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Journal of Clinical Microbiology, October 2004, p. 4545-4551, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4545-4551.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genotyping Hepatitis C Virus by Heteroduplex Mobility Analysis Using Temperature Gradient Capillary Electrophoresis

Rebecca L. Margraf,1* Maria Erali,1 Michael Liew,1 and Carl T. Wittwer2

ARUP Institute for Clinical and Experimental Pathology,1 Department of Pathology, University of Utah Medical School, Salt Lake City, Utah2

Received 12 December 2003/ Returned for modification 2 April 2004/ Accepted 14 June 2004

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.

* Corresponding author. Mailing address: Advanced Technology Group, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108. Phone: (801) 583-2787. Fax: (801) 584-5114. E-mail: 18298{at}aruplab.com.

Journal of Clinical Microbiology, October 2004, p. 4545-4551, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4545-4551.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Antonishyn, N. A., Ast, V. M., McDonald, R. R., Chaudhary, R. K., Lin, L., Andonov, A. P., Horsman, G. B. (2005). Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis. J. Clin. Microbiol. 43: 5158-5163 [Abstract] [Full Text]  


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