Genotyping simplified: rationally designed antisense oligonucleotide-mediated PCR amplification-free colorimetric sensing of viral RNA in HCV genotypes 1 and 3.

Molecular diagnosis of viral genotyping devoid of polymerase chain reaction (PCR) amplification in clinical cohorts has hitherto been challenging. Here we present a simplified molecular diagnostic strategy for direct genotyping of hepatitis C virus (HCV) 1 and 3 (prevalent worldwide) using a combination of rationally designed genotype-specific antisense oligonucleotides (ASOs) and plasmonic gold nanoparticles. The ASOs specific to genotypes 1 and 3 have been designed from the nonstructural region 5A (NS5A) of the viral genome using the ClustalW multiple sequence alignment tool. A total of 79 clinical samples including 18 HCV genotype 1, 18 HCV genotype 3, one HIV positive, one HBV positive, and 41 healthy controls have been tested against both the designed ASOs. The study reveals 100% specificity and sensitivity with the employed samples and thereby opens up new avenues for PCR-free direct genotyping of other viruses as well, through the rational design of ASOs.

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

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.

Effects of free patchy ends in ssDNA and dsDNA on gold nanoparticles in a colorimetric gene sensor for Hepatitis C virus RNA.

The presence or absence and nature of the free patchy ends in DNA sequences has a decisive effect on the performance of colorimetric sensors based on the use of gold nanoparticles (Au NPs). The authors have designed two unmodified gene probes (probe 1: a 19-mer; probe 2: an 18-mer). They are complementary to either half of a 37-mer target derived from the conserved region of Hepatitis C Virus (HCV) RNA. Each probe has further been modified with 10-mer poly(A) and thiol-functionalized 10-mer poly(A) at the 5' positions. Nine combinations of probe and HCV RNA were then designed to investigate the effect of free patchy ends on the stability of citrate-modified Au NPs against salt-induced aggregation which lead to color change from red to blue. The aggregation of Au NPs can be monitored by ratiometric spectroscopy at wavelengths of 520 and 700 nm. The differentiation between HCV RNA and control has also been studied by varying the concentration of probe and analyte. The particle size and zeta potentials were determined before and after aggregation. It is demonstrated that the change in surface charge density of the Au NPs governs the critical coagulation concentration of NaCl. The method presented here can be used to quantify HCV RNA in the 370 nM to 3 µM concentration range, and the detection limit is 500 nM. The results obtained with Au NPs that are chemically non-conjugated with the oligonucleotides have been found to be valuable in rationally devising the design rules for rapid and efficient colorimetric sensing of oligonucleotides. Graphical abstract Schematic representation of the nine combinatorial pairs of oligonucleotides that vary in the length of patchy ends and their position to unearth their effect in rapid gold nanoparticle-based colorimetric gene sensing without time-consuming and expensive thiol-conjugation step.