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Journal of Clinical Microbiology, November 2005, p. 5685-5689, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5685-5689.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid and Accurate Genotyping of YMDD Motif Variants in the Hepatitis B Virus Genome by an Improved Reverse Dot Blot Method

Zhi-Ying Ou,1,2 Na Liu,1,2 Chang-Jie Chen,1,2 Gang Cheng,2 and Yun-Shao He1,2*

Department of Anatomy,1 DaAn Gene Diagnostic Center, Zhongshan Medical College of Sun Yat-Sen University, Guangzhou, Guangdong, People's Republic of China2

Received 4 March 2005/ Returned for modification 2 May 2005/ Accepted 12 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic hepatitis B is caused by hepatitis B virus (HBV) infection, which remains a major public health problem worldwide. Chronic, persistent HBV infection develops to end-stage liver disease, such as liver cirrhosis and hepatocellular carcinoma (21). More than 380 million people have been infected by chronic HBV in the world, and 1 to 2 million people have died from it (7, 15). Antiviral therapy to treat chronic HBV infection aims at interrupting the progress and clinical outcome of the disease. The nucleoside analogue lamivudine, which has antiviral activity by inhibiting viral DNA synthesis (13), is extensively used in patients with chronic hepatitis B. Despite a good safety profile and initial efficacy, lamivudine-induced decreases in viral load are difficult to sustain for a long time due to the occurrence of viral drug resistance. The antiviral effects of the drug are gradually reversed in most cases, and rebound occurs. Many patients became drug resistant after 6 months of lamivudine therapy. In lamivudine-resistant patients, serum HBV DNA and alanine and aspartate aminotransferases increased rapidly (4, 22). Some sequence analysis revealed the emergence of a specific mutation in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HBV polymerase gene, where the methionine was replaced with either an isoleucine or a valine (8, 14). It is important to detect the YMDD motif mutation during lamivudine treatment. At present, many methods have been developed to monitor HBV drug resistance, such as mass spectrometric analysis (11), fluorescence polarization (3), peptide nucleic acid clamping (17), restriction fragment length polymorphism (RFLP) (2), LiPA (18), fluorescence quantitative PCR (19), microarray (10), and sequencing. Most of these techniques are accurate but time-consuming, labor intensive, and hard to adapt to high-throughput screening. They are only amenable to the analysis by those who are well trained and well equipped, which is not suitable for small hospitals. RFLP, simple and available in most hospitals, is mainly used for detecting mono-infection but cannot detect samples with infection by mixed virus populations, especially those in which 5% to 10% are mutant virus (2, 16). The procedure is long and fussy, with multiple PCRs and multiple enzyme digestions. A skillful technician is required because a specific endonuclease reaction mixture must be developed for each mutant analyzed. Such mixtures only tend to be "home brews" (6, 12).

The reverse dot blot (RDB) method can be used to detect many mutants simultaneously. Samples differing only in one nucleotide are easily distinguished (5, 19). We combined "flow-through hybridization" technology with RDB assay to develop a rapid RDB method that can simultaneously detect the YMDD wild type and its mutants. Compared with the conventional passive hybridization process that required hours or even overnight hybridization, the "flow-through hybridization" takes only several minutes to complete by directing the flow of the target molecules toward the immobilized probes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Equipment and reagent. The "flow-through hybridization" was performed on KaiPu DNA hybriMax Rapid Hybridization Machine (Hong Kong DNA Ltd., Hong Kong, China). Taq DNA polymerase was from Fermentas MBI (Jingmei Biotech Co., Ltd.). The membrane strip was a Bigdye C membrane (Pall Co.).

Primer and probe design. We aligned the HBV genome DNA sequences extracted from the international DNA data banks (GenBank/EMBL/DDBJ) and selected the conserved region to design primers. Primers and probes (Table 1) were designed with the aid of bio-software Primer Express 2.0 (Applied Biosystem, Inc., Co.), DNAMAN 4.0 (Lynnon BioSoft), and Oligo 6.31 (Molecular Biology Insights, Inc., Co.). The reverse primer was labeled with biotin at the 5' end, and the hybridization probes were labeled with amino group at the 5' end. In order to judge the validity of hybridization process, we designed a color control probe (Table 1) for the hybridization control, which was labeled with a biotin group at the 5' end and an amino group at the 3' end. The color control probe can bind only with the chromogen but not with the targeting molecule.


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  		TABLE 1. Preferentially selected PCR primers for amplification of YMDD motif and probes for RDB analysis

 
Sample preparation. Six DNA samples with the YMDD wild type and two mutant types from the Affiliated Hospital of AnHui Medical University (HeFei, China) were used to develop the method. Each of these known serum HBV DNA sequences was previously characterized by DNA sequence analysis. One hundred archived serum samples from Guangzhou Liver Hospital (Guangzhou, China) were used to test this method; all of the sera were HBV DNA positive, with viral loads from 1 x 103 to 1 x 108 copies/ml. Eighteen samples had a viral load of 1 x 103 copies/ml. Forty-nine blood samples for clinical detection came from patients with clinically suspected lamivudine resistance in Guangzhou Liver Hospital based on the following criteria. (i) The patients had a virological (serum viral load of at least 1 x 103 copies/ml detected with an HBV fluorescence quantitative PCR detection kit from DaAn Gene Corporation, Guangzhou, China) and biochemical (alanine and aspartate aminotransferases) evidence of treatment failure. (ii) All patients were treated with lamivudine for at least 8 months, with an average of 16.9 months. Of the 49 clinic serum samples, 5 had a viral load of 1 x 103 copies/ml. All clinically suspected resistant samples were further tested by DNA clonal sequencing analysis.

Serum HBV DNA extraction and PCR conditions. From each serum sample, 50 µl was incubated at 100°C for 10 min in the same volume of DNA extraction buffer containing 50 mM NaOH, 10 mM Tris-HCl (pH 8.0), 1% Triton X-100, 1% NP-40, and 0.5 mM EDTA (pH 8.0), which was then centrifuged at 10,000 rpm for 5 min. Five microliters of the supernatant was amplified by PCR in 50 µl of 1x PCR buffer containing 200 µM of each deoxynucleoside triphosphate (dNTP), 2 U of Taq DNA polymerase, and 0.2 µM of each primer. In order to prevent contamination, we replaced dTTP with dUTP and added 0.5 U of uracil-DNA glycosylase (UDG) to the PCR system. The amplification was performed by using an Applied Biosystems 9700 thermal cycler (Perkin-Elmer) under the following conditions: incubation at 50°C for 3 min before an initial denaturation step at 93°C for 3 min, followed by 40 cycles of 93°C for 30 s, 55°C for 30 s, and 72°C for 35 s. A final extension was performed at 72°C for 5 min.

PCR products, clones, and sequencing. The PCR products were purified by phenol-chloroform (1:1) extraction, precipitated, and dissolved in water; fragments 233 bp in length were cloned into pMD 18-T simple vector (TaKaRa, Japan) according to the manufacturer's instructions. Cloned plasmids were sequenced at BioAsia Corporation (Shanghai, China).

RDB and RFLP analysis. RFLP was used according to Allen et al. (2). We used NdeI and NlaIII to discriminate the YMDD motif and YVDD motif, respectively. Based on the method of Zhang et al. (20), the improved RDB was used according to the principle of "flow-through hybridization," which was performed on the KaiPu DNA hybriMax Rapid Hybridization Machine. Its detailed steps are as follows. (i) Make the hybrid membrane strip by activating the membrane with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (Sigma-Aldrich, commercial grade) and then dotting the YMDD, YIDD, and YVDD motif probe and the color control probe at the given positions on the membrane; the amino group of the probe may bind with the carboxyl group of the membrane. (ii) Denature the PCR products. (iii) Prehybridize the membrane. (iv) Hybridize the target PCR products with the specific probes. (v) Wash the unhybridized PCR products. (vi) Combine peroxidase (POD) with the biotin group on the PCR products or on the color control probe. (vii) Wash the membrane to eliminate the uncombined POD. (viii) Color with 3,3',5,5'-tetramethylbenzidine (TMB) chromogen. We set positive and negative controls for all detection. The machine works on the basis of the particular principle of "flowthrough hybridization"; there is a negative pressure under the airproof hybridization membrane, which was produced by pumping. All of the hybridization solution, washing solution, POD solution, and coloring solution flows through the membrane automatically. The improved RDB method actively directs the flow of the targeting molecules toward the immobilized probes within the membrane fibers. The complementary molecules will be hybridized and form duplexed DNA; at the same time, any unbound molecules will be removed by passing through the membrane. This speeds up the interaction between the complementary molecules, reduces the hybridization time from hours down to minutes, and provides results hundreds of times faster than traditional passive hybridization methods.

Determination of the optimum probe concentration and hybridization temperature for the RDB assay. We used different concentrations of YMDD, YIDD, and YVDD motif-specific probes (0.5, 2, 5, 10, 15, and 20 µmol/liter, respectively) on the same membrane strip to decide the optimal probe concentration. The RDB experiments were carried out at different hybridization temperatures (38, 40, 42, 44, and 46°C) to determine the optimal hybridization temperature.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of RDB and sequence analysis. To enable the unequivocal interpretation of data according to RDB analysis, probes used in the RDB assay were selected and optimized to produce a clear and specific dot pattern. The probe concentration and hybrid temperature were optimized for the RDB analysis (Fig. 1). The results indicated that all of them had nonspecific hybridization blue dots that appeared at 38°C and were reduced with the increase of temperature. A clear hybridization blue dot and clean background appeared at 42°C; YMDD and YIDD had a weak hybridization dot at 44°C and a weaker dot at 46°C. This indicated that 42°C was the optimal temperature for hybridization. The hybridization dots became bright and clear with the increase in probe concentration. At a concentration of 5 µmol/liter, all of them had a clear blue signal that was similar to the results at 10, 15, and 20 µmol/liter. So we concluded that 5 µmol/liter was an economically optimal concentration for hybridization.



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  		FIG. 1. Dynamic RDB assay profiles of the YMDD wild type and mutants at different probe concentrations and hybridization temperatures. The numbers (0.5, 2, 5, 10, 15, and 20) represent the probe concentrations (µmol/liter) of the HBV YMDD, YIDD, and YVDD motifs; the left dot in the last row in each hybrid membrane is the color control probe (Ctrl); the concentration is 2 µmol/liter.

 
Four samples with YMDD mutations and two samples with the YMDD wild type previously characterized by DNA sequence analysis were used to develop the assay. Each of those two mutant types was represented in this panel of samples. Figure 2 showed the representative RDB assay profiles for the different motifs of HBV YMDD infection in serum. The results indicated that the assay could clearly distinguish each YMDD wild type and mutant in one RDB profile. For all detections, they were included in the following possible RDB profiles: (i) YMDD, YIDD, or YVDD mono-infection (Fig. 2A, B, and C); (ii) mixed infection with two of the three YMDD motifs, such as YMDD plus YIDD (Fig. 2D), YIDD plus YVDD (Fig. 2E), or YMDD plus YVDD (Fig. 2F); or (iii) mixed infection with YMDD, YIDD, and YVDD (Fig. 2G).



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  		FIG. 2. Representative RDB profiles of infection with different YMDD motifs. (A) HBV YMDD. (B) HBV YIDD. (C) HBV YVDD. (D) Mixed HBV YMDD plus YIDD. (E) Mixed HBV YIDD plus YVDD. (F) Mixed HBV YMDD plus YVDD. (G) Mixed HBV YMDD plus YIDD plus YVDD. (H) Probes in the aligned mode in the hybridization membrane strip. Circled numbers: 1, HBV YMDD probe; 2, HBV YIDD probe; 3, HBV YVDD probe; and 4, color control probe.

 
Blind analysis. To evaluate the accuracy of RDB for detecting YMDD motif mutations, we performed a comparison of this assay with sequencing and RFLP in a blind analysis. A total of 100 serum HBV DNA archival samples that had been previously characterized by RFLP or DNA clonal sequencing were detected. The RFLP and sequencing results were unknown until the genotypes obtained by RDB were scored. All of the samples tested by RDB were found to be in 100% (100/100) concordance with the sequencing data (Table 2) but only 83.0% (83/100) concordance with RFLP.


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  		TABLE 2. Concordant results obtained by blind analysis by three methods of 100 archival serum samples

 
Specificity and sensitivity. Compared to DNA sequencing and RFLP, the specificities of RDB were 100% and 83.0%, respectively (Fig. 3). As to the sensitivity, the improved RDB method can successfully detect all of the serum HBV with a virus load from 1 x 103 to 1 x 108 copies/ml; i.e., all of the positive HBV serum can be successfully tested.



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  		FIG. 3. Comparison of the accuracy and specificity of RDB and RFLP assays (n = 100; P < 0.01).

 
Clinical detection. To evaluate the application of this assay, 49 patients highly suspected of clinical lamivudine resistance, including 5 subjects with a serum HBV viral load of 1 x 103 copies/ml, were detected. All of the samples were successfully detected by the methods; they were all included in the RDB assay profiles described above. There were 4 YMDD infections, 2 YIDD infections, 10 YVDD infections, 1 mixed YMDD and YIDD infection, 26 mixed YMDD and YVDD infections, 2 mixed YIDD and YVDD infections, and 4 mixed YMDD, YIDD, and YVDD infections (Table 3). The results were all confirmed by the DNA clonal sequencing analysis.


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  		TABLE 3. Cases used in clinical detection

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
After long periods of antiviral treatment with lamivudine in HBV carriers, there develops a high risk of HBV drug resistance (8, 14). The sensitivity of lamivudine decreased after the emergence of resistance mutants leading to treatment failure (1). With the development of modern medicine, detailed virus serological and molecular biology information is required to know the status of virus variation, which is important to guide clinical practice. The method developed in this study can be used in detection of HBV YMDD, YIDD, or YVDD mono-infection and mixed infections. It can provide individual pieces of key molecular virology information for lamivudine therapy.

In this study, we have described a simple, rapid, semiautomatic, reliable, and contamination-proof approach for genotyping the YMDD motif variants in the HBV genome. We developed a commercially prepared HBV YMDD motif variant detection kit equipped with the KaiPu DNA HybriMax Rapid Hybridization Machine. The machine was designed based on the particular principle of "flow-through hybridization." There is a negative pressure under the airproof hybridization membrane that was produced by pumping, so the improved method can actively direct the flow of the target molecules toward the immobilized probes within the membrane fibers, which enables rapid hybridization to occur. The dominant characteristic of the improved RDB method is that all of the PCR products, washing buffer, binding solution, and coloring solution flow through the hybrid membrane quickly and directly with the help of negative pressure, which is semiautomated and is essentially different from the traditional method. The complementary molecules will be hybridized and form duplexed DNA; at the same time, any unbound molecules will be removed through the membrane. This speeds up the interaction between the complementary molecules, reduces the hybridization time from hours down to minutes, and provides results hundreds of times faster than the traditional passive hybridization methods. For the study, we designed and optimized not only the specific probes for the specific virus types but also the color control probe for the hybridization operation to reach 100% specificity. The color control probe can bind only with the chromogen, but it can't bind with the target molecule, which helps to judge the validity of hybridization. Instead of using dTTP, we used dUTP and UDG in the PCR system to prevent PCR products from contaminating. As to the sensitivity, the improved method can detect a positive HBV serum DNA level as low as the viral load of 1 copy per microliter (i.e., 103 copies/ml), which is the critical value to judge if it is a positive HBV serum in HBV fluorescence quantitative PCR detection. All of these findings indicate the method is sensitive, specific, and ensures quality in clinical tests.

In addition, the improved RDB method is clean, versatile, and less expensive than traditional hybridization. PCR-based direct nucleotide sequence analysis (9) is considered as the gold standard for mutation detection. DNA sequencing is highly sensitive and specific for mutational scanning but is time-consuming and costly. The new RDB assay directs all of the PCR products and solution to directly flow through the hybrid membrane, which increases the diffusivity and local reaction concentration of the nucleic acid molecule and occurs in three-dimensional volumes rather than the conventional two-dimensional surface. Compared with the traditional RDB method and the commercially available Inno-LiPA assay, which require several hours to complete the molecular hybridization process and large volumes of sample and reagent, hybridization by the new technique can be done within 7 min with very small samples and reagent volumes, which increases the hybridization efficiency markedly and reduces the running cost. The cost of this assay is low: the KaiPu DNA hybriMax Rapid Hybridization Machine costs only $2,000, and the total cost is about $1.40 for a sample. It's available for most small hospitals. With the new method, the background of the hybridization membrane strip is very clean and nonspecific hybridization is reduced. The method can be used to rapidly detect not only a large number of clinical samples and genotypes simultaneously, but also single-base mutations. (Fifteen or more samples can be detected simultaneously; more than 24 probes can be used for a given sample.) All of the probes are integrated in a small hybridization membrane just like a low-density microarray, which makes high-throughput detection possible. The improved method can also be extensively used to rapidly detect other clinical microbes or genetic disorders if we change the probes in the membrane strip.

As for RFLP, we used two kinds of restriction enzymes to discriminate the YMDD, YIDD, and YVDD motifs. RFLP is a fussy and tedious technique, in which it is easy to make mistakes and hard to discriminate the small DNA fragments digested by the endonuclease. Skilled personnel are required because a specific endonuclease reaction mixture has to be developed for each mutant. In addition, gel electrophoresis can discriminate limited concentrations of PCR products with much lower sensitivity than the RDB, which includes a cascade amplification reaction. The RFLP often leaves out the samples with mixed virus populations and the low-component virus population (Table 2), which is suited only for mono-infection; this may explain the lower specificity and sensitivity of the RFLP analysis.


   ACKNOWLEDGMENTS
 
This work was supported by the National High Technology Research and Development Program of China (863 Program) grant no. 2002AA215018 from the Ministry of Science and Technology.

We thank Shao-Jing Wei and Mei-Fang Huang, Liver Institute of Guangzhou Liver Hospital (Guangzhou, SouthChina), for their advice on the study and kindly collecting and contributing samples. The authors also thank Qiu-Hua Mo, First Military Medical University (Guangzhou, South China), for his constructive suggestions on the RDB method and manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: DaAn Gene Diagnostic Center, Zhongshan Medical School of Sun Yat-Sen University, 74 Zhongshan Rd. 2, Guangzhou, People's Republic of China 510080. Phone: 86 20 88288735. Fax: 86 20 8733 3435. E-mail: ouzhiying{at}yahoo.com.cn. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Allen, M. I., M. Deslauriers, C. W. Andrews, G. A. Tipples, K. A. Walters, D. L. Tyrrell, N. Brown, L. D. Condreay et al. 1998. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Hepatology 27:1670-1677.[CrossRef][Medline]
  2. Allen, M. I., J. Gauthier, M. DesLauriers, E. J. Bourne, K. M. Carrick, F. Baldanti, L. L. Ross, M. W. Lutz, and L. D. Condreay. 1999. Two sensitive PCR-based methods for detection of hepatitis B variants associated with reduced susceptibility to lamivudine. J. Clin. Microbiol. 37:3338-3347.[Abstract/Free Full Text]
  3. Bai, Y. J., J. R. Zhao, G. T. Lv, W. H. Zhang, Y. Wang, and X. J. Yan. 2003. Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization. World J. Gastroenterol. 9:2344-2347.[Medline]
  4. Bartholmeusz, A., R. F. Schinazi, and S. A. Locarnini. 1998. Significance of mutations in the hepatitis B virus polymerase selected by nucleoside analogues and implications for controlling chronic disease. Viral Hep. Rev. 4:167-187.
  5. Cane, P. A., P. Cook, D. Ratcliffe, D. Mutimer, and D. Pillay. 1999. Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus. Antimicrob. Agents Chemother. 43:1600-1608.[Abstract/Free Full Text]
  6. Chayama, K., Y. Suzuki, M. Kobayashi, A. Tsubota, M. Hashimoto, Y. Miyano, H. Koike, M. Kobayashi, I. Koida, Y. Arase, S. Saitoh, N. Murashima, K. Ikeda, and H. Kumada. 1998. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology 27:1711-1716.[CrossRef][Medline]
  7. Clarke, B., and S. Bloor. 2002. Molecular genotyping of hepatitis B virus. J. Clin. Virol. 25:S41-S45.
  8. Dienstag, J. L., E. R. Schiff, T. L. Wright, R. P. Perrillo, H. W. Hann, Z. Goodman, L. Crowther, L. D. Condreay, M. Woessner, M. Rubin, and N. A. Brown. 1999. Lamivudine as initial treatment for chronic hepatitis B in the United States. N. Engl. J. Med. 341:1256-1263.[Abstract/Free Full Text]
  9. Engelke, D. R., P. A. Hoener, and F. S. Collins. 1988. Direct sequencing of enzymatically amplified human genomic DNA. Proc. Natl. Acad. Sci. USA 85:544-548.[Abstract/Free Full Text]
  10. Heo, J., M. Cho, H. H. Kim, Y. M. Shin, H. J. Jang, H. K. Park, C. M. Kim, G. H. Kim, D. H. Kang, G. A. Song, and U. S. Yang. 2004. Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection. J. Korean Med. Sci. 19:541-546.[Medline]
  11. Hong, S. P., N. K. Kim, S. G. Hwang, H. J. Chung, S. Kim, J. H. Han, H. T. Kim, K. S. Rim, M. S. Kang, W. Yoo, and S. O. Kim. 2004. Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J. Hepatol. 40:837-844.[CrossRef][Medline]
  12. Jardi, R., M. Buti, F. Rodriguez-Frias, M. Cotrina, X. Costa, C. Pascual, R. Esteban, and J. Guardia. 1999. Rapid detection of lamivudine-resistant hepatitis B virus polymerase gene variants. J. Virol. Methods 83:181-187.[CrossRef][Medline]
  13. Lai, C. L., R. N. Chien, N. W. Leung, T. T. Chang, R. Guan, D. I. Tai, K. Y. Ng, P. C. Wu, J. C. Dent, J. Barber, S. L. Stephenson, and D. F. Gray. 1998. A one-year trial of lamivudine for chronic hepatitis B. N. Engl. J. Med. 339:61-68.[Abstract/Free Full Text]
  14. Leung, N. W. Y., C. L. Lai, T. T. Chang, R. Guan, C. M. Lee, K. Y. Ng, S. G. Lim, P. C. Wu, J. C. Dent, S. Edmundson, L. D. Condreay, and R. N. Chien. 2001. Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy. Hepatology 33:1527-1532.[CrossRef][Medline]
  15. Maynard, J. E. 1990. Hepatitis B: global importance and need for control. Vaccine 8(Suppl. 1):S18-S20.
  16. Niesters, H. G. M., R. A. De Man, S. D. Pas, E. Fries, and A. D. M. E. Osterhaus. 2002. Identification of a new variant in the YMDD motif of the hepatitis B polymerase gene selected during lamivudine therapy. J. Med. Microbiol. 51:695-699.[Abstract/Free Full Text]
  17. Ohishi, W., and K. Chayama. 2003. Rare quasispecies in the YMDD motif of hepatitis B virus detected by polymerase chain reaction with peptide nucleic acid clamping. Intervirology 46:355-361.[CrossRef][Medline]
  18. Stuyver, L., C. Van Geyt, S. De Gendt, G. Van Reybroeck, F. Zoulim, G. Leroux-Roels, and R. Rossau. 2000. Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy. J. Clin. Microbiol. 38:702-707.[Abstract/Free Full Text]
  19. Whalley, S. A., D. Brown, C. G. Teo, G. M. Dusheiko, and N. A. Saunders. 2001. Monitoring the emergence of hepatitis B virus polymerase gene variants during lamivudine therapy using the LightCycler. J. Clin. Microbiol. 39:1456-1459.[Abstract/Free Full Text]
  20. Zhang, Y., M. Y. Coyne, S. G. Will, C. H. Levenson, and E. S. Kawasaki. 1991. Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides. Nucleic Acids Res. 19:3929-3933.[Abstract/Free Full Text]
  21. Zoulim, F. 2001. Detection of hepatitis B virus resistance to antivirals. J. Clin. Virol. 21:243-253.[CrossRef][Medline]
  22. Zoulim, F., and C. Trépo. 1998. Drug therapy for chronic hepatitis B: antiviral efficacy and influence of hepatitis B virus polymerase mutations on the outcome of therapy. J. Hepatol. 29:151-168.[CrossRef][Medline]

Journal of Clinical Microbiology, November 2005, p. 5685-5689, Vol. 43, No. 11
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.11.5685-5689.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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