Journal of Clinical Microbiology, February 2000, p. 477-482, Vol. 38, No. 2
Virology Division, Department of
Microbiology, SEALS, Prince of Wales Hospital, Randwick, Sydney,
New South Wales 2031, Australia,1 and
Beijing Biochemical and Immune Reagents Centre (BBIRC),
Yang Fang Cun, An Ding Men, Beijing 100012, China2
Received 16 July 1999/Returned for modification 16 September
1999/Accepted 22 October 1999
Heteroduplex mobility analysis (HMA) was used to genotype hepatitis
C viruses (HCV) with PCR fragments derived from the 5' untranslated
region (5'-UTR) or the NS5b region. HCV 5'-UTR fragments were amplified
from 296 serum samples by use of a combined reverse transcription-PCR
assay, and the genotypes of isolates were determined by sequencing. HCV
genotype distributions in Australia were 39% for genotype 1a, 15% for
1b, 3% for 1a/b, <1% for 2a/c, 5% for 2b, 34% for 3a, <1% for
3b, and 1% for 4, and 1% of patients were infected with more than one
genotype. Pairwise HMA of subtypes 1a, 1b, 2a/c, 2b, 3a, 3b, 4a, and 6a
demonstrated that five distinct heteroduplex patterns were formed
between the eight subtypes. A reference panel that contained a
representative of each pattern (1a, 2b, 3a, 4a, and 6a) was used for
genotyping. The pattern of heteroduplexes formed when a test isolate
was mixed with the five reference isolates was correlated with the
genotype, as determined by sequencing. Genotypes determined by HMA
correlated exactly with sequencing results within the groups 1, 2, 3a,
3b/4, and 6. HMA was also used to simplify the identification of mixed
infection with two HCV genotypes. In further studies, with amplicons
from the NS5b region, HMA classified isolates into their respective subtypes, and the heteroduplex mobility ratio correlated closely with
nucleotide sequence variation at the isolate, subtype, and genotype
levels. HMA provides an adaptable, inexpensive, and rapid method of
genotyping HCV that requires fewer resources than DNA sequencing.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simplified Hepatitis C Virus Genotyping by
Heteroduplex Mobility Analysis
*
Corresponding author. Mailing address: Virology
Division, Department of Microbiology, SEALS, Prince of Wales Hospital,
Randwick, New South Wales 2031, Australia. Phone: 61 2 9382 9096. Fax:
61 2 9398 4275. E-mail: whitepa{at}sesahs.nsw.gov.au.
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