Determination of hepatitis C virus genotype by Pyrosequencing

被引:40
作者
Elahi, E
Pourmand, N
Chaung, R
Rofoogaran, A
Boisver, J
Samimi-Rad, K
Davis, RW
Ronaghi, M
机构
[1] Stanford Univ, Stanford Genome Technol Ctr, Palo Alto, CA 94304 USA
[2] Univ Tehran, Fac Sci, Tehran, Iran
[3] Stanford Univ, Vet Amer Hosp, Palo Alto, CA 94304 USA
[4] Univ Tehran, Sch Publ Hlth, Tehran, Iran
关键词
Pyrosequencing; multiplex; sequencing; typing; hepatitis C;
D O I
10.1016/S0166-0934(03)00068-5
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping. (C) 2003 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:171 / 176
页数:6
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