title: Population genotyping of hepatitis C virus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of short DNA fragments.
abstract: BACKGROUND
  Identifying hepatitis C virus (HCV) genotypes has become increasingly important for determining clinical course and the outcome of antiviral therapy. Here we describe the development of restriction fragment mass polymorphism (RFMP) analysis, a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay suitable for high-throughput, sensitive, specific genotyping of multiple HCV species.
  
  
  METHODS
  The assay is based on PCR amplification and mass measurement of oligonucleotides containing genotype-specific motifs in the 5' untranslated region, into which a type IIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products led to excision of multiple oligonucleotide fragments representing variable regions whose masses were determined by MALDI-TOF MS.
  
  
  RESULTS
  The RFMP assay identified viral genotypes present at concentrations as low as 0.5% and reliably determined their relative abundance. When sera from 318 patients were analyzed, the RFMP assay exhibited 100% concordance with results obtained by clonal sequencing and identified mixed-genotype infections in 22% of the samples, in addition to several subtype variants.
  
  
  CONCLUSIONS
  The RFMP assay has practical advantages over existing methods, including better quantitative detection of mixed populations and detection of genotype variants without need for population-based cloning, enabling reliable viral genotyping in laboratories and efficient study of the relationship between viral genotypes and clinical outcome.
authors.affiliation: Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Yoon Jun
authors.initials: YJ
authors.lastname: Kim
authors.forename: Soo-Ok
authors.initials: SO
authors.lastname: Kim
authors.forename: Hyun Jae
authors.initials: HJ
authors.lastname: Chung
authors.forename: Mi Sun
authors.initials: MS
authors.lastname: Jee
authors.forename: Byeong Gwan
authors.initials: BG
authors.lastname: Kim
authors.forename: Kang Mo
authors.initials: KM
authors.lastname: Kim
authors.forename: Jung-Hwan
authors.initials: JH
authors.lastname: Yoon
authors.forename: Hyo-Suk
authors.initials: HS
authors.lastname: Lee
authors.forename: Chung Yong
authors.initials: CY
authors.lastname: Kim
authors.forename: Sukjoon
authors.initials: S
authors.lastname: Kim
authors.forename: Wangdon
authors.initials: W
authors.lastname: Yoo
authors.forename: Sun Pyo
authors.initials: SP
authors.lastname: Hong
mesh_terms: D020121:5' Untranslated Regions; D004279:DNA, Viral / Q000097:blood / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D020121:5' Untranslated Regions; D004279:DNA, Viral