmid template per test was as low as 10 copies. The results of the novel HBV-LAMP-AuNPs-LFB assay were consistent with those of fluorescent quantitative PCR in clinical application.

Conclusion The HBV-LAMP-AuNPs-LFB assay established in the study is easy to use with high specificity and sensitivity, and without relying on expensive instrument, and can be used as a new laboratory diagnostic method for the treatment, prevention and control of HBV infection.