title: 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.
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan; Research and development department, SRL, Inc., 5-6-50 Shin, Hino, Tokyo 191-0002, Japan.
authors.forename: Motokazu
authors.initials: M
authors.lastname: Mukaide
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Sugiyama
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan. Electronic address: dmkorenaga@hospk.ncgm.go.jp.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Korenaga
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan.
authors.forename: Kazumoto
authors.initials: K
authors.lastname: Murata
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kanto
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan.
authors.forename: Naohiko
authors.initials: N
authors.lastname: Masaki
authors.affiliation: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohonodi, Ichikawa, Chiba 272-8516 Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
mesh_terms: D000328:Adult; D000368:Aged; D000596:Amino Acids / Q000235:genetics*; D003062:Codon; D005260:Female; D005787:Gene Frequency; D016174:Hepacivirus / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods*
chemical_list: D000596:Amino Acids; D003062:Codon; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus
keywords: Core a.a.70; HCV; Ultra-deep pyrosequencing; ddPCR