Volume 41, Issue 4, Pages 659-666 (October 2004) Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis  Shiou-Hwei Yeh, Ching-Yi Tsai, Jia-Horng Kao, Chun-Jen Liu, Ti-Jung Kuo, Ming-Wei Lin, Wen-Ling Huang, Shu-Fen Lu, Jane Jih, Ding-Shinn Chen, Pei-Jer Chen  Journal of Hepatology  Volume 41, Issue 4, Pages 659-666 (October 2004) DOI: 10.1016/j.jhep.2004.06.031 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 The underlying principle (panel A) and the representative results (panel B) illustrating quantification and genotyping of HBV in one-tube reaction with the set 1 primers/probes. The detailed principle is described in the text. In panel B, the thick solid line indicates the genotype C infected sample, the thick dotted lines indicates the genotype B infected sample, and thin dotted lines indicate standards. Step 1 is the quantification step with real-time fluorescence detection accompanying with the standards ranging from 102 to 1011copies/ml. The crossing point (Cp) values of the tested samples are converted to concentrations expressed as copy/ml, using the equation derived from the standards. Step 2 is the genotyping step conducted by melting curve analysis. Melting peak at 55°C indicates genotype C and that at 61°C indicates genotype B; F1, fluorescence 1; F2, fluorescence 2; −d(F2)/dT, negative derivative of fluorescence with respect to temperature; LC-red, LightCycler Red 640 Fluorescent Dye. [This figure appears in colour on the web.] Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 The representative results for genotyping of 20 samples, containing 10 samples with genotype B and 10 samples with genotype C using set 1 primers/probes. The melting temperature (Tm) for each genotype has been indicated with vertical lines; −d(F2)/dT, negative derivative of fluorescence with respect to temperature. [This figure appears in colour on the web.] Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 The standard curve used for quantification of the virus. Linear regression of the standards raging from 102 to 1011copies/ml is determined by using the ‘second derivative maximum’ method. The standard curve showed regression coefficient r=−1.00; mean squared error=0.199; intercept=51.79; and slope=−3.681. Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 The comparison of the quantification assay by set 1 amplicon with the three methods currently used for HBV quantification, including Roche Amplicor assay, NGI SuperQuant assay, and QUANTIPLEX bDNA assay. Panel A showed the quantification results of samples from either the HBV genotype panel (left panel) or from the standards of bDNA kit (right panel). The solid bar indicates results from LightCycler analysis, slash bar indicates results from Amplicor assay, dotted bar indicates results from SuperQuant assay, and the bar filled with diagonals indicates results by bDNA assay. The standard deviation (SD) is derived from the 6 individual experiments. The linear regression comparison has been performed individually between LighCycler/Roche Amplicor assay (panel B), between LightCycler/ NGI SuperQuant (panel C), and between LighCycler/bDNA assay (panel D). [This figure appears in colour on the web.] Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 The determination of HBV mixed infection by the melting curve analysis. Panel A showed three clinical samples with mixed infected detected in this study as indicated by arrows. Panel B showed results with different proportion of mixtures of genotypes B and C plasmids as indicated. [This figure appears in colour on the web.] Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 The workflow used for differentiating the HBV genotypes A, B, C, and F, with the three primer/probe sets. The Tm for each genotype has been indicated in the parenthesis. Journal of Hepatology 2004 41, 659-666DOI: (10.1016/j.jhep.2004.06.031) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions