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J Clin Microbiol. Dec 2006; 44(12): 4491–4497.
Published online Oct 4, 2006. doi:  10.1128/JCM.01375-06
PMCID: PMC1698380

Simultaneous Quantification and Genotyping of Hepatitis B Virus for Genotypes A to G by Real-Time PCR and Two-Step Melting Curve Analysis[down-pointing small open triangle]

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 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.

Hepatitis B virus (HBV) infection is an important public health problem chronically infecting more than 350 million people worldwide. The infection can cause acute and chronic liver disease, including cirrhosis and hepatocellular carcinoma. HBV has a circular genome of approximately 3,200 base pairs and is divided into genotypes A to H, with an intergenotypic diversity of at least 8% in the full genome sequence (1, 10, 12, 13, 17). The genotypes have distinct geographic distributions, and accumulating evidence indicates that genotyping is of clinical importance, because the genotypes correlate with the severity of liver disease. HBV genotype C is associated with more severe liver disease than genotype B (3, 5, 6), and patients infected with genotype B have a lower rate of positive hepatitis B virus e antigen (HBeAg) and 1 decade earlier spontaneous HBeAg seroconversion than genotype C-infected patients (14). Moreover, patients infected with genotypes C and D seem to have a lower response rate to alpha interferon than those infected with genotypes A and B (5), and the risk of emergence of lamivudine resistance-associated mutations has been reported to be higher in genotype A-infected patients than in genotype D-infected patients (2, 20).

Several methods for genotyping HBV have been reported, including melting curve analysis (MCA) (16, 19), restriction fragment length polymorphism (RFLP) (7, 8), post-PCR hybridization or line probe assay (4), PCR with genotype-specific primers (9), and enzyme-linked immunosorbent assay-based methods (18). One genotyping method was shown to also quantitate HBV viremia (19), but that method was limited to distinguishing between genotypes B and C, which are prevalent in Asia. In this study, an efficient PCR-based method was developed to combine quantification of HBV DNA and genotyping with differentiation of HBV genotypes A to G. The results showed consistency with currently available viral-load quantification and efficient genotyping. The PCR-based method thus provides a useful tool for rapid and cost-effective diagnosis in areas with different geographical distributions of HBV genotypes.

MATERIALS AND METHODS

Study subjects and samples.

A total of 441 HBV DNA-positive serum samples from Taiwan, Spain, Japan, and Sweden were used for the evaluation of our genotyping system. Among these samples, 380 were from the outpatient clinic at National Cheng Kung University Hospital in Taiwan. Stored samples (−80°C) from these patients were analyzed in the study. All samples were genotyped by PCR-RFLP (7), and the HBV DNA levels of 110 samples were determined by the Roche Amplicor HBV Monitor Test (11).

Principle of real-time PCR genotyping by melting curve analysis.

The genotyping method was based on MCA with LightCycler hybridization probes as shown in Fig. Fig.1A.1A. The primers and hybridization probes were designed by analyzing 369 full-length HBV nucleotide sequences from the GenBank database, which by alignments and phylogenetic analysis using the Clustal X1.81, GeneDoc2.6.002, and Mega2 programs had been classified into eight genotypes, from A to H. These alignments included, after excluding the sequences of inconclusive genotypes, 69 full-length HBV sequences of type A, 93 of type B, 101 of type C, 54 of type D, 6 of type E, 27 of type F, 10 of type G, and 9 of type H isolates (1). Thus, a few conserved signature single-nucleotide polymorphisms that could differentiate all genotypes were identified. These polymorphisms were targeted by the LightCycler sensor probes to allow genotype identification by melting temperature (Tm) analysis.

FIG. 1.
Primers and hybridization probes on melting curve analysis. (A) The workflow used for differentiating the seven HBV genotypes A to G with two-step melting curve analysis. With the set 1 amplicon, real-time PCR was used for quantification, with simultaneous ...

A two-step melting curve analysis was used to differentiate HBV genotype groups into single genotypes. The first step, using the set 1 amplicon (the ACDG/BEF set), simultaneously used real-time PCR for quantification of the viral titer and melting curve analysis for genotyping into groups (B, E, F) and (A, C, D, G). The second step used melting curve analysis with set 2-1 (B/E/F set) and set 2-2 (A/CD/G set and C/D set) amplicons for differentiating each group into single genotypes. Thus, seven genotypes could be clearly differentiated by showing distinct Tm values (Fig. (Fig.1A).1A). The anchor probes were labeled at the 5′ ends with LC-Red 640 dye, and sensor probes covering the single-nucleotide polymorphisms were labeled at the 3′ ends with fluorescence. The 3′ ends of the anchor probes were also phosphorylated.

Real-time PCR amplification of HBV using LightCycler.

The serum HBV DNA was extracted with a Viogene extraction kit, and real-time PCR was then performed on a LightCycler instrument (Roche Diagnostics Applied Science) using primers and probes described in Table Table11 and Fig. Fig.1B.1B. The PCR was run in a total volume of 10 μl containing 2.5 μl of DNA template, 1 μl of LightCycler FastStart DNA Master Hybridization Mixture (Taq DNA polymerase, PCR buffer, 10 mM MgCl2, and a deoxynucleoside triphosphate mixture) (Roche Diagnostics Applied Science), 1.2 μl of 25 mM MgCl2, 0.075 μl of 20 μM (each) of the probes, and 0.5 μl of 5 μM of each primer. The amplification using set 1 (ACDG/BEF set) and set 2-1 (B/E/F set) amplicons was performed as follows: initial hot-start denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 53°C for 10 s, and extension at 72°C for 15 s. The programmed temperature transition rate was 20°C/s for denaturation/annealing and 3°C/s for extension. Real-time PCR monitoring was achieved by measuring the fluorescence at the end of the annealing phase for each cycle. After PCR, a melting curve was generated by holding the reaction mixture at 95°C for 10 s and then lowering the temperature to 48°C at a transition rate of 20°C/s and holding it for 60 s. This was followed by heating the reaction mixture slowly at a transition rate of 0.1°C/s to 80°C with continuous collection of fluorescence at 640 nm. The melting curve and quantitative analyses were conducted by using LightCycler analysis software version 3.5 following the manufacturer's instructions (Roche Diagnostics Applied Science). For the set 2-2 amplicon (the A/CD/G set plus the C/D set), the PCR was the same as that for set 1 and set 2-1, except for extension at 72°C for 20 s.

TABLE 1.
PCR primers and probes in the amplicons used for HBV quantification and genotyping

Quantification of HBV DNA by real-time PCR.

Amplification by set 1 primers and probes (Table (Table1)1) was used for the assessment of viremia. The HBV DNA levels were calculated by comparing each sample's threshold cycle value to a standard curve obtained in each run. The quantification standard was derived from plasmid pGEM-HBV (with an HBV insert from genotype B) purified with the plasmid Gene-SpinTM Miniprep Purification Kit (Protech). The concentration of the plasmid was determined spectrophotometrically and expressed as copies/ml. Serial dilutions of the plasmid ranging from 1 × 102 to 1 × 1013 copies/ml were used to prepare the standard curve for quantification of HBV DNA from patient specimens. The reproducibility was evaluated by analyzing triplicates of two serum samples with HBV DNA levels at x and y, respectively, on two occasions. The mean values, standard deviations, and coefficient of variation were calculated.

Alternative genotyping of HBV genotypes A, C, D, and G by type-specific multiplex PCR.

Because the accuracy of the second-step melting curve analysis was probably influenced by complicated conditions during differentiation of the group of four genotypes (A, C, D, and G), an alternative genotyping method using type-specific multiplex PCR was designed. This PCR utilized one universal forward primer (5′ nucleotides [nt] 2305 to 2325) and a mixture of four HBV genotype-specific reverse primers: 5′-TAGGGGACCTGCCTCGGTC-3′ (nt 2413 to 2395) for genotype A, 5′-TTCATTAACTGTAAGAGGGCCYAAAT-3′ (nt 2659 to 2634) for genotype C, 5′-GATTGCTGGTGGAAAGATTCTGC-3′ (nt 2952 to 2907) for genotype D, and 5′-ACTAACATTGGGAAGCTGGAGATGC-3′ (nt 2497 to 2473) for genotype G. Multiplex PCR was carried out in a total volume of 50 μl, which contained 0.5 μl of each primer (20 mM), 2 ml deoxynucleoside triphosphate (5 mM), 5 μl 10× Tag reaction buffer, and 10 μl template and water for a total volume of 50 μl. The thermocycler (GeneAmp PCR System 9600; Perkin-Elmer) was programmed to incubate the samples for 5 min at 94°C, followed by 40 cycles consisting of 94°C for 1 min, 59°C for 1 min, and 72°C for 2 min. After the PCR, electrophoresis of the amplified products was completed on a 4% agarose gel, which was stained with ethidium bromide and evaluated under UV light. The multiplex PCR produced different amplicon sizes for genotypes A, C, D, and G, with 109, 349, 609, and 187 bp, respectively.

Direct-sequencing analyses.

For samples with discordant results between the PCR-based method and PCR-RFLP, direct sequencing of the HBV complement genome and phylogenetic analysis was conducted (13, 15). HBV amplicons were then subjected to cycle sequencing using ABI PRISM Big-Dye kits (Applied Biosystems, Foster City, CA) and subsequent reading of the sequence using an ABI 3100 Genetics Analyzer. If the genotyping results suggested a mixed infection, the HBV isolates were cloned into a pGEM plasmid and then analyzed by sequencing of five clones.

Statistical methods.

The interclass correlation between three duplication experiments in each individual run was calculated to evaluate the reproducibility of the developed genotype quantification method. The Pearson correlation between different runs was also calculated. The correlation between our quantification method and the Roche Amplicor HBV monitor test was evaluated by Pearson correlation. All statistics analyses were conducted using the SPSS statistical package, version 11.0.

RESULTS

Quantification of HBV by the set 1 amplicon (the ACDG/BEF set).

The standard calibration curve (serial dilution of plasmid HBV) was linear in a range from 102 to 1013 copies/ml (Fig. (Fig.2).2). Reproducibility as measured by dual testing of triplicates of two serum samples was acceptable, with coefficients of variation at 7.9% and 12.9%. Analysis of 110 serum samples showed good correlation between real-time PCR quantification and the Amplicor HBV Monitor test (Roche), with an R2 value of 0.93 and a gamma of >0.80 (Fig. (Fig.2).2). The correlation was also good at levels above the range quantification of the Amplicor test, i.e., when samples had been prediluted prior to being tested with Amplicor (but not prior to real-time PCR).

FIG. 2.
Quantification of the virus by real-time PCR with set 1. (A) Linear regression of the standards ranging from 102 to 1013 copies/ml was determined by using the “second derivative maximum” method. The standard curve showed a regression coefficient, ...

Genotyping by MCA.

In an initial evaluation of the genotyping, 62 samples representative of all of the genotypes from A to G were analyzed. By MCA with the set 1 amplicon, all seven of the genotypes could be correctly differentiated into two groups (ACDG/BEF) on the basis of the Tm, which was 56.7°C for genotypes A, C, D, and G and 62.6°C for genotypes B, E, and F (Fig. (Fig.3A).3A). With the set 2-1 amplicon (the B/E/F set), samples of genotypes B, E, and F could be further classified with Tm characteristics of 66.5°C, 63.4°C, and 61.4°C for genotypes B, E, and F, respectively (Fig. (Fig.3B).3B). With the set 2-2 amplicon (the A/CD/G set and the C/D set), using two channels (F2 and F3), identification of genotypes A, C, D, and G could be conducted with Tm characteristics of 66.3°C, 62.6°C, and 57.8°C for genotypes C/D, A, and G, respectively, in the F2 channel and 57.6°C and 62.6°C for genotypes C and D, respectively, in the F3 channel (Fig. (Fig.3C).3C). The Tm differences in each set were significant enough for clear differentiation of these genotypes (Fig. (Fig.1A1A).

FIG. 3.
Representative results for melting curve analysis of clinical samples with genotypes A to G. (A) Differentiation into the group of genotypes (B, E, F) and genotypes (A, C, D, G) with the set 1 amplicon. (B) Differentiating genotypes B, E, and F into single ...

After confirming the reproducibility of the amplifications and melting point analyses, with few cases showing a Tm shift, the accuracy of the genotyping method was further evaluated by analyzing 441 clinical samples whose genotypes were also tested by PCR-RFLP for comparison.

When the results by melting point and PCR-RFLP genotyping were congruent, they were considered accurate. When the results were conflicting, accuracy was assessed by comparison with sequencing. Assessed in this way, the overall accuracy of the melting curve genotyping was 92.3%, compared to 87.0% for RFLP (Table (Table2).2). The melting point typing, however, was more accurate with the set 1 amplicon and the set 2-1 amplicon than with the set 2-2 amplicon (99.1% and 97.0% versus 87.9%) because of the higher genotypic specificity of the polymorphisms at nt 225 and nt 706. Tm shift from expected values was relatively common, particularly in genotype C, and sequencing showed that this was due to 1- or 2-nucleotide variations in the segment targeted by the sensor or the anchor probes. Accordingly, identification of genotypes B, E, F, and G was more accurate (98.7%, 100%, 100%, and 100%) than that of genotypes A, C, and D (88.2%, 80.9%, and 85.7%).

TABLE 2.
Comparison between HBV genotypic analysis using two-step melting curve analysis with different amplicons and PCR-RFLP

Typing of genotypes A, C, D, and G by multiplex PCR.

A proportion (12%) of samples identified as A, C, D, or G by the set 1 amplicon could not be separated by MCA using the set 2-2 amplicon due to Tm shift. However, the correct genotype could be identified by type-specific multiplex PCR in the majority of these cases (94.44%) (Table (Table22).

Detection of HBV mixed infections with melting curve analysis.

Although there were no cases of mixed-genotype infection among the samples, the power of the set 1 amplicon in detecting mixed viral infections was evaluated. By mixing two plasmids corresponding to genotypes B and G with various proportions of mixtures ranging from 1:1 to 1:9 and a total concentration of 108copies/ml, the detection sensitivity was 10% for the minor population (Fig. (Fig.44).

FIG. 4.
Determination of HBV mixed infection by melting curve analysis. Different proportions of mixtures of genotype B and G plasmids are indicated.

DISCUSSION

Here, we present a new method for large-scale HBV genotyping and simultaneous quantification of viremia. The method is based on real-time PCR in the LightCycler format, with probes targeting segments of the HBV genome that include nucleotide polymorphisms strongly linked to the genotype. The genotype is identified by analyzing probe melting points, and quantification of HBV DNA is obtained by first-step real-time PCR.

The accuracy of genotyping was verified by analyzing 441 clinical samples representing genotypes A to G. Overall, the MCA typing provided an accuracy of 92.3%, which was better than that observed for RFLP (87%). For differentiating genotypes B, E, and F by MCA, the accuracy was even higher, up to 96.1%. Due to the appearance of point mutations that altered the melting point, differentiation of genotypes A, C, and D was uncertain in some cases (overall accuracy, 88%). Therefore, an alternative typing method based on multiplex PCR was developed and applied to these samples, allowing correct typing of 99%.

An advantage with this new method is that the genotype is obtained directly from the PCR without further processing. Moreover, the method was found to have higher precision in genotyping and sensitivity for mixed-genotype detection equal to or higher than that of RFLP. A limitation of the method is that the genotype is not identified by a single reaction. We suggest that typing be done in a stepwise manner, the first step separating genotype ACDE from BFG, and the second identifying the genotype within the ACDE or BFG group. The higher cost-effectiveness of this approach is important when the assay is run on LightCycler instruments with only 32 reaction capillaries, particularly if larger sets of samples are genotyped, for example, in clinical studies. If amplification is instead run in the 96-well format with three reaction mixtures (set 1, set 2-1, and set 2-2 amplicons) in parallel, e.g., on a LightCycler 96 instrument, the genotype may alternatively be obtained directly. This would require inclusion of a 72°C extension step (which is otherwise used only in the set 2-2 PCR), but it would allow genotyping of 32 samples per run.

Genotype H was recently identified in Central America. After analyzing genotype H isolates from GenBank, we predict that genotype H will have the same melting point as genotype F, and would thus be typed as F by MCA. However, in most regions of the world, this limitation of the method would be of minor importance.

An additional advantage of the method was the simultaneous quantification, performed with the set 1 amplicon amplification, which targets conserved genomic segments. Previously, a similar method (16) for simultaneous HBV quantification and genotyping was described. However, that method is mainly used for differentiation of genotypes B and C, which are the major HBV genotypes in Asia. The method described in this study allows quantification in parallel with typing of all of the genotypes A to G. The quantification was linear over a broad range of viremia, and its accuracy was documented by comparison with the Roche Amplicor assay. Although the comparison with Amplicor indicated that quantification is accurate for all genotypes, this needs to be confirmed by further analysis of reproducibility panels and clinical samples. An advantage of the Amplicor assay compared to our method is the inclusion of internal inhibition controls and output in IU (by calibration to a WHO standard). On the other hand, our real-time PCR method has a much broader detection range and lower reagent costs. It should be attractive for monitoring patients during antiviral treatment and for research projects when output in the IU format is considered less important.

In summary, this study provides a quick and useful HBV quantification and large-scale genotyping method for genotypes A to G suitable for research and clinical diagnostics in all regions of the world.

Acknowledgments

The study was supported by grants from the National Science Council, Taiwan, Republic of China (NCS 95-2314-B-006-030).

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 October 2006.

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