Rapid genotyping of hepatitis C virus by dideoxy fingerprinting

S.A. Fox, R.R. Lareu, Nigel Swanson

Research output: Contribution to journalArticle

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Abstract

A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha-interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non-coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a P-33-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6% acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a and 1b); a ddC reaction confirmed 1a and 1b typing. Standard denaturing gels gave superior results than a variety of non-denaturing ('SSCP') gels. Our results show that dideoxy fingerprinting is a reliable and efficient alternative to direct sequencing for HCV genotyping which is adaptable to semi-routine screening.
Original languageEnglish
Pages (from-to)1-9
JournalJournal of Virological Methods
Volume53
DOIs
Publication statusPublished - 1995

Fingerprint

Hepacivirus
Gels
Thiamine
Cytosine
DNA Sequence Analysis
Cinnarizine
Genotype
RNA
Single-Stranded Conformational Polymorphism
Polymerase Chain Reaction
Acrylamide
Guanidine
Chronic Hepatitis C
Virus Diseases
Phenol
Autoradiography
Interferon-alpha
Sequence Analysis
Urea
Electrophoresis

Cite this

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abstract = "A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha-interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non-coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a P-33-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6{\%} acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a and 1b); a ddC reaction confirmed 1a and 1b typing. Standard denaturing gels gave superior results than a variety of non-denaturing ('SSCP') gels. Our results show that dideoxy fingerprinting is a reliable and efficient alternative to direct sequencing for HCV genotyping which is adaptable to semi-routine screening.",
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Rapid genotyping of hepatitis C virus by dideoxy fingerprinting. / Fox, S.A.; Lareu, R.R.; Swanson, Nigel.

In: Journal of Virological Methods, Vol. 53, 1995, p. 1-9.

Research output: Contribution to journalArticle

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AU - Fox, S.A.

AU - Lareu, R.R.

AU - Swanson, Nigel

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AB - A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha-interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non-coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a P-33-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6% acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a and 1b); a ddC reaction confirmed 1a and 1b typing. Standard denaturing gels gave superior results than a variety of non-denaturing ('SSCP') gels. Our results show that dideoxy fingerprinting is a reliable and efficient alternative to direct sequencing for HCV genotyping which is adaptable to semi-routine screening.

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