A Genotype-Independent Real-Time PCR Assay for Qua

Copyright©2007,American Society for Microbiology.All Rights Reserved.

A Genotype-Independent Real-Time PCR Assay for Quantification of

Hepatitis B Virus DNAᰔ

Ying Liu,Munira Hussain,Stephen Wong,Scott K.Fung,Hyung Joon Yim,and Anna S.F.Lok* Division of Gastroenterology,University of Michigan Medical Center,Ann Arbor,Michigan

Received4April2006/Returned for modification14June2006/Accepted22November2006

Accurate quantification of hepatitis B virus(HBV)DNA levels is important for monitoring patients with

chronic HBV infection and for assessing their responses to antiviral therapy.This study aimed to develop a

real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the

known HBV genotypes.An“in-house”real-time PCR assay using primers and a TaqMan probe in a highly

conserved region of the HBV surface gene was designed.The assay was standardized against a WHO standard

and validated against plasmids of HBV genotypes A through H.The linear quantification range was approx-

imately5؋100to2.0؋109IU/ml.Results of samples from patients infected with HBV genotypes A through

H tested using our real-time“in-house”PCR assay showed an excellent correlation with those of the Cobas

Amplicor HBV Monitor(R2؍0.9435)and the Cobas TaqMan HBV(R2؍0.9873)tests.We have established

a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA

levels.Further studies of additional samples are ongoing to validate the genotype independence of our assay.

The availability of PCR assays that can detect copy numbers

as low as1,000copies/ml of hepatitis B virus(HBV)DNA led

to the recognition that HBV persists,albeit at low levels,

throughout the course of chronic HBV infection(6,15,16,21).

The level of viremia is highly variable among HBV carriers,

and serum HBV DNA levels within the same person canfluc-

tuate from undetectable to more than10log

10IU/ml.Quan-

tification of serum HBV DNA levels is important to determine the level of viral replication,to assess antiviral response,and to monitor antiviral resistance.However,HBV DNA assays used in clinical practice were not calibrated to WHO standards until recently,and the lower limit of detection as well as the range of linearity varies from one assay to another(5,19).

The Amplicor assay is the most widely used commercial PCR assay for quantifying HBV DNA.The automated version, Cobas Amplicor HBV Monitor test,has a lower limit of de-tection of60IU/ml but a limited range of linearity,up to 38,000IU/ml,based on the manufacturer’s package insert. Given that many patients with chronic hepatitis B,particularly those who are hepatitis B e antigen(HBeAg)positive,have HBV DNA levels that are109IU/ml or higher,retesting serial dilutions up to1:1,000,000may be necessary.Thus,the possi-bility of errors in quantifying very high HBV DNA levels is tremendous.In addition,the Cobas Amplicor HBV Monitor test,like other conventional PCR assays,measures the amount of input DNA at the end of the amplification reaction and is, therefore,subject to errors caused by a“plateau”effect(3). Various assays utilizing real-time PCR technology have been recently developed to quantify HBV DNA(1,4,18,24,26,27). Real-time PCR technology is based on the evaluation of a threshold cycle when the amplification products are detected for thefirst time.It is more precise than conventional PCR because results are obtained when the PCR is still in the exponential phase(10,11).Many real-time PCR HBV DNA assays were developed in-house and have not been standard-ized or validated(1,4,18,27).The Cobas TaqMan HBV test, the most commonly used commercial real-time PCR HBV DNA assay,has a lower limit of detection of6IU/ml and a range of linearity up to780,000,000IU/ml,based on the man-ufacturer’s brochure.This assay is fully automated and incor-porates an internal quantification standard,but it is expensive (8,23,25).

There are eight recognized HBV genotypes,designated A to H,based on a nucleotide difference ofϾ8%in whole-genome sequencing(2,7).Discrepancies in quantifying the hepatitis C virus(HCV)RNA in samples from patients infected with var-ious HCV genotypes have been reported(9,17,20,22).Data for the performance of HBV DNA assays across HBV geno-types are scanty.

Here we describe the development and evaluation of a TaqMan-based real-time in-house PCR assay validated for HBV genotypes A through H.The lower limit of detection and the dynamic range of the assay were determined,and the performance results of this assay of clinical samples was com-pared to those of two commercially available assays,Cobas Amplicor HBV Monitor test and Cobas TaqMan HBV test.

MATERIALS AND METHODS

DNA extraction,PCR amplification,and detection.A QIAamp DNA blood minikit(QIAGEN,Valencia,CA)was used to extract nucleic acids.Two hun-dred microliters of serum was used,and DNA was eluted in50␮l of DNase/ RNase-free water and stored atϪ20°C until used.Real-time PCR was performed in a50-␮l reaction mixture containing25␮l TaqMan Universal PCR master mix (Applied Biosystems)with0.2␮M primers,0.1␮M probes,and10␮l extracted DNA.An initial2-min incubation at50°C to activate uracil-N-glycosylase and to destroy potential amplicon carryover was followed by a10-min incubation at 95°C,which activates the thermostable DNA polymerase and denatures the uracil-N-glycosylase.Subsequently,50cycles of95°C for15s and60°C for1 min were performed with an iCycler iQ multicolor real-time PCR detection

*Corresponding author.Mailing address:Division of Gastroenter-ology,University of Michigan Medical Center,1500East Medical Center Dr.,3912Taubman Center,Box0362,Ann Arbor,MI48109-0362.Phone:(734)615-4628.Fax:(734)936-7392.E-mail:aslok

@umich.edu.

ᰔPublished ahead of print on20December2006.

553system(Bio-Rad Laboratories,Hercules,CA)and the results analyzed with sequence detector software(version3.0)according to the manufacturer’s instructions.

The primers and afluorogenic5Ј-end nuclease probe(TaqMan)were selected from a highly conserved region of the S gene after careful analysis of27full-length HBV sequences belonging to genotypes A through H deposited in the GenBank database and synthesized and purified by Integrated DNA Technolo-gies(Coralville,IA).Comparisons of the sequences of the primers and probe to consensus sequences of each genotype are shown in Fig.1.

Preparation of plasmids and calibration of the in-house real-time PCR assay against a WHO standard and plasmids of HBV genotypes A through H.DNA extracted from200-␮l serum samples from patients who were previously deter-mined to be infected with HBV genotypes A through H and DNA from a WHO international HBV DNA standard(NIBSC code97/746,genotype A,serotype adw2)were PCR amplified.The PCR was conducted with a50-␮l reaction mixture containing AmpliTaq Gold DNA polymerase(0.25U;Applied Biosys-tems,Foster,CA)and buffer,MgCl2(5mM),800nM of PCR nucleotide mix(10 mM of each deoxynucleoside triphosphate)(Promega,Madison,WI),200nM

of FIG.2.Calibration of the HBV genotype A plasmid DNA against that of the WHO HBV international standard.Amounts of WHO HBV standard used in each reaction were20,2ϫ102,2ϫ103,2ϫ104,2ϫ105,and2ϫ106IU/ml.(A)The threshold cycle was plotted against input HBV DNA concentrations (standard curve).(B)Standard curve obtained from serial dilutions of HBV genotype A plasmid:5,10,20,2ϫ103,2ϫ105,2ϫ107,and2ϫ109

IU/ml. FIG.1.Comparison of real-time PCR TaqMan probe and primer sequences with GenBank database sequences for HBV genotypes(accession numbers X02763,X51970,and AF090842),B(accession numbers D00329,AF100309,AB033554,D00330,and AB073858),C(accession numbers X04615,AB014381,M12906,and AB033556),D(accession numbers X65259,M32138,X85254,and X02496),E(accession numbers X75657and AB032431),F(accession numbers X69798,AB036910,and AF22396),G(accession numbers AF160501,AB0310,and AF405706),and H (accession numbers AY090454,AY090457,and AY090460).

554LIU ET AL.J.C LIN.M ICROBIOL.

each primer,and 5␮l of DNA extract.Thermal cycling was performed using the following conditions:an initial incubation at 95°C for 10min and then 45cycles of 95°C for 30s,55°C for 30s,and 72°C for 60s.The amplified region corre-sponded to a 681-bp region of the S gene (nucleotide [nt]15to 2244;genotype A,serotype adw2).The PCR products were cloned into a pGEM-T-Easy vector (Promega,Madison,WI).The recombinant plasmids were purified,and the concentration of HBV DNA was determined using a spectrophotometer.

Serial dilutions of the WHO standard,with concentrations ranging from 20to 2.0ϫ106IU/ml,were tested with the real-time PCR assay to obtain a standard curve.Subsequently,serial dilutions of HBV S gene plasmid DNA from a genotype A sample with concentrations ranging from 2.0ϫ102to 2.0ϫ105IU/ml were calibrated against the WHO standard.Finally,serial dilutions of the WHO standard and S gene plasmids of HBV genotypes A through H with concentrations ranging from 2.0ϫ102to 2.0ϫ107IU/ml were tested in parallel with the real-time PCR assay to compare the amplification efficiencies across these genotypes.

Patients and serum samples.A total of 169serum samples collected from patients with chronic HBV infection seen in the Liver Clinics at the University of Michigan Hospital were studied.These patients were participants in one of three studies:the natural history of chronic HBV infection,antiviral resistance sur-veillance,and prevention of recurrent HBV infection after liver transplantation.All three studies were approved by our institutional review board,and all pa-tients provided written informed consent for sample collection.Sera were di-vided into aliquots and stored at Ϫ70°C until used.

HBV DNA quantification.All samples were tested using our in-house real-time PCR assay,without knowledge of the results of commercial assays.Each assay included the following:HBV S plasmid standards (genotype A,concentrations of 5ϫ100to 2.0ϫ109IU/ml),two human serum samples that had previously tested positive for HBV DNA using a Cobas Amplicor HBV Monitor test (positive controls),water,and two serum samples from healthy subjects who were HBsAg negative (negative controls).

All samples were also tested with a Cobas Amplicor HBV Monitor version 2(Roche Molecular Diagnostics,Branchburg,NJ),which has a linear dynamic range from 60to 38,000IU/ml.All samples with values of Ն38,000IU/ml were diluted 1:1,000to 1:100,000to obtain a valid result.Forty-nine samples collected between June and December 2005were further tested using a Cobas TaqMan HBV test (Roche Molecular Diagnostics,Branchburg,NJ),which has a linear dynamic range from 6to at least 780,000,000IU/ml.

HBV genotyping.The HBV genotypes of samples with HBV DNA detectable by a Cobas Amplicor Monitor assay were determined using a line probe assay,INNO-LiPA HBV Genotyping (Innogenetics NV,Ghent,Belgium),as described previously (14).

Statistical analyses.iCycler iQ TM sequence detector software (version 3.0)(Bio-Rad Laboratories,Hercules,CA)was used to determine real-time PCR results.Precision was calculated with the use of JMP statistical software (SAS Institute,Cary,NC).Assay reproducibility was assessed by the coefficient of variation (CV).Pearson’s correlation coefficient was used to assess the linear regression between the log-transformed values of HBV DNA using our in-house real-time PCR assay and Cobas Amplicor and Cobas TaqMan assays.

RESULTS

Serial dilutions of the WHO HBV DNA standard were tested along with serial dilutions of HBV genotype A plasmid DNA.The amplification efficiency of the HBV genotype A plasmid DNA was the same as that of the WHO HBV DNA standard,as judged by the identical slope of the superimposed calibration curves (Fig.2A).Triplicate testing confirmed these results,enabling us to use our panel of genotype A plasmid DNA dilutions,ranging from 5ϫ100to 2.0ϫ109IU/ml,as standards for future assays.

To determine the range of linearity,eight duplicate serial dilutions from the HBV genotype A plasmid DNA standard,with concentrations from 1ϫ100to 2.0ϫ109IU/ml,were tested.A linear relationship was observed over the range of 5ϫ100to 2.0ϫ109IU/ml (Fig.2B).To determine the lower limit of detection,DNA was extracted from HBV genotype A plasmid at concentrations of 1,2,5,10,20,and 200IU/reaction on four occasions,and each DNA extract was tested in dupli-cate in three different assays.Plasmid DNA concentrations above 5IU/ml were detected on all 24occasions,and concen-trations at 5IU/ml were detected on 20of 24occasions.

The precision of our in-house real-time PCR assay was de-termined by assessing the intra-and interassay CV.For in-traassay variability,serum samples from 12patients with chronic HBV infection including virus genotypes A through H and two samples from healthy subjects who were HBsAg

neg-

FIG.3.Coamplification of plasmids of HBV genotypes A through H and the WHO standard at dilutions of 20,2ϫ103,2ϫ105,and 2ϫ107IU/ml.Correlation coef ficient,0.995;PCR ef ficiency,111.9%.Genotype A,azure;B,pink;C,crimson;D,blue;E,purple;F,yellow;G,red;H,grass green;WHO standard,green.

V OL .45,2007GENOTYPE-INDEPENDENT REAL-TIME PCR ASSAY FOR HBV DNA 555

ative were tested in triplicate in the same assay.The mean CV was 0.082(range,0.051to 0.099).For interassay variability,DNA was extracted from each of 10serum samples on three separate occasions.Each DNA extract was tested in duplicate in three different PCR assays.The HBV DNA values were found to be normally distributed on a logarithmic scale.The mean CV for all 10samples tested was 0.16(range,0.036to 0.216).

To determine whether plasmid HBV DNA can be used as the standard for testing serum samples,we tested three dilu-tions of the WHO standard in human serum at concentrations of 3ϫ106,3ϫ104,and 3ϫ102IU/ml,three serum samples from HBV patients that had the same HBV DNA concentrations based on Cobas Amplicor HBV Monitor test results,and three serum samples,which were spiked with the same concentra-tions of HBV genotype A plasmid DNA,from HBsAg-nega-tive subjects.DNA was extracted and HBV DNA quantified with our in-house real-time PCR assay using HBV genotype A plasmid DNA as the standard.DNA extraction from serum samples versus that from plasmids had a negligible effect on the results.

To determine if our in-house real-time PCR assay is equally accurate in quantifying HBV genotypes A through H,HBV genotype A through H plasmid DNA at concentrations from 2ϫ101to 2.0ϫ107IU/reaction were coamplified with the WHO standard.Figure 3shows that amplification efficiencies were comparable for these eight genotypes.We then tested 32patient samples (HBV genotype A,5samples;B,5samples;C,5samples;D,5samples;E,3samples;F,2samples;G,2samples;and H,1sample)with our in-house real-time PCR assay using genotype A through D plasmids as standards.Fig-ure 4shows that the results of samples from all eight genotypes (A through H)were similar regardless of which genotype (A through D)plasmid was used as the standard.These results indicate that the genotype A plasmid can be used as the stan-dard for testing patient samples with other HBV genotypes.Forty-nine samples (HBV genotype A,9samples;B,6sam-ples;C,9samples;D,8samples;F,2samples;and not avail-able,15samples)previously tested by a Cobas TaqMan HBV

test were retested using our in-house real-time PCR assay.Correlation between the two assays was excellent,with an R 2value of 0.9873over the shared 9log 10dynamic range (Fig.5).All 46samples with detectable HBV DNA according to the Cobas TaqMan HBV test results had detectable values in our real-time PCR assay.Similarly,none of the three samples with undetectable HBV DNA in the Cobas TaqMan HBV test had detectable values in our assay.Of the eight samples with HBV DNA values of Ͼ7log 10IU/ml determined with the Cobas TaqMan HBV test results,our in-house real-time PCR assay reported values that were within 0.5log 10IU/ml in six samples,a 0.5-to 1.0-log 10-IU/ml-lower value in one sample,and a 0.5-to 1.0-log 10-IU/ml-higher value in one sample.

One hundred sixty-nine samples (HBV genotype A,38sam-ples;B,20samples;C,35samples;D,25samples;E,8samples;F,7samples;G,6samples;H,1sample;and not available,29samples)previously tested by the Cobas Amplicor HBV Mon-itor were retested using our in-house real-time PCR assay.The overall correlation of the two assays was very good,with an R 2of 0.9435,regardless of HBV genotype (Fig.6),and the cor-relation was excellent for HBV DNA concentrations from 2to 5log 10IU/ml (R 2ϭ0.9754).Correlations between the

two

FIG.4.Comparison of HBV DNA levels of 32serum samples with HBV genotypes A through H tested with our in-house real-time PCR assay using DNA from plasmids of HBV genotypes A through D as

standards.

FIG.5.Correlation of HBV DNA values from 49serum samples measured by our in-house real-time PCR assay and by the Cobas TaqMan HBV test.

556LIU ET AL.J.C LIN .M ICROBIOL .

assays were comparable for all the genotypes,including geno-type F.Six of 13samples with undetectable HBV DNA ac-cording to the Cobas Amplicor Monitor test had detectable HBV DNA with our in-house real-time PCR assay,with values from 60to 70IU/ml (Table 1).All eight samples that had detectable but nonquantifiable results with the Cobas Ampli-cor Monitor test had quantifiable results with our in-house real-time PCR assay,with values from 25to 60IU/ml.Of the 16samples with HBV DNA results of Ͼ7log 10IU/ml as de-termined with the Cobas Amplicor Monitor test,our in-house real-time PCR assay reported values that were within 0.5log 10IU/ml in 12samples,a 0.5-to 1.0-log 10-IU/ml-lower value in 1sample and a Ͼ1-log 10-IU/ml-lower value in 3(19%)samples.

DISCUSSION

We have established a sensitive “in-house”real-time PCR assay for the quantification of HBV DNA,with a lower limit of detection of 5IU/ml and a dynamic range of up to 2.0ϫ109IU/ml.Our assay is reproducible,with limited intra-and inter-assay variability and similar amplification efficiencies across HBV genotypes A through H.We demonstrated that the re-sults of our assay correlated very well with the two commonly used commercial assays,with an R 2of 0.9435for the Cobas Amplicor HBV Monitor test and an R 2of 0.9873for the Cobas

TaqMan test.Our assay was 1log 10IU more sensitive and had a dynamic range of 5log 10IU wider than the Cobas Amplicor HBV Monitor test.Furthermore,our assay costs approxi-mately $60per sample,which is less than one-third of the charge for the Cobas TaqMan HBV test.

The availability of HBV DNA assays with an improved sen-sitivity and a wider dynamic range will improve our ability to assess viral response to antiviral therapy for chronic hepatitis B.One-step testing not only saves time but also eliminates the risk of errors introduced by the dilution process.The potential for errors when samples need to be diluted 100,000-fold or more is substantial.The tendency of the Cobas Amplicor HBV Monitor test to overestimate samples with high HBV DNA levels may explain why recent clinical trials of antiviral therapy for chronic hepatitis B reported baseline serum HBV DNA levels as high as 13log 10IU/ml (13,14).Falsely high pretreat-ment serum HBV DNA levels may inflate the potency of an-tiviral therapies.

A major problem with HBV DNA assays is the lack of standardization.This has prompted recommendations to stan-dardize HBV DNA assays against WHO or other international standards and to express results in international units (IU).Our assay was initially standardized against the WHO interna-tional HBV DNA standard (NIBSC code 97/746;genotype A,serotype adw2).However,the WHO standard was available only in concentrations of up to 2.0ϫ107IU/ml;therefore,we calibrated it against an HBV genotype A plasmid,which was used to establish a standard dilution panel with concentrations of up to 2ϫ109IU/ml for testing clinical samples.We showed that similar results were obtained for plasmid DNA or negative serum spiked with plasmid DNA,indicating that plasmid DNA can be used as the standard for testing patient samples despite potential differences in DNA extraction.Furthermore,we demonstrated that results were similar when a panel of geno-type A through H patient samples was tested using genotype A through D plasmids as standards,indicating that our genotype A plasmid can be used as a standard for serum samples from patients infected with various HBV genotypes.

Several studies in the 1990s reported that some PCR assays used to quantify HCV RNA do not have the same accuracy across all HCV genotypes (16–19).One study reported the validation of the Cobas TaqMan HBV test against plasmids of HBV genotypes A through G (12).In that study,57

HBsAg-

FIG.6.Correlation of HBV DNA values of 169serum samples measured by our in-house real-time PCR assay and by the Cobas Amplicor HBV Monitor test.

TABLE 1.Comparison of results of 169serum samples tested by our in-house real-time PCR assay and

the Cobas Amplicor HBV Monitor test

Concn detected by Cobas Amplicor HBV Monitor

(IU/ml)a

No.of serum samples in which HBV DNA was detected by in-house real-time PCR at indicated concn (IU/ml)Not detected

Ͻ60

60to 2ϫ102

2ϫ102to 2ϫ104

2ϫ104to 2ϫ107

2ϫ107to 2ϫ109

Ͼ2ϫ109

Not detected (n ϭ13)7

5b 1b Ͻ60(n ϭ8)

8

60to 2ϫ102(n ϭ8)

6

22ϫ102to 2ϫ104(n ϭ83)8132ϫ104to 2ϫ107(n ϭ41)2

392ϫ107to 2ϫ109(n ϭ11)2

9Ͼ2ϫ109(n ϭ5)

2

3

a n ,number of samples.

b

Six of 13samples with undetectable HBV DNA according to Cobas Amplicor Monitor results had detectable HBV DNA with our in-house real-time PCR assay,with values from 60to 70IU/ml.

V OL .45,2007GENOTYPE-INDEPENDENT REAL-TIME PCR ASSAY FOR HBV DNA 557

Our assay has similar amplification efficiencies across all known HBV genotypes,including genotype H;and intra-and interassay variabilities were also comparable across these eight genotypes. Correlation between the results of our assay and those of the Cobas TaqMan HBV test was excellent for the genotypes tested (A,B,C,D,and F).Unfortunately,due to a limitation in sample availability,we were not able to compare these two assays for HBV genotypes E,G,and H samples.Likewise,the correlation between our assay and the Cobas Amplicor HBV Monitor test was excellent across genotypes A through H for samples with results that fall within the dynamic range of the Amplicor test. Of the seven genotype F samples tested with our assay and with the Cobas Amplicor HBV Monitor test,all seven had values

within0.5log

10IU/ml in the two assays.Two of these samples

were also tested with the Cobas TaqMan HBV test,and results

were within0.5log

10IU/ml in all three assays.

There are limitations with our in-house assays.The lack of an internal control does not allow us to rule out false-negative results due to the presence of inhibitors to PCR amplification. The limit of detection and the upper limit of the dynamic range are approximates,as a lot more replicates and lot-to-lot testing would be necessary to verify these values.Sequence variations in the primer target and probe regions that are not present in the27sequences from our GenBank search can affect quanti-fication accuracy.There are also multiple challenges in maintain-ing quality control,including the prevention of cross-contamina-tion,the maintenance of a dilution panel calibrated against the WHO standard,and the validation of genotype independence as new genotypes and new variants within known genotypes are identified.

In summary,we have developed an in-house real-time PCR assay that is sensitive,has a wide dynamic range,and has comparable amplification efficiencies across all known HBV genotypes.Our assay results have excellent correlation with those of the Cobas TaqMan HBV test and the Cobas Amplicor HBV Monitor test,regardless of HBV genotypes.Further studies to test additional samples with more replicates to con-firm the genotype independence of our assay of clinical sam-ples are ongoing.

ACKNOWLEDGMENTS

This study was supported in part by NIH grant U01-DK57577(to A.S.F.L.).Cobas Amplicor HBV Monitor test kits were provided by Roche Molecular Diagnostics and Inno-Lipa kits for HBV genotype were provided by Innogenetics.

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