title: A reliable multiplex genotyping assay for HCV using a suspension bead array.
abstract: The genotyping of the hepatitis C virus (HCV) plays an important role in the treatment of HCV because genotype determination has recently been incorporated into the treatment guidelines for HCV infections. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. We therefore developed a multiplex genotyping assay to determine HCV genotypes using a bead array. Synthetic plasmids, genotype panels and standards were used to verify the target-specific primer (TSP) design in the assay, and the results indicated that discrimination efforts using 10 TSPs in a single reaction were extremely successful. Thirty-five specimens were then tested to evaluate the assay performance, and the results were highly consistent with those of direct sequencing, supporting the reliability of the assay. Moreover, the results from samples with mixed HCV genotypes revealed that the method is capable of detecting two different genotypes within a sample. Furthermore, the specificity evaluation results suggested that the assay could correctly identify HCV in HCV/human immunodeficiency virus (HIV) co-infected patients. This genotyping platform enables the simultaneous detection and identification of more than one genotype in a same sample and is able to test 96 samples simultaneously. It could therefore provide a rapid, efficient and reliable method of determining HCV genotypes in the future.
authors.affiliation: Food and Drug Administration, Ministry of Health and Welfare, Taipei, Taiwan; Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Yang
authors.forename: Der-Yuan
authors.initials: DY
authors.lastname: Wang
authors.forename: Hwei-Fang
authors.initials: HF
authors.lastname: Cheng
authors.forename: Eric Y
authors.initials: EY
authors.lastname: Chuang
authors.forename: Mong-Hsun
authors.initials: MH
authors.lastname: Tsai
mesh_terms: D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers