Population genotyping of hepatitis C virus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of short DNA fragments.
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.
Kim
Yoon Jun
YJ
Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Kim
Soo-Ok
SO
Chung
Hyun Jae
HJ
Jee
Mi Sun
MS
Kim
Byeong Gwan
BG
Kim
Kang Mo
KM
Yoon
Jung-Hwan
JH
Lee
Hyo-Suk
HS
Kim
Chung Yong
CY
Kim
Sukjoon
S
Yoo
Wangdon
W
Hong
Sun Pyo
SP
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
D020121:5' Untranslated Regions; D004279:DNA, Viral