High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70

Abstract

The next-generation droplet digital polymerase chain reaction (ddPCR) assay employs an emulsion-based endpoint to quantitate the amount of target DNA and is more robust than real-time PCR when analyzing sequence variations. However, no studies have applied this technique to quantitate mutations in polymorphic viral genomes. To develop this approach, a ddPCR-based assay was designed to quantitate with high-throughput and sensitivity mutations and their frequencies in codon 70 of the hepatitis C virus (HCV) gene that encodes the Core protein. The assay was linear from 2.5 to 10(5) copies per assay, and the limit of detection of mutants in the presence of a 20,000-fold excess of wild type was 0.005%. The results correlated well with those obtained using the COBAS(®) TaqMan(®) HCV Test, which is a real-time PCR assay for the quantitative detection of HCV RNA in human serum (n=87; range, 2.3-7.7log10IU/mL; Pearson's R(2)=0.9120; p<0.0001). The median frequencies of mutations by ddPCR were 0.262% (n=55; range, 0-37.951%) and 99.687% (n=32; range, 52.191-100%) for the wild-type and mutant sequences, respectively, by direct sequencing. The ddPCR assay should be useful for quantitating mutations in other polymorphic viral genomes.

Keywords: Core a.a.70; HCV; Ultra-deep pyrosequencing; ddPCR.