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Population genotyping of hepatitis C virus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of short DNA fragments

Cited 40 time in Web of Science Cited 39 time in Scopus
Authors
Kim, Yoon Jun; Kim, Soo-Ok; Chung, Hyun Jae; Jee, Mi Sun; Kim, Byeong Gwan; Kim, Kang Mo; Yoon, Jung-Hwan; Lee, Hyo-Suk; Kim, Chung Yong; Kim, Sukjoon; Yoo, Wangdon; Hong, Sun Pyo
Issue Date
2005-05-14
Publisher
American Association for Clinical Chemistry
Citation
Clin Chem 2005;51:1123-31
Keywords
5' Untranslated RegionsDNA, Viral/blood/*geneticsHepacivirus/*geneticsHepatitis C, Chronic/geneticsHumansPolymerase Chain ReactionReproducibility of ResultsSensitivity and SpecificitySequence Analysis, DNASpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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.
ISSN
0009-9147 (Print)
Language
English
URI
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15890891

http://hdl.handle.net/10371/23301
DOI
https://doi.org/10.1373/clinchem.2004.047506
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College of Medicine/School of Medicine (의과대학/대학원)Internal Medicine (내과학전공)Journal Papers (저널논문_내과학전공)
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