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.