Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

被引:15
作者
Margraf, RL
Erali, M
Liew, M
Wittwer, CT
机构
[1] Univ Utah, Sch Med, Adv Technol Grp, ARUP Inst Clin & Expt Pathol, Salt Lake City, UT 84108 USA
[2] Univ Utah, Sch Med, Dept Pathol, Salt Lake City, UT 84108 USA
关键词
D O I
10.1128/JCM.42.10.4545-4551.2004
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.
引用
收藏
页码:4545 / 4551
页数:7
相关论文
共 22 条
[1]  
Bullock GC, 2002, CLIN CHEM, V48, P2147
[2]  
*CDCP, 2003, HEPATITIS C
[3]   Hepatitis C virus genotyping:: Interrogation of the 5′ untranslated region cannot accurately distinguish genotypes 1a and 1b [J].
Chen, ZY ;
Weck, KE .
JOURNAL OF CLINICAL MICROBIOLOGY, 2002, 40 (09) :3127-3134
[4]   GENETIC-RELATIONSHIPS DETERMINED BY A DNA HETERODUPLEX MOBILITY ASSAY - ANALYSIS OF HIV-1 ENV GENES [J].
DELWART, EL ;
SHPAER, EG ;
LOUWAGIE, J ;
MCCUTCHAN, FE ;
GREZ, M ;
RUBSAMENWAIGMANN, H ;
MULLINS, JI .
SCIENCE, 1993, 262 (5137) :1257-1261
[5]   Rapid genotyping of hepatitis C virus by direct cycle sequencing of PCR-amplified cDNAs and capillary electrophoresis analysis [J].
Doglio, A ;
Laffont, C ;
Thyss, S ;
Lefebvre, JC .
RESEARCH IN VIROLOGY, 1998, 149 (04) :219-227
[6]   Determination of hepatitis C virus genotype by direct sequence analysis of products generated with the amplicor HCV test [J].
Germer, JJ ;
Rys, PN ;
Thorvilson, JN ;
Persing, DH .
JOURNAL OF CLINICAL MICROBIOLOGY, 1999, 37 (08) :2625-2630
[7]  
Li QB, 2002, ELECTROPHORESIS, V23, P1499, DOI 10.1002/1522-2683(200205)23:10<1499::AID-ELPS1499>3.0.CO
[8]  
2-X
[9]   Hepatitis C genotyping by denaturing high-performance liquid chromatography [J].
Liew, M ;
Erali, M ;
Page, S ;
Hillyard, D ;
Wittwer, C .
JOURNAL OF CLINICAL MICROBIOLOGY, 2004, 42 (01) :158-163
[10]   Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C [J].
McHutchison, JG ;
Gordon, SC ;
Schiff, ER ;
Shiffman, ML ;
Lee, WM ;
Rustgi, VK ;
Goodman, ZD ;
Ling, MH ;
Cort, S ;
Albrecht, JK .
NEW ENGLAND JOURNAL OF MEDICINE, 1998, 339 (21) :1485-1492