Naked-eye colorimetric detection of HCV RNA mediated by a 5′ UTR-targeted antisense oligonucleotide and plasmonic gold nanoparticles

Abstract

The increasing incidence of hepatitis C viral (HCV) infection worldwide is a major concern for causing liver cirrhosis and hepatocellular carcinoma, leading to increased morbidity and mortality. Currently, the prevalence of HCV infection is estimated to be in the range of ∼3%. According to the World Health Organization, antiviral drugs can cure more than 95% of the HCV infected cases, if timely diagnosis and treatment are provided. The gold standard RT-qPCR assay is expensive and requires a minimum turnaround time of 4 h. Hence, a rapid and cost-effective detection assay that can be used even in resource-limited settings would be highly beneficial for mass level screening. Herein, we present an Au NP based facile strategy for rapid, early-stage, and sensitive detection of HCV RNA in clinical samples which avoids thiol tagging to the antisense oligonucleotide and expensive infrastructure. This technique utilizes the hybridization of a short-chain antisense oligonucleotide from the 5′ untranslated region (UTR) of the viral genome with the isolated HCV RNA samples. Using a specific sequence universal to all HCV genotypes—obtained through the NCBI BLASTn tool—the HCV positive samples have stabilized the citrate capped Au NPs against salt-induced aggregation, retaining their red color. On the other hand, negative controls, including HBV and HIV positive samples, do not stabilize the Au NPs, which results in purple coloration. Besides, the assay is successfully tested with a RNase A enzyme-treated HCV positive sample, which does not stabilize the Au NPs, thus confirming the role of the viral HCV RNA in this strategy. This Au NP based assay takes about 30 min using the viral RNA isolate and has high specificity with a detection limit of 100 IU mL⁻¹, which is ∼10 fold lower than the state-of-the-art Au NP based strategy.