title: Development and validation of a real-time, reverse transcription PCR assay for rapid and low-cost genotyping of hepatitis C virus genotypes 1a, 1b, 2, and 3a.
abstract: Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98-100% and 85-98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.
authors.affiliation: University of British Columbia Centre for Disease Control, Vancouver, British Columbia, Canada.
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Olmstead
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada.
authors.forename: Tracy D
authors.initials: TD
authors.lastname: Lee
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada.
authors.forename: Ron
authors.initials: R
authors.lastname: Chow
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada.
authors.forename: Kingsley
authors.initials: K
authors.lastname: Gunadasa
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Auk
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Mel
authors.initials: M
authors.lastname: Krajden
authors.affiliation: British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address: agatha.jassem@bccdc.ca.
authors.forename: Agatha N
authors.initials: AN
authors.lastname: Jassem
mesh_terms: D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D009656:North America; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: Genotyping; Hepatitis C virus; Line probe assay; PCR