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

J

OURNAL OF

C

LINICAL

M

ICROBIOLOGY

, Dec. 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

䌤

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

_and

Institute of Molecular Medicine,

8

_{Medical College, and Department of Statistics, Management College,}

7

_{National Cheng Kung University,}

and Institute of Computer Science of Information Education, National University of Tainan,

6

_{Tainan, Taiwan, Republic of China;}

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

_{; and Department of Clinical Virology,}

Go

¨teborg University, Guldhedsgatan 10B, Goteborg, Sweden

4

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 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.

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

clin-ical importance, because the genotypes correlate with the

se-verity 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

hy-bridization 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

geno-typing 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 geno-typing method was based on MCA with LightCycler hybridization probes as shown in Fig. 1A. The primers and hybridization probes were designed by analyzing 369 full-length HBV nucleotide sequences from the GenBank data-base, 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

* 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@mail

.ncku.edu.tw.

䌤

_{Published ahead of print on 4 October 2006.}

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targeted by the LightCycler sensor probes to allow genotype identification by

melting temperature (Tm) analysis.

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 distinctTmvalues (Fig. 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 1 and Fig. 1B. The PCR

was run in a total volume of 10␮l containing 2.5␮l of DNA template, 1␮l

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␮l of 25 mM MgCl2, 0.075

␮l of 20␮M (each) of the probes, and 0.5␮l of 5␮M of each primer. The

[image:2.585.110.476.68.486.2]

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 temper-ature 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 Diag-nostics Applied Science). For the set 2-2 amplicon (the A/CD/G set plus the

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 melting curve

analysis for differentiating genotypes into two groups. According to the distinct melting temperature, single genotypes can be further differentiated

with either set 2-1 or set 2-2 reaction mixtures. (B) The relevant portions of the HBV genome with binding sites for the different primers and

probes.

3

, forward primer;

4

, reverse primer;

}

, sense probe;

F

, anchor probe.

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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.

Quantification of HBV DNA by real-time PCR.Amplification by set 1 primers and probes (Table 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-SpinTMMiniprep Purification Kit (Protech). The

concentra-tion of the plasmid was determined spectrophotometrically and expressed as

copies/ml. Serial dilutions of the plasmid ranging from 1⫻102_{to 1⫻10}13

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 atxandy, 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⬘-ACTAACATTGGGAAGCTGGAG

ATGC-3⬘(nt 2497 to 2473) for genotype G. Multiplex PCR was carried out in a

total volume of 50␮l, which contained 0.5␮l of each primer (20 mM), 2 ml

deoxynucleoside triphosphate (5 mM), 5␮l 10⫻Tag reaction buffer, and 10␮l

template and water for a total volume of 50␮l. 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 infec-tion, 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 ex-periments 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

[image:3.585.50.539.80.419.2]

Quantification of HBV by the set 1 amplicon (the ACDG/

BEF set).

The standard calibration curve (serial dilution of

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

Amplicon Sequence (5⬘–3⬘) Sense/

antisensea

Amplicon size (bp)

Position

(nt)b Tmc

Set 1 (ACDG/BEF)

关

Surface/polymerase

兴

340

Genotype BEF,

62.2°C

Forward primer

5

⬘

-TGCTGGTGGCTCCAGTTC-3

⬘

S

58–75

Genotype ACDG,

56.7°C

Reverse primer

5

⬘

-TGATAAAACGCCGCAGACAC-3

⬘

As

398–379

BEF-Sen probe

5

⬘

GTTGACAAAAATCCTCACAATACC-3

⬘

-FL

S

217–240

Genetic-polymorphism site

A/G, nt225

ACDG/BEF-Anc probe

5

⬘

-LC Red 640-CAGAGTCTAGACTCGTGGTGG

ACTTCTCTCA-3

⬘

-PH

S

242–272

Set 2-1 (B/E/F)

关

Surface/polymerase

兴

262

Genotype B, 66.5°C

Forward primer

5

⬘

-TCAAGGAACCTCTATGTTTCCCTC-3

⬘

S

538–561

Genotype E, 63.4°C

Reverse primer

5

⬘

-ACAGCGGCATAAAGGGACTC-3

⬘

As

800–781

Genotype F, 61.4°C

B-Sen probe

5

⬘

-ATTTGTTCAGTGGTTCGTAGGGCTT-3

⬘

-FL

S

688–712

Genetic-polymorphism site

T/G, nt 702; T/C, nt 705; A/C, nt 706

BEF-Anc probe

5

⬘

-LC Red 640-CCCCCACTGTCTGGCTTTCAG

TTATATGGATGAT-3

⬘

-PH

S

714–747

Set 2-2 (A/C/D/G)

关

Surface/polymerase

兴

481

Genotype CD, 66.3

A/CD/G

Forward primer

5

⬘

-CTATGGGAGTGGGCCTCAG-3

⬘

S

638–656

Genotype A, 62.6°C

Reverse primer

5

⬘

-AAAGGCCTTGTAAGTTGGCG-3

⬘

As

1119–1100

Genotype G, 57.8°C

CD-Sen probe

5

⬘

-CATCATCCATATAACTGAAAGCCAAACA

GTG-3

⬘

-FL

As

748–718

Genetic-polymorphism site

A/G, nt 735; A/G, nt 724

ACDG-Anc probe

5

⬘

-LC Red 640-GGGAAAGCCCTACGAACCAC

TGAACAAATGGC-3

⬘

-PH

As

716–685

Genotype D, 62.6°C

C/D

283

Forward primer

5

⬘

-CGCTGGATGTGTCTGCG-3

⬘

S

371–387

Genotype C, 57.6°C

Reverse primer

5

⬘

-GAGGCCCACTCCCATAGG-3

⬘

As

654–637

D-Sen probe

5

⬘

-CAACATACCTTGATAGTCCAGAAGA-3

⬘

-FL

As

466–442

Genetic-polymorphism site

A/G, nt 454

CD-Anc probe

5

⬘

-LC Red 705-CCAACAAGAAGATGAGGCAT

AGCAGCAGGATGAAGAGG-3

⬘

-PH

As

440–403

a_{S, sense; As, antisense.}

b_{The nucleotide position is based on the HBV genotype B sequence (NCBI accession no. AB106884).}

c_{⌬Tmis the difference between the melting temperatures of two groups or genotypes.⌬Tm}

BEF/ACDG⫽5.5°C⫾0.3°C,⌬TmB/E⫽3.1°C⫾0.2°C,⌬TmE/F⫽2.0°C⫾

0.05°C,⌬TmB/F⫽5.1°C⫾0.2°C,⌬TmCD/A⫽3.7°C⫾0.3°C,⌬TmA/G⫽4.8°C⫾0.2°C,⌬TmCD/G⫽8.3⫾0.3°C, and⌬TmC/D⫽5.0°C⫾0.2°C.

V

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QUANTIFICATION AND GENOTYPING OF HBV BY REAL-TIME PCR

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plasmid HBV) was linear in a range from 10

2

_{to 10}

13

_copies/ml

(Fig. 2). Reproducibility as measured by dual testing of

tripli-cates 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

quantifica-tion and the Amplicor HBV Monitor test (Roche), with an

R

2

value of 0.93 and a gamma of

⬎

0.80 (Fig. 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).

Genotyping by MCA.

In an initial evaluation of the

geno-typing, 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

T

m

, which was

56.7°C for genotypes A, C, D, and G and 62.6°C for genotypes

B, E, and F (Fig. 3A). With the set 2-1 amplicon (the B/E/F

set), samples of genotypes B, E, and F could be further

clas-sified with

T

m

characteristics of 66.5°C, 63.4°C, and 61.4°C for

genotypes B, E, and F, respectively (Fig. 3B). With the set 2-2

amplicon (the A/CD/G set and the C/D set), using two

chan-nels (F2 and F3), identification of genotypes A, C, D, and G

could be conducted with

T

m

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,

respec-tively, in the F3 channel (Fig. 3C). The

T

m

differences in each

set were significant enough for clear differentiation of these

genotypes (Fig. 1A).

After confirming the reproducibility of the amplifications

and melting point analyses, with few cases showing a

T

m

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

genotyp-ing 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 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

poly-FIG. 2. Quantification of the virus by real-time PCR with set 1. (A) Linear regression of the standards ranging from 10

2

_{to 10}

13

_{copies/ml was}

determined by using the “second derivative maximum” method. The standard curve showed a regression coefficient,

r

, of

⫺

1.00; a mean squared

error of 0.138; an intercept of 47.03; and a slope of

⫺

3.293. (B) Comparison of the quantification assay with the Roche Amplicor assay for HBV

quantification.

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[image:4.585.117.474.71.430.2]

morphisms at nt 225 and nt 706.

T

m

shift from expected values

was relatively common, particularly in genotype C, and

se-quencing showed that this was due to 1- or 2-nucleotide

vari-ations 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%).

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

T

m

shift. However, the correct genotype

could be identified by type-specific multiplex PCR in the

ma-jority of these cases (94.44%) (Table 2).

Detection of HBV mixed infections with melting curve

anal-ysis.

Although there were no cases of mixed-genotype infection

among the samples, the power of the set 1 amplicon in

detect-ing mixed viral infections was evaluated. By mixdetect-ing two

plas-mids corresponding to genotypes B and G with various

pro-portions of mixtures ranging from 1:1 to 1:9 and a total

concentration of 10

8

_{copies/ml, the detection sensitivity was}

10% for the minor population (Fig. 4).

DISCUSSION

[image:5.585.101.487.70.508.2]

Here, we present a new method for large-scale HBV

geno-typing and simultaneous quantification of viremia. The method

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 genotypes with the

set 2-1 amplicon. (C) Differentiating genotypes A, C, D, and G into single genotypes with the set 2-2 amplicon. In addition, the representative

results of each genotype (left), and the results of many clinical samples (right) are shown. The melting temperature for each genotype is indicated

with a vertical line.

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QUANTIFICATION AND GENOTYPING OF HBV BY REAL-TIME PCR

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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

geno-types 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

iden-tifying 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

reac-tion capillaries, particularly if larger sets of samples are

geno-typed, for example, in clinical studies. If amplification is

in-stead 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

ex-tension 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

simulta-neous quantification, performed with the set 1 amplicon

amplification, which targets conserved genomic segments.

Pre-viously, a similar method (16) for simultaneous HBV

quanti-fication 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

ac-curate for all genotypes, this needs to be confirmed by further

analysis of reproducibility panels and clinical samples. An

ad-TABLE 2. Comparison between HBV genotypic analysis using

two-step melting curve analysis with different amplicons and PCR-RFLP

HBV genotyping method Accuracy (%)

Two-step genotypic analysis

Step 1

Differentiation into two genotype groups (A, C,

D, G) and (B, E, F) with set 1 amplicon

a

_{...99.1 (437/441)}

Step 2

Differentiating genotypes B, E, and F with

set 2-1 amplicon

a

_{...97.0 (223/230)}

Differentiating genotypes A, C, D, and G

with set 2-2 amplicon

a

_{...87.9 (174/198)}

Differentiating genotypes A, C, D, and G with

alternative type-specific multiplex PCR ...94.4 (187/198)

Combination of step 1 and step 2 for

differentiating all genotypes

Two-step melting curve analysis with set 1

and set 2-1/set 2-2 ...92.3 (407/441)

Using alternative type-specific multiplex PCR

instead of melting curve analysis with set 2-2 ...98.4 (434/441)

PCR-RFLP

b

_{...87.0 (369/424)}

a_{These amplicons were used for melting curve analysis.}

[image:6.585.43.284.90.281.2]

b_{The PCR-RFLP method was designed by Lindh et al. (7).}

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

indicated.

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[image:6.585.125.460.530.703.2]

vantage 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

geno-types A to G suitable for research and clinical diagnostics in all

regions of the world.

ACKNOWLEDGMENT

The study was supported by grants from the National Science

Coun-cil, Taiwan, Republic of China (NCS 95-2314-B-006-030).

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