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Bai, et al. "rapid and high throughput detection of hbv ymdd mutants with fluorescence polarization"

P.O.Box 2345, Beijing 100023,China World J Gastroenterol 2003;9(10):2344-2347Fax: +86-10-85381893 World Journal of Gastroenterology E-mail: [email protected] Copyright 2003 by The WJG Press ISSN 1007-9327 • BRIEF REPORTS • Rapid and high throughput detection of HBV YMDD mutants with
fluorescence polarization

Yui-Jie Bai, Jin-Rong Zhao, Guan-Ting Lv, Wen-Hong Zhang, Yan Wang, Xiao-Jun Yan Yui-Jie Bai, Jin-Rong Zhao, Guan-Ting Lv, Wen-Hong Zhang,
chronic hepatitis[6-10]. Because interferon alfa shows significant Yan Wang, Xiao-Jun Yan, Institute of Genetic Diagnosis, Fourth
dose-dependent side effects, lamivudine has emerged as the Military Medical University, Xi'an 710032, Shannxi Province, China first therapeutic agent[11-13]. Chinese patients are immunotolerant Supported by Shen Zhen Syno Gene Digital Co. Ltd
to interferon alfa because of acquisition of the disease during Correspondence to: Dr. Yu-Jie Bai, Institute of Genetic Diagnosis,
early childhood. The efficacy of interferon alfa in Chinese is Fourth Military Medical University, Xi'an 710032, Shannxi province, lower than in white patients[14]. However, the efficacy of China. [email protected]
Telephone: +86-29-3216587 Fax: +86-29-3285729
lamivudine is equal in Chinese and white patients.
Received: 2003-04-09 Accepted: 2003-05-19
Lamivudine is an antiviral nucleoside analogue. It caninhibit HBV reverse transcriptase activity and also act as aviral DNA chain terminator[15]. It is an inactive form before activated in hepatocytes by addition of phosphate group[2].
Lamivudine has two pathways of viral suppression. First, the AIM: To develop a simple and rapid detection of HBV gene
active triphosphate metabolite is incorporated into newly variants and prediction of lamivudine-resistance in patients.
synthesized HBV DNA to stop the chain extension. Second, itinhibits HBV reverse transcriptase. Its clinical use has resulted METHODS: Initial y, plasmids harboring the wild-type or
in HBeAg seroconversion and undetectable HBV DNA[16].
mutant HBV DNA fragments were used in a model system.
Unlike some nucleoside analogues, lamivudine is well tolerated The technique was then applied to clinical samples for an and has an excellent safety profile. It has little or no effect on analysis of YMDD mutations. The sera were extracted from bone marrow, hepatocytes, kidney, or muscle tissues. However, chronic hepatitis patients who had received lamivudine long-term lamivudine treatment has led to emergence of HBV treatment for more than one year. P region gene of HBV variants resistant to lamivudine therapy in some patients. The was amplified by polymerase chain reaction. The excess major sites of mutation are situated in the highly conserved primers and dNTPs in PCR products were removed by motif, tyrosine (Y), methionine (M), aspartate (D), aspartate cleaning-up reagents. Template-directed dye-terminator (D) (YMDD), of the catalytic (C) domain of the reverse incorporation reaction was performed and R110 or TAMRA transcriptase. Mutations consist of an amino acid substitution labeled acyclo-terminator was added on the 3' end of TDI- from M to either valine (A739G, Met552 Val552) or isoleucine primer specifically. Fluorescence polarization value was Ile552). Those mutations at the YMDD motif measured with Victor 2 multilabel counter and the genotypes could render HBV resistant to lamivudine[17].
of HBV were analyzed.
Fluorescence polarization assay (FPA) is a well establishedmethod used to analyze associations and dissociations between RESULTS: The YMDD genotypes in recombined positive
molecules in solution. This technique relates the change in the plasmid and 56 serum samples of HBV infected patients molecular size of a fluorophore to a change in the fluorescence were analyzed by using our TDI-FP method and the specificity polarization value (mp unit). The value changes indicate and sensitivity were confirmed by DNA sequencing. Five of molecular associations or dissociations, so that FPA have been 56 serum samples showed YVDD phenotype (9 %), including used to examine the interactions between molecules, such as 1 YMDD and YVDD mixed infection. Four of 56 showed YIDD protein-protein interactions, DNA-protein interaction and phenotype (7.1 %).
DNA-DNA hybridization.
Based on the FP detection method and the combined CONCLUSION: This is a simple, rapid, low cost and high
template-directed dye-terminator incorporation technique, a throughput assay to detect HBV polymerase gene variants novel SNP (single nucleotide polymorphism) detection system and suitable for large-scale screening and prediction of the (TDI-FP) has been developed[18]. It relies on the ability of lamivudine-resistance in clinical samples.
AcycloPolTM, a novel mutant thermostable polymerase fromthe Archeon family, to extend accurately an annealed probe Bai YJ, Zhao JR, Lv GT, Zhang WH, Wang Y, Yan XJ. Rapid and by a single dye-terminator that is complementary to the high throughput detection of HBV YMDD mutants with opposite strand. It performs an assay to determine the base at fluorescence polarization. World J Gastroenterol 2003; 9(10): a SNP site with many advantages over the other methods, such as homogeneity, low cost, high accuracy, high throughput and ready adaptation to automation. This method has been used togenotyping and SNP detections but has not, to our knowledge,been used to microbial mutant analysis yet.
Routine detection of HBV YMDD mutation after lamivudinetreatment will be required to increase the efficacy. In this study, Hepatitis virus B (HBV) is the causative agent of acute and we undertook such a study using TDI-FP to detect the YMDD chronic hepatitis. Approximately 400 million people worldwide variants using mutagenesis plasmids and serum samples of 56 are chronically infected with HBV. There are 170 million patients. This new method is a rapid, accurate and high people infected in China[1,2]. HBV infection can lead to cirrhosis throughout method to analyses the YMDD mutations. It will
and primary hepatocellular carcinoma[3-5]. To date, interferon be a very useful tool for the clinical diagnosis and monitor of alfa and lamivudine are the only two agents approved for the lamivudine resistance.

Bai YJ et al. Rapid detection of lamivudine -resistance HBV mutants MATERIALS AND METHODS
cycles containing denaturation at 94 for 30 s, annealing at for 30 s and extension at 72 for 1 min. After a final Fluorescence polarization values were determined with the extension step at 72 for 10 minutes, the reactions were VICTOR2 multilabel counter (Perkin-Elmer, USA). The until further use. The PCR reaction was carried out in a Touchgene gradient termal cycler (TECHNE Co. USA).
excitation and emission wavelengths were 544 nm and 595 nmfor R110 and 485 nm and 535 nm for TAMRA, respectively.
In order to evaluate the amplification, each 5 µL of PCR The samples were measured in 384 well PCR plates (MSP- products was separated on a 20 g/L agarose gel in 0.5× TBEbuffer. The products were visualized and photographed under 3862 MJ Research, USA).
ultraviolet (UV) light.
Taq DNA polymerase, DNA extractor kit were obtained from Following PCR amplification, 2 µL clean-up reagent Hua-mei Bio-tech Co., LTD. AcycloPrimeTM-FP detection kit (including shrimp alkaline phosphatase and exonuclease I and (including AcycloPolTM, AcycloTeminatorsTM labeled withR110 and TAMRA, shrimp alkaline phosphatase, exonuclease reaction buffer) was added into 5 µL of PCR product andincubated at 37 for 60 min to remove unincorporated dNTPs I) was product of Perkin-Elmer Co., Ltd.
and free primers. The enzymes were then heat-inactivated at for 30 min.
TDI-FP was performed according to the protocol supplied A set of PCR primers was designed to amplify the P region of by the manufacturer (Perkin-Elmer Life Sciences, Inc, USA).
HBV gene including the YMDD motif encoding sequences. 2 The reaction system consisted of 13 µL of AcycloPrime-FP TDI-primers were also designed to encompass 17-21 bp 5' mixture (containing 0.05 µL of AcycloPolTM polymerase, 2 µL (A739Gtdi, sense) or 3' (G741T, antisense) to the nucleotide of 10× reaction buffer, 1 µL of Acyclo-Terminator Mix, adjacent to the mutation sites using DNAStar software. 3 sets 0.5 µL mutant detection primer and 9.45 µL of water) and 7 µL of primers for the site-directed mutagenesis were also designed of amplified and processed target DNA. After an initial using DNAStar software. All the primers, whose sequences denaturation at 95 for 2 min, 25 cycles at 95 are shown in Table 1, were synthesized using 391 DNA for 30 s were performed on a black 384-well PCR synthesizer (Perkin-Elmer, USA) by Bao Tai Ke Biotech Co.
plate (MSP-3862, MJ Research). The fluorescence polarization values were measured on the 384-well plate using Victor2multilabel counter directly.
The site-directed HBV YMDD mutants were made usingTakara MutanBEST Kit by TaKaRa Biotechnology Co., Ltd.
The A719G mutation was created using primers A739Gfor and A739Grev. The G741T mutation was created using primers G741Tfor and G741Trev. The A739G/G741T double mutants were made using primers diMutfor and diMutrev with the electrophoresis (Figure 1). A single band corresponding to a plasmid pMD/HBV as a template respectively. (All the molecular size of 200 bp was detected, which was in sequences of primers are shown in Table 1). The mutagenesis concordance with expectation.
was detected according to the manufacture's manual andverified by DNA sequencing.
Table 1 Primer sequences
Figure 1 Electrophoretic analysis of PCR products. 1: plasmid
pMD/HBV-A739G as PCR template, 2-6: serum samples as PCR template, 7: water as template (negative control), M: DNA molecular weight marker.
5' -CCAGACAGTGGGGGAAAGC 3' 5'- CCAGACAGTGGGGGAAAGC 3' Following PCR amplification, excess primers and dNTPs wereremoved. In the TDI extension reaction, 729tdi primer and dye-terminators mix (R110-acyGTP/ TAMRA-acyATP) were 5' -CCAGACAGTGGGGGAAAGC 3' used to detect the A739G mutation (Figure 2). 741tdi primerand R110-acyCTP/TAMRA- acyATP) were used to detect the G741T mutation. The dye-terminator was incorporated and Venous blood (1 ml) was obtained from 56 patients with HBV the TDI-primer was extended by one base complementary to chronic hepatitis B in Tangdu Hospital and Second Hospital the specific mutation site. The fluorescence intensity was of Jiaotong University, Xi'an.
measured using Victor 2 multilabel counter and the FP valuewas calculated by the instrument software automatically. The Amplification of P region of HBV gene
graph was created from an Excel workbook and the cluster of PCR amplification was performed in a total volume of 50 µL, AcycloPrime-FP data was obtained by plotting the TAMRA containing 1.5U Taq polymerase, PCR buffer, 0.1 mmol/L polarization vs the R110 polarization. The FP reading of the dNTPs, 1 µL of DNA sample (10 ng plasmid DNA or 100 ng samples was clustered into four distinct groups (Figure 3). As genome DNA), 100 nmol/L PCRfor and PCRrev primer each.
expected, for HBV YVDD samples, the values for R110- The amplification procedure consisted of an initial denaturation acyGTP were high and the values for TAMRA-acyATP were and enzyme activation step at 94 for 5 min, followed by 40 low, reflecting incorporation of the R110-acyGTP but not 2346 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol October 15, 2003 Volume 9 Number 10 TAMRA-acyATP. The genotypes were represented by the YMDD variant was detected in 46 (82 %), the YVDD variant cluster in the lower left. Conversely, the YMDD genotypes in 5 (8.9 %) and the YIDD in 4 (7.1 %). In one patient (1.8 %), appeared in the upper right. YMDD and YVDD double both YMDD and YVDD variants were detected, which might mutations or mixed infection samples appeared in the upper reflect a mixed infection.
right. Negative controls were in the lower left cluster (Figure 3).
For the G741T detection, the YIDD samples appeared in the upper left, YMDD samples in the lower right and the negativecontrol in the lower left.
As reported before, the major drawback to the therapy oflamivudine is the emergence of drug-resistance due to the HBVDNA mutations. The patients who have lamivudine resistant HBV variants infection are more likely to develop more severe liver damage than those who do not have drug-resistant mutants. The mutation at YMDD (YMDD YIDD) is considered to be the major lamivudine-resistance related variants. DNA sequencing is considered as the ideal method for characterizing DNA mutants, but it can not be afforded by most laboratories, especially clinical laboratories due to the cost and equipment requirement. Type-specific PCR, PCR-RLFP and PCR-SSCP have been used to detect HBV YMDD mutants. However, all the methods have drawbacks, e.g. lack of accuracy, difficulty in determining the mutation 20 40 60 80 100 120 140 160 180 200 220 240 sites exactly or detecting mixed infection[19].
We reported here, for the first time, the successful applicationof the TDI-FP to detect the HBV YMDD mutation in hepatitis Figure 3 TDI-FP analysis data of YMDD mutation.
B patients. As a homogenous assay, the FP value reflectedthe total sum of free and incorporated dye-terminators. Two Detection of YMDD mutation in clinical samples
major factors affecting the result were faced: (1) The To evaluate the utility and accuracy of the TDI-FP in clinical concentration of the first PCR product in TDI extension was samples, 56 serum samples from patients with chronic hepatitis too high or too low, which would create misincorporation or B were analyzed. Among the sera analyzed, the wild-type lower incorporation of dye-terminators, (2) the excess primers Clean-up, inactive enzymes Acyclo-Terminator mix Acyclo-Pol polymerase TTTCAGTTAT A G
R110 polarization TAMRA polarization Figure 2 The scheme of TDI assay of YMDD.
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