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Journal of Clinical Microbiology, December 2006, p. 4491-4497, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01375-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Simultaneous Quantification and Genotyping of Hepatitis B Virus for Genotypes A to G by Real-Time PCR and Two-Step Melting Curve Analysis

Wen-Chun Liu,¹ Masashi Mizokami,² Maria Buti,³ Magnus Lindh,⁴ Kung-Chia Young,⁵ Koun-Tem Sun,⁶ Yun-Chan Chi,⁷ Hsi-Hsien Li,⁸ and Ting-Tsung Chang^1,9*

Institute of Basic Medical Sciences,¹ Department of Medicine,⁹ Department of Medical Laboratory Science and Biotechnology,⁵ Institute of Molecular Medicine, Medical College,⁸ Department of Statistics, Management College, National Cheng Kung University,⁷ Institute of Computer Science of Information Education, National University of Tainan, Tainan, Taiwan, Republic of China,⁶ Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan,² Liver Unit, Hospital General Universitari Vall d'Hebron, Barcelona, Spain,³ Department of Clinical Virology, Göteborg University, Guldhedsgatan 10B, Goteborg, Sweden⁴

Received 5 July 2006/ Returned for modification 8 September 2006/ Accepted 22 September 2006

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 10² to 10¹³ 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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* Corresponding author. Mailing address: Department of Internal Medicine, National Cheng Kung University Hospital, No. 138 Sheng-Li Road, Tainan City 704, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5389. Fax: 886-6-2095233. E-mail: ttchang{at}mail.ncku.edu.tw.

Published ahead of print on 4 October 2006.

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Journal of Clinical Microbiology, December 2006, p. 4491-4497, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01375-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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