Rapid, visual, label-based biosensor platform for identification of hepatitis C virus in clinical applications

Abstract

ObjectivesIn the current study, for the first time, we reported a novel HCV molecular diagnostic approach termed reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticles-based lateral flow biosensor (RT-LAMP-AuNPs-LFB), which we developed for rapid, sensitive, specific, simple, and visual identification of HCV.MethodsA set of LAMP primer was designed according to 5’untranslated region (5’UTR) gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a, which are prevalent in China. The HCV-RT-LAMP-AuNPs-LFB assay conditions, including HCV-RT-LAMP reaction temperature and time were optimized. The sensitivity, specificity, and selectivity of our assay were evaluated in the current study. The feasibility of HCV-RT-LAMP-AuNPs-LFB was confirmed through clinical serum samples from patients with suspected HCV infections.ResultsAn unique set of HCV-RT-LAMP primers were successfully designed targeting on the 5’UTR gene. The optimal detection process, including crude nucleic acid extraction (approximately 5 min), RT-LAMP reaction (67℃, 30 min), and visual interpretation of AuNPs-LFB results (~ 2 min), could be performed within 40 min without specific instruments. The limit of detection was determined to be 20 copies per test. The HCV-RT-LAMP-AuNPs-LFB assay exhibited high specificity and anti-interference.ConclusionsThese preliminary results confirmed that the HCV-RT-LAMP-AuNPs-LFB assay is a sensitive, specific, rapid, visual, and cost-saving assay for identification of HCV. This diagnostic approach has great potential value for point-of-care (POC) diagnostic of HCV, especially in resource-challenged regions.