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Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

ABSTRACT: Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1-7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5' untranslated region (5'UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. •A new one-step RT-PCR method for the amplification of the 5' untranslated region and partial Core region of hepatitis C virus was established.•HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool.•The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills.

SUBMITTER: Virtanen E

PROVIDER: S-EPMC6060086 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

Virtanen Elina E Mannonen Laura L Lappalainen Maija M Auvinen Eeva E

MethodsX 20180416

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1-7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic ...[more]

PMID: 30050759

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Project description:BackgroundThe September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus.MethodThis genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors.ResultEach WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA.ConclusionThe molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories.Keywordssingle nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit.

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Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

Publications

Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

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