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Evaluation of a Novel Domestic Hepatitis B Viral DNA QuantitativeFluorescence Diagnostic Kit

SHEN Tao1, LONG Lu1, DENG Zhong-ping2, Wang Jie1, DAI Li- zhong2, CHEN De-xi3, TAN De-ming4,

ZHUANG Hui1 and LU Feng-min1*

Department of Pathogen Biology, School of Basic Medicine, (former Infectious Diseases Research Center),

Peking University, Beijing, P. R. China1; Sansure Biotech Inc. Changsha, Hunan Province, P. R. China 2; Beijing

YouAn Hospital, Capital Medical University, Beijing, P. R. China 3; XiangYa Hospital, Central South University,

Changsha, Hunan Province, P. R. China4

*Correspondeing author, E-mail: lufengmin@hsc.pku.edu.cn

Chin J Lab Med, 2013,36:280-285【Abstract】Objective A comparative study was performed between a novel domestic HBV DNA

quantitative fluorescence diagnostic kit (Sansure) and its counterpart Roche Cobas Ampliprep/COBAS

Taqman HBV test kit in order to assess their performance characteristics. Methods Firstly, WHO

international standard for HBV-DNA was diluted to 50, 200, 2000, 20 000 IU/ml separately, and then all

diluted samples were detected by both the novel domestic HBV DNA quantitative fluorescence diagnostic

kit and Roche Cobas Ampliprep/COBAS Taqman HBV test kit to perform traceability analysis and to

determine kits’ reliability. Secondly, pre-diluted series of standard HBV B and C genotype plasma (25, 50,

200, 2000, 20 000, 200 000 IU/ml) were simultaneously performed 216 times for three-center detection by

use of the domestic and Roche HBV DNA test kits separately. Accuracy and precision of those two types of

HBV DNA kits were comparatively analyzed. Finally, a total of 42 clinical plasma samples including 5

negative HBV DNA and different concentration levels of 37 positive HBV DNA were detected by the above

two types of kits to perform clinical quality evaluation. Results Traceability results showed that both

HBV DNA test kits agreed with the HBV DNA WHO standard across all four titers tested and all absolute

differences (observed mean HBV HBV-expected HBV DNA) were within 0.3 log IU/ml. Accuracy results

indicated that the deviation of all observed values at 6 titers (both of HBV genotyping B and C) tested were

within the acceptance criteria (0.005-0.280 lg IU/ml). Comparative performance of coefficient of variation of

PCR assays to HBV genotypes B and C measured by both kits showed that there were no differences of

precision found ( genotyping B: t = 1.226, P = 0.275; genotyping C: t = 2.319, P = 0.07). The clinical

performance of the domestic assay compared to the COBAS assay had been assessed on a panel of 42

clinical specimens. The qualitative results indicated that the total concordant results between two tests were

determined in 97.62% (41/42) of samples. Also, a significant correlation was observed between values tested

by two HBV DNA kits(R2= 0.934, P < 0.0001). Conclusion No significant differences of the traceability,

accuracy, precision and clinical detection are found between two kits. （Chin J Lab Med, 2013,36:280-285)

【Key words】Hepatitis B virus; DNA, viral; Reagent kits, diagnostic; Evaluation studies

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This research was funded by the following projects: (1) Research on Hepatitis B and C Diagnosis and

Clinical Monitoring, “12th Five-year Plan” special project, the Prevention and Treatment of Major Infectious

Diseases Including AIDS and Viral Hepatitis （2012ZX10002005); (2) the “863 Project”, Highly Sensitive

Test Kits and Genome-Based Diagnostic and Therapeutic Technology, the “12th five-year plan” of the

National Ministry of Science and Technology（2012AA022605）.

HBV infection is one of the major causes of cirrhosis and hepatocellular carcinoma.[1-2] The HBV DNA

present in HBV-infected human plasma (serum）is an important indicator for understanding HBV replication

in liver, diagnosing HBV infection, monitoring efficacy and providing proper treatment. Recently,

although domestic quantitative test kits for HBV DNA detection have been dramatically improved,

compared to imported test kits, a wide gap still exists in terms of sensitivity, accuracy and reproducibility,.

Major international HBV DNA quantitative test kits such as those made by Roche have a detection limit of

12-20 IU/ml; while main domestic HBV DNA quantitative test kits mostly based on a boiling method for

nucleic acid extraction have a detection limit in the range of 500 - 1000 IU/ml and cannot meet the needs of

clinical diagnostic. With the support from national projects for prevention and control of infectious diseases

included in the 11th and 12th Five-Year Plans, domestic HBV DNA quantitative test kits based on

magnetic-bead method for nucleic acid extraction have got mainstream research support. In this study, we

evaluated the novel HBV DNA quantitative fluorescence diagnostic kit from the Sansure Biotech Inc.

(hereinafter referred as “novel quantitative test kit”) in the aspects of traceability, accuracy, precision and

clinical quality. The evaluation samples included WHO HBV DNA standard, standard HBV B genotype

plasma from the Clinical Laboratory Center of the Ministry of Health of China , standard HBV C genotype

plasma from National Institutes for Food and Drug Control, and the clinical serum panel established by our

laboratory for HBV DNA quantitative test kit evaluation during the 11th Five-Year Plan.

Materials and MethodsMaterialsWHO HBV DNA standard (genotype A2)

TheWHO standard for HBV DNA quantification (code 10/264) was purchased from the National

Institute for Biological Standards and Control (NIBSC). The standard has HBV concentration of 850 000

IU/ml,and 0.5 ml, stored at -70℃. It is re-suspended with sterilized double distilled DNA-free water at 4℃

before use. This WHO HBV standard was diluted to 50, 200, 2000 and 20 000 IU/ml (4 key concentrations

in HBV antiviral treatment) separately with HBV-negative plasma containing EDTA anticoagulant.

Standard HBV B genotype plasma

The HBV B standard with catalog number GBW09150 was provided by the Clinical Laboratory Center

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of theMinistry of Health at no cost with concentration at 1.2×106 IU/ml, stored at -70℃. It was re-thawed by

sterilized double distilled DNA-free water at 4℃ before use. A 0.25 ml of re-suspended standard was added

to 14.75 ml of negative plasma and mixed to achieve 20 000 IU/ml. The standard plasma was further diluted

with negative plasma to make 25, 50, 200, 2000, 20 000, 200 000 IU/ml HBV B plasma.

Standard HBV C genotype plasma

The HBV C standard of with concentration at 2×108 IU/ml was provided by the National Institutes for

Food and Drug Control at no cost and stored at -70℃. After suspended with sterilized water, 0.1 ml of

standard was added to 0.9 ml negative plasma and mixed to achieve 2×107 IU/ml HBV C genotype plasma.

The standard plasma was further diluted with negative plasma to achieve 25, 50, 200, 2000, 20 000, 200 000

IU/ml HBV C plasma.

Negative plasma for dilution

The negative plasma was both HBsAg and anti-HBc negative, and contained EDTA anticoagulant.

Clinical serum panel

Total 42 clinical serum samples were tested, which included 5 HBV DNA negative serum samples

(HBsAg and anti-HBc negative) and 37 HBV DNA positive serum samples (detected positive by Abbott

RealTime HBV Test) with different HBV titers. This serum panel was established by our laboratory for HBV

DNA quantitative test kit evaluation during the 11th Five-Year Plan.

Roche quantitative test kit

Roche COBAS AmpliPrep device and Roche HBV DNA test kit were used to process samples

automatically. COBAS Taqman 48 device was used to automatically amplify and detect nucleic acid. The

Roche quantitative test kit has a detection limit of 16.4 IU/ml for HBV DNA detection. Threshold setting: if

the threshold cycle (Ct) is greater than the upper limit of detection or Ct value is not detected, then it is HBV

DNA negative; if detected < 20 IU/ml, it is HBV DNA positive and < 20 IU/ml; if detected ≥ 20 IU/ml

and ≤ 1.7 ×108 IU/ml, it’s in the detection linear range; if it is detected ≥1.7×108 IU/ml , the sample needs

to be diluted further until it falls in the linear range.

The novel quantitative test kit

With the novel Sansure HBV test kit, HBV DNA in serum samples was extracted with automatic

nucleic acid extraction platform using magnetic-bead technology and quantified using fluorescence

quantitative PCR device. HBV DNA internal control with HEX-labeled TaqMan probe was used to monitor

the presence of PCR inhibitors in samples to prevent false negative results. UNG + dUTP

contamination-proof system was used to avoid a false positive result. The ROX (passive reference) dye was

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also used in the kit to eliminate variations among tubes to ensure accuracy of quantification. For samples

detected between 20 IU/ml and 2.0×109 IU/ml, the copy number results are reported directly. For samples

detected > 2×109 IU/ml, it is reported as > 2.0×109 IU/ml. If more accurate quantification is required, the

sample needs to be diluted 1000 times and tested again. For samples detected ≥10 IU/ml and <20 IU/ml, if

internal control is also detected positive with Ct≤40, it indicates a low viral load and the result is for

reference only. For samples detected < 10 IU/ml with positive internal control (Ct≤40) , it’s reported that

HBV DNA is lower than the detection limit of the quantitative test kit..

MethodsTraceability evaluation

The WHO HBV DNA standard was used to evaluate the traceability of the novel quantitative test kit

and Roche HBV DNA test kit. WHO standard was diluted with HBV negative plasma to 50, 200, 2000 and

20 000 IU/ml, which are the 4 key concentrations for HBV antiviral treatment. One reaction of each diluted

sample was tested on one device each day (including negative control tubes). All diluted samples were tested

total 3 times for each concentration in 3 days.

Accuracy and precision evaluation

HBV negative plasma was used to dilute standard plasma with HBV B and C genotype to 25, 50, 200,

2000, 20 000 and 200 000 IU/ml. One reaction of the 6 diluted samples was tested on one device each day.

All diluted samples were tested total 3 times for each concentration in 3 days. These tests were conducted at

three different test centers, including the Department of Microbiology of Peking University, Beijing Youan

Hospital of Capital Medical University and Xiangya Hospital of Central South University,to evaluate the

accuracy and precision of the two test kits. The accuracy was evaluated according to the relative deviation at

all detection points (the mean logarithmic value of detected value - the logarithm of theoretical value) which

should be within the accepted range of ± 0.3 log10 IU/ml. The precision was evaluated according to the

coefficient of variation (CV) from 9 parallel detections at the same detection point. The less the CV is, the

more precise it is.

Clinical serum panel evaluation

Roche HBV DNA test kit and the novel quantitative test kit were used to test the clinical serum panel to

evaluate the concordance and correlation between the two kits.

Sequencing and evolutionary tree analysis

With the Sansure HBV quantitative test kit, magnetic-bead technology was used to extract HBV DNA

in plasma and nested PCR was used to amplify HBV genomic fragment. The sequence of the outer forward

primer is: 5’-GCTTTGGGGCATGGACATTGACCCGTATAA- 3’, and the outer reverse primer is

5’-CTGACTACTAATTCCCTGGATGCTGGGTCT-3’. The sequence of the inner foward primer is

5’-GACGAATTCCATTGACCCGTATAAAGAATT-3’, and the inner reverse primer is 5’ - ATGGGA

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TCCCTGGATGCTGGGTCTTCCAAA-3’. The evolutionary tree was analyzed using the MEGA5 software.

Neighbor-jioning method and Kimura method were used to build model. Bootstrap was used to repeat the

verification 1000 times. The HBV sequences of different genotypes from GenBank were used as standards,

including HB-J1444AF for genotype A;, AH2 for genotype B; HCC-2-NY, TRF08111, MRK89073 and

NAB1 for genotype C; GRS08457 for genotype D;, HB-J1411F for genotype E;, HBV - BL592 for genotype

F;, HB-J1444GF for genotype G;, and MEX912M for genotype H.

Statistical Analysis

Graphpad prism was used for statistical analysis. The mean value of measurement data, variance,

standard deviation and CV are verified by the use of non-parametric and paired-t tests. The correlation

between the two kits was analyzed using Spearman rank correlation coefficient test. If P <0. 05, the

difference is statistically significant.

ResultsTraceability Analysis Results

In Table 1, it is shown that when using the novel quantitative test kit and Roche quantitative test kit to

test WHO HBV DNA diluted standards, the difference between the detected log10 value at the 4 detection

points of 50, 200, 2 000 and 20 000 IU/ml and the theoretical log value of WHO HBV DNA standard is

within ± 0.3 log10IU/mL (from -0.121 to -0.271 log10IU/ml). This shows that the difference in traceability

between the two diagnostic kits is not statistically significant (t = 0.8639，P > 0.05).

Table 1 Traceability Analysis of the WHO HBV DNA Standard measured by the 2 HBV DNAQuantitative Test Kits

Note: (log10 (detected value) - log10 (theoretical value)) < ±0.3 lg IU/ml is within accepted range.

Accuracy Analysis

The novel quantitative test kit (Sansure) and Roche quantitative test kit were used to test standard

plasma in order to evaluate the two kits' accuracy. As shown in Table 2: for standard genotype B and C

Name of test kitTheoretical value(IU/ml)

Theoreticalvalue(log10 IU/ml)

Detected value( log10 IU/ml)

Relativedeviation( log10 IU/ml)

Standarddeviation( log10IU/ml)

The novel quantitative testkit (Sansure)

50 1.699 1.524 -0.176 0.207

200 2.301 2.119 -0.182 0.205

2000 3.301 3.126 -0.175 0.095

20 000 4.301 4.145 -0.156 0.103

Roche quantitative test kit

50 1.699 1.579 -0.121 0.029

200 2.301 2.056 -0.245 0.092

2 000 3.301 3.030 -0.271 0.131

20 000 4.301 4.135 -0.166 0.202

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plasma, the relative deviation (mean value of detected value - theoretical value) of all detection points

between the two kits was within ±0.3 log10 (IU/ml) (0.005 - 0.28 log10 IU/ml), see Figure 1. This suggested

that the test results of the novel quantitative test kit were very stable with a relative deviation within ± 0.1

log10 IU/mL. Although Roche quantitative test kit’s test results are within the accepted range, the standard

genotype B serum had a relative deviation essentially within 0.2 - 0.3 log10 IU/ml. For standard genotype C

plasma (Figure 1b), the accuracy of Roche test kit was better than that for standard genotype B plasma

(Figure 1b), but less stable than the novel quantitative test kit.

Figure 1 Relative deviation analysis of standard HBV genotype B and C plasma samples measured with

Roche and Sansure HBV test kits.

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Table 2 Accuracy and precision analysis of the standard HBV genotype B and C plasma measured with two test kits

HBVgenotype

Test kits to beevaluated

Theoreticalvalue(IU/ml)

Theoreticalvalue(log10IU/ml)

Testtimes

(log10IU/ml)Detectedvalue

Detectedvalue(IU/ml)

Relativedeviation(log10 IU/ml)

Variance (standarddifference）(log10 IU/ml)

LognormalCV%

HBVgenotypeB

Sansure HBVDNA Test Kit(PCR-Fluorescen-ce Probing)

25 1.398 9 1.362 23.7 -0.036 0.013 (0.114) 8.3850 1.699 9 1.704 52.5 0.005 0.017 (0.130) 7.61200 2.301 9 2.341 226 0.04 0.012 (0.110) 4.742000 3.301 9 3.361 2350 0.06 0.009 (0.097) 2.7020 000 4.301 9 4.342 22 600 0.041 0.012 (0.108) 2.49200 000 5.301 9 5.328 216 000 0.027 0.006 (0.079) 1.49

Roche HBVDNA Test Kit(COBAS 2.0)

25 1.398 9 1.642 46.9 0.244 0.027 (0.167) 10.1750 1.699 9 1.945 88.7 0.246 0.003 (0.054) 2.80200 2.301 9 2.491 317 0.19 0.011 (0.106) 4.272000 3.301 9 3.518 3320 0.217 0.003 (0.056) 1.5920 000 4.301 9 4.581 38400 0.28 0.003 (0.059) 1.28200 000 5.301 9 5.398 251000 0.097 0.002 (0.048) 0.89

HBVgenotypeC

Sansure HBVDNA Test Kit(PCR-Fluorescen-ce Probing)

25 1.398 9 1.325 22.8 -0.073 0.027 (0.166) 12.5250 1.699 9 1.693 52.6 -0.006 0.025 (0.157) 9.29200 2.301 9 2.348 237 0.047 0.024 (0.155) 6.582000 3.301 9 3.295 2080 -0.006 0.023 (0.153) 4.5320 000 4.301 9 4.304 20400 0.003 0.006 (0.077) 1.80200 000 5.301 9 5.329 217000 0.028 0.007 (0.083) 1.56

Roche HBVDNA Test Kit(COBAS 2.0)

25 1.398 9 1.540 39 0.142 0.049 (0.222) 14.4150 1.699 9 1.785 64.5 0.086 0.024 (0.156) 8.75200 2.301 9 2.277 206 -0.024 0.035 (0.188) 8.242000 3.301 9 3.351 2380 0.05 0.024 (0.154) 4.6020 000 4.301 9 4.371 25800 0.07 0.035 (0.188) 4.30200 000 5.301 9 5.325 223000 0.024 0.023 (0.152) 2.85

Note: (log10 (detected value) - log10 (theoretical value)) < ± 0.3 lg IU/ml is considered to be within accepted range.

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Figure 2 Comparative CV analysis of standard HBV B (a) and C (b) plasma samples at different HBV-DNA

concentrations

Figure 3 Comparative CV analysis of low viral load (25, 50, 200 IU/ml) and high viral load plasma samples

(2000, 20000, 200000 IU/ml) with HBV B (a) and C(b) genotypes

Precision Analysis Results

The two quantitative test kits were used to detect standard plasma samples of different viral loads with

HBV B and C genotype at different test centers to evaluate the precision of the kits. See Figure 2 for results.

The results showed that the difference of precision between the two kits was not statistically significant

(genotype B: t = 1.226，P > 0.05; genotype C: t = 2.319，P > 0.05) . Meanwhile, the CV of the two test kits

was compared at different detection point. When the two test kits were used to test low viral load plasma

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samples, the CV was significantly higher than high viral load plasma (genotype B: t = 3.863，P = 0.0031;

genotype C: t = 5.067，P = 0.0005), see Figure 3.

Evaluation Results for Clinical Serum Panel

The clinical serum panel was composed of 42 HBV negative plasma samples and HBV positive plasma

samples with different levels of HBV DNA. Roche test kit and the novel test kit (Sansure) were used to test

the 42 clinical samples separately. See Table 3 for results. Among the 42 samples, 41 samples had

concordant results and the concordance rate reached 97.62%. For the 37 positive samples, the results of the

two test kits were significantly correlated (R2= 0.934，P < 0.0001). See Figure 4. Among the samples, two of

them (sample No. 236 and 283) were tested positive by Roche test kit and was < 20 IU/ml. When they were

tested by the novel test kit (Sansure), the results were about 10 IU/ml. For these 2 samples, we designed

primers for nested PCR to amplify a region flanking the target gene region in the novel test kit (Sansure)

with both positive and negative controls. The amplified products were cloned and sequenced (at least 3

clones per sample). The results proved that the sample number 236 and 283 had HBV gene sequence and the

PCR product size was 258 bp which matched the theoretical sequence length. Evolutionary tree analysis was

conducted on the sequencing results with HBV genotypes A-H standard sequences (Figure 5) and showed

both samples were genotype C but belonged to two subtypes.

Table 3 Comparative results of the 42 samples from clinical serum panel tested with Roche and Sansure

HBV DNA test kits

Roche Test Kit The novel test kit

Positive Negative

Positive 37 0

Negative 1 4

Aggregate 38 4

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Figure 4 Correlation of log10 quantitative HBV DNA concentrations of samples from the clinical serumpanel tested with Roche and Sansure HBV test kits. .

Note: HBV standard sequences are HCC-2-NY, TRF08111, MRK89073, NAB1, HB-J1444AF, AH2,

GRS08457, HB-J1411F, HBV-BL592, HB-J1444GF and MEX912M, and corresponding GenBank No. are

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AB113876, AB554015, AB554025, AB644280, AP007263, AB602818, AB554023, AP007262, AB166850,

AP007264 and AB516394.

Figure 5 Evolutionary tree analysis on sequencing results of NO. 236 and 283 samples from the clinical

serum panel.

DiscussionQuantification of HBV DNA is of great significance for the analysis of HBV infection progress,

evaluation of the efficacy of antiviral treatment and drug resistance monitoring [4]. Currently, there are

various domestic HBV DNA quantitative test kits which vary dramatically in sensitivity, linear range, and

precision of low copy sample tests [5-6]. When some chronic hepatitis B patients with antiviral treatment were

tested for HBV, their HBV DNA level was shown to be lower than the detection limit of domestic kits but

can be detected by imported test kits, which indicates the low sensitivity of domestic HBV DNA test kits.[7]

Roche HBV test kit has been internationally recognized as a reference test kit for HBV DNA quantification

with sound sensitivity, accuracy, reproducibility and wide linear range. By comparison, it’s apparent that low

sensitivity and poor reproducibility are caused by poor nucleic acid extraction and purification processes. For

instance, HBV DNA in plasma samples with low viral load are usually difficult to detect and test results can

not reflect the actual level of HBV DNA[5]. By introducing advanced nucleic acid test technologies from

Europe and America, Sansure Biotech Inc. has developed a nucleic acid extraction technology based on

magnetic-bead method and a high-precision nucleic acid quantification system with independent intellectual

rights. The magnetic-bead extraction system is also suitable for automation, which provides a simple

operation with quality being close to or reaching international standard.

In this study, the traceability analysis showed that the concordance of the detected values from both

Sansure and Roche kits and the theoretical values of the WHO standards are very good, with relative

deviations ＜ ± 0.3 log10 IU/ml, which is within the accepted range. The novel test kit from Sansure also

showed high accuracy in testing standard HBV genotypes B and C plasma samples compared to Roche test

kit (relative deviation within ±0.3 log10 IU/ml). However, when testing standard genotype B plasma sample,

the novel test kit (Sansure) showed a better accuracy than Roche test kit. This could be due to that the

primers and probes in the novel test kit are designed mainly to target the domestic HBV epidemic strains

(B/C genotype) in China. For precision analysis, the CV of both Roche and the novel test kit is < 15%, of

which the difference is not statistically significant. However, for both kits, the test CV of the low viral load

samples was higher than high viral load samples, which suggested that they all need to improve the precision

of testing low viral load samples. In clinical serum panel evaluation, the overall concordance of the novel test

kit and Roche test kit reached 97.62%. The two kits were highly correlated (R2= 0.937，P < 0.0001). When

testing low viral load samples, the two kits were essentially the same in performance. One sample showed

discrepant result, which was detected positive by the Sansure HBV test kit with viral load < 10 IU/ml, but

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negative by the Roche HBV test kit. We used both kits to extract nucleic acid separately and repeated

experiment showed both negative results, which indicates the stability of the Sansure HBV test kit needs to

be improved.

Our study did not conduct rigorous evaluation on the linear range of ultra-high viral load clinical

samples ( >2.0×108 IU/ml) because these samples were not collected in ample quantity. Furthermore, Roche

test kit could be stored at 2 - 8℃ for 18 months but the novel test kit (Sansure) could be stored at -20±5℃

for 9 months. The novel test kit (Sansure) should be improved in storage condition and the term of validity.

In summary, the Sansure HBV test kit shows no significant difference from the Roche test kit in terms

of traceability, accuracy and precision. The study proves the high sensitivity and reliability of the Sansure

HBV test kit for HBV DNA quantification. Due to the competitive price, it is worthwhile to promote its use

in large scale clinical application.

Declaration: The novel test kit is an important outcome of the national major projects during the 12th 5-year

plan period. Sansure Biotech Inc. is involved in primer designing, sequencing and evolutionary tree analysis.

This study has no interest relations with Sansure Biotech Inc.

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[2] Franco E, Bagnato B, Marino MG, et al. Hepatitis B: Epidemiology and prevention in developing

countries. World J Hepatol, 2012,4:74-80.

[3] Garson JA, Grant PR, Ayliffe U, et al. Real-time PCR quantitation of hepatitis B virus DNA

using automated sample preparation and murine cytomegalovirus internal control. J Virol

Methods, 2005,126(1-2):207-213.

[4] Hochberger S, Althof D, Gallegos de Schrott R, et al. Fully automated quantitation of Hepatitis B virus

(HBV) DNA in human plasma by the COBAS® AmpliPrep/COBAS® TaqMan® System. J Clin

Virol, 2006,35:373-380.

[5] 蒋素贞，鲁凤民，庄辉. 慢性乙型肝炎病毒 DNA定量检测的临床意义. 中华检验医学

杂志，2012,35: 117-121.

[6] 李美忠，王敏，乐晓华，等.四种 HBV DNA荧光定量 PCR试剂比较及其结果分析. 中

华实验和临床感染病杂志 (电子版), 2008,1:7-12.

[7] 叶剑荣，袁利群，范旭，等.两种 HBV DNA荧光定量试剂盒检测结果比较. 国家检验

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（Date of receival: December 21, 2012）

（Edited by Li Jian-ling）