Simultaneous quantification and genotyping of hepatitis B virus for genotypes A to G by real-time PCR and two-step melting curve analysis |
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| Authors: | Liu Wen-Chun Mizokami Masashi Buti Maria Lindh Magnus Young Kung-Chia Sun Koun-Tem Chi Yun-Chan Li Hsi-Hsien Chang Ting-Tsung |
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| 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. |
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| 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. |
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