title: Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis.
abstract: Both the viral titer and the genotype significantly determine clinical outcomes and responses to antiviral treatment in chronic hepatitis B virus (HBV) infection. A method was developed for large-scale A-to-G genotyping with simultaneous viral quantification. The assay was run on a LightCycler instrument using hybridization probes. The genotype was determined from the melting points of the probes in a two-step manner. Set 1 amplicons differentiated genotypes B, E, and F from A, C, D, and G and simultaneously quantified viremia by real-time PCR. Melting curve analysis using the set 2-1 amplicon or the set 2-2 amplicon reaction mixture was then used to differentiate these genotype groups into single genotypes. HBV DNA quantification was consistent with that of the Amplicor assay and linear in a range from 10(2) to 10(13) copies/ml. By comparison with the restriction fragment length polymorphism method, 92.3% of 441 samples were accurately genotyped by the current assay. The method should be useful for genotyping and quantification of HBV DNA in areas where all genotypes exist.
authors.affiliation: Institute of Basic Medical Sciences, Department of Medicine, National Cheng Kung University, Tainan City 704, Taiwan, Republic of China, and Liver Unit, Hospital General Universitari Vall d'Hebron, Barcelona, Spain.
authors.forename: Wen-Chun
authors.initials: WC
authors.lastname: Liu
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizokami
authors.forename: Maria
authors.initials: M
authors.lastname: Buti
authors.forename: Magnus
authors.initials: M
authors.lastname: Lindh
authors.forename: Kung-Chia
authors.initials: KC
authors.lastname: Young
authors.forename: Koun-Tem
authors.initials: KT
authors.lastname: Sun
authors.forename: Yun-Chan
authors.initials: YC
authors.lastname: Chi
authors.forename: Hsi-Hsien
authors.initials: HH
authors.lastname: Li
authors.forename: Ting-Tsung
authors.initials: TT
authors.lastname: Chang
mesh_terms: D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics; D005838:Genotype; D006509:Hepatitis B / Q000175:diagnosis / Q000821:virology*; D006515:Hepatitis B virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D019562:Viral Load
chemical_list: D017931:DNA Primers; D004279:DNA, Viral