2103

대한진단검사의학회지제 28 권제 2 호 2008 Korean J Lab Med 2008;28:144-50 DOI 10.3343/kjlm.2008.28.2.144 원저 진단유전학 실시간정량적중합효소연쇄반응을이용한 Abbott RealTime HBV Quantification Kit의성능평가 김명희 차충환 안동희 최성은 오흥범 울산의대서울아산병원진단검사의학과 Performance Evaluation of Abbott RealTime HBV Quantification Kit for HBV Viral Load by Real-Time PCR Myeong-Hee Kim, M.D., Choong-Hwan Cha, M.D., Dongheui An, M.D., Sung-Eun Choi, M.D., and Heung-Bum Oh, M.D. Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, Korea Background : Hepatitis B virus (HBV) DNA quantification is necessary for starting and monitoring of antiviral therapy in patients with chronic hepatitis B. This study was intended to assess the clinical performance of Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA). Methods : The performance was evaluated in terms of precision, linearity, detection sensitivity, cross-reactivity, and carry-over. A correlation with the Real-Q HBV Quantification kit (BioSewoom Inc., Korea) was also examined using serum samples from 64 patients diagnosed with chronic hepatitis B and underwent lamivudine therapy in Asan Medical Center. We verified the trueness of the system by comparing the outputs with the assigned values of the BBI panel (BBI Diagnostics, USA). Results : Within-run and between-run coefficients of variation (CV) were 3.56-4.71% and 3.03-4.98%, respectively. Linearity was manifested ranging from 53 to 10 9 copies/ml and the detection sensitivity was verified to be 51 copies/ml. None of hepatitis C virus showed cross-reactivity. No cross-contamination occurred when negative and positive samples were alternatively placed in a row. It showed a good correlation with the Real-Q HBV (r 2 =0.9609) and the test results for the BBI panel were also well agreed to the assigned values (r 2 =0.9933). Conclusions : The performance of Abbott RealTime HBV Quantification kit was excellent; thus, it should be widely used in starting and monitoring of antiviral therapy in Korean patients with chronic hepatitis B. (Korean J Lab Med 2008;28:144-50) Key Words : Real-time PCR, Hepatitis B virus, Performance, Evaluation 서 론 항바이러스제치료를받는만성 B 형간염환자에게혈중 B 형 접 수 : 2008년 1월 3일 접수번호 : KJLM2103 수정본접수 : 2008년 1월 30일 게재승인일 : 2008년 2월 22일 교신저자 : 오흥범 우 138-736 서울시송파구풍납동 388-1 울산의대서울아산병원진단검사의학과 전화 : 02-3010-4505, Fax: 02-478-0884 E-mail: hboh@amc.seoul.kr 간염바이러스 (hepatitis B virus, HBV) DNA의측정은치료모니터링을위한필수적인검사이다. 항바이러스제치료를시행하면혈중바이러스양의급격한감소와간기능개선이나타나지만, 장기간사용시에는약제내성주의출현으로인해다시혈중바이러스가급증하는것으로알려져있다. 최근여러연구들에서항바이러스제치료시작후 24-48주에혈중 HBV DNA 농도를측정하면항바이러스제에대한내성출현을예측할수있다고하였다 [1-3]. 또한 HBV의혈중농도는간경화나간세포암종과같은합병증의발생위험과직접관련이있다는연구 144

Evaluation of RealTime HBV Quantification Kit 145 도있어 HBV DNA 농도측정은환자를치료하고예후를예측하는데있어매우중요한검사가되었다 [4]. 국내에서 HBV DNA 정량을위해주로이용되고있는검사법은 branched DNA (bdna) 기술을이용하는 versant HBV DNA 3.0 (Bayer Diagnostics, Emeryville, CA, USA) 과 Digene HBV Hybrid-Capture II (Murex Diagnostics, Dartfold, UK) 등이다 [5]. bdna와 hybrid capture 방법은개발시기에따라차이는있으나일반적으로 10 5 copies/ml 이상의고농도에서는정확한정량이가능하나저농도의바이러스혈증은검출하지못한다 [6, 7]. 과거에는 HBV DNA의혈중농도가 10 5 copies/ml 이하인경우비활동성보유자 (inactive carrier) 로간주되어임상적으로관심이낮았으나, 저농도범위에대해서검사가가능한 Cobas Amplicor HBV Monitor kits (Roche Molecular System, Pleasanton, CA, USA) 로혈중 HBV DNA를정량측정했을때 200 copies/ml 이하에서는재발률이 37%, 1,000 copies/ml 이상인경우는 73% 에서재발한다는보고가있고 [8], 최근미국간학회의만성 B형간염치료가이드라인에서도 10 4-10 5 copies/ml인경우지속적인추적검사와함께필요한경우치료가필요하다고하여 [9], 10 5 copies/ml 이상뿐아니라그이하에서도정확한정량이필요하게되었다. 실시간중합효소연쇄반응은기존의 PCR 방법과달리증폭산물을형광의양으로실시간정량검출할수있는방법으로, 주로 TaqMan probe원리가이용된다 [10]. 이는형광표지자가부착된탐식자와형광공명에너지전이현상 (fluorescence resonance energy transfer, FRET) 을이용하는것이다. 탐식자내보고색소 (reporter dye; FAM, VIC, TET, HEX) 의형광발현을억제하는억제색소 (quencher dye; TAMRA) 를핵산증폭과정에서 DNA polymerase로제거함으로써증폭주기마다증폭산물에비례하여형광이발생하도록하고, 증가하는형광의강도를실시간으로측정함으로써원검체에서의핵산농도를산출하는것이그원리이다 [11-13]. 본연구에서는 Abbott RealTime HBV 정량검사시약에대해정밀도, 직선성, 검출한계, 상관성, 교차반응등의성능을평가하고자하였다. 재료및방법 1. 성능평가대상시약 Abbott m2000rt (Abbott Molecular Inc., Abbott Park, IL, USA) 기기를이용하여 Abbott RealTime HBV Quantification kit (Abbott Molecular Inc.) 를평가하였다. Abbott m2000sp System (Abbott Molecular Inc.) 을이용하여혈청 200 L에서 HBV DNA 를추출하였다. 이때내부대조 (internal control, IC) DNA를첨가하여추출함으로써핵산추출및증폭과정상의오류를확인할수있도록하였다. 기기내에서추출한 HBV DNA에 50 L의반응혼합물 (master mix) 을혼합하고반응 plate에옮긴후 Abbott m2000rt로증폭과정을시행하였다. 증폭및검출과정은 Abbott m2000rt 기기내에서이루어지는데, HBV DNA sequence와내부대조가동시에증폭이이루어진다. 각증폭주기마다목표로하는 target DNA에가서붙은 probe의형광을측정함으로써정량할수있다. IC DNA에붙는 probe는 target DNA와는다른형광을내는것으로각주기마다 target DNA와같이정량할수있다. 매반응마다 PCR 의오염을확인하기위해음성대조에서음성의결과가나오는지를확인하며, 저농도및고농도정도관리물질을동시검사하여지정된범위내에드는지를확인하였다. 본검사의결과는 International Units (IU)/mL 또는 copies/ml로보고가능하며, 본연구에서는 copies/ml로결과를산출하였다. 2. 정밀도평가 CLSI EP A2의 precision verification기준에따라 1 10 6 과 1 10 3 copies/ml의양성대조물질을사용하여 20일간하루에 1회의검사를시행하였고, 매회마다세번반복측정하였다. 이결과를이용하여제조사에서제시한검사차례내정밀도 (within-run precision) 와검사일간정밀도 (between-day precision) 를평가하였다. 3. 직선성평가 Biosewoom HBV RQ-PCR kit (Biosewoom Inc., Seoul, Korea) 를이용하여이미농도를알고있는 HBV DNA 고농도환자검체를이용하여 10배수로연속희석하여 53-10 9 copies/ ml 범위에서각농도마다 2회씩반복측정하여직선성을평가하였다. 4. 검출한계검증검출한계를검증하기위하여 ACCURUN 325 HBV DNA positive control (BBI Diagnostics, Milford, MA, USA) 을 HBV 음성혈장으로희석하여 270, 135, 90, 67.5 및 33.75 copies/ml 농도의희석액을만들어 4-10회반복검사를실시하였다.

146 김명희 차충환 안동희외 2 인 5. 상관성비교서울아산병원에서라미부딘치료를받고있는만성간염환자 64명의검체를이용하여 Biosewoom HBV RQ-PCR kit[14] 와선형회귀모델을이용하여검사간상관성을평가하였다. 검사는당일 HBV DNA정량검사가의뢰된환자혈청을이용하여두기기모두에서당일내검사가이루어졌다. 64명의바이러스농도의분포는 2-4 log의경우 22 검체 (34.4%), 4-6 log 13검체 (20.3 %), 6-8 log 20검체 (31.3%), 8 log 이상의경우 9검체 (14.1%) 이었다. 또한 10개의검체를가지는 BBI panel (BBI diagnostics) 을이용하여 BBI panel에서제시하는값과비교하였다. 6. 교차반응평가 Architect (Abbott Laboratories) 에서 hepatitis B surface antigen (HBsAg) 음성이면서 hepatitis C virus (HCV) RNA 양성인 20개검체를이용하여평가하였다. 직선성을평가하기위해다항식회귀분석 (polynomial regression) 을시행하고, t-test로검정하였다. 검사간상관성분석은선형회귀분석을이용하였다. P<0.05일때통계적으로유의한것으로판정하고통계소프트웨어는 R 프로그램 verion 2.5.1 (R Foundation for Statistical Computing, Vienna, Austria) 을사용하였으며그래프는 Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA, USA) 을이용하여작성하였다. 결과 1. 정밀도 7.8 10 3, 1 10 6 copies/ml 두농도에대해검사차례내정밀도의변이계수 (within-run CV) 는 3.56-4.71% 이었으며검사일간정밀도의변이계수 (between-day CV) 는 3.03-4.98% 이었다. 2. 직선성 7. 검체간오염 (Carry-over) 평가 유럽연합체외진단의약품에대한공통성능기준 (Commission Decision of 7 May 2002 on common technical specifications for in vitro-diagnostic medical device) 에따라강양성검체 5개와음성검체 5개를교대로배치하여 2회검사하여평가하여검체간오염 (carry-over) 을평가하였다. Estimated HBV DNA concentration (Log10 copies/ml) 8. 통계분석 11 10 9 8 7 6 5 4 y=0.9045x+0.8316 R 2 =0.999 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 Nominal HBV DNA concentration (Log10 copies/ml) Fig. 1. The linearity range of Abbott RealTime HBV Quantification kit. Claimed linearity was verified with a result ranging from 53 to 10 9 copies/ml. Abbott RealTime HBV Quantification kit의직선성은 53-10 9 copies/ml 범위의경우선형회귀모델 (y=a+b 1 X) 에서 b 1 = 0.9045이면서 r 2 =0.999로직선성이유지되었다 (Fig. 1). 3. 검출한계검증 검출한계를검증하기위해시행한검사에서 Abbott Real- Time HBV는 33.75 copies/ml의 HBV DNA를 100% 의양성률로검출할수있었다 (Table 1). 따라서, 검출한계는제조사에서제시하는 51 copies/ml 이하인것을검증할수있었다. 4. 상관성비교 BBI panel에대해서는목표값 (target value) 이 160 copies/ ml 미만인 4개의검체중 3개의검체에대해서는양성의결과 Table 1. Detection limit of Abbott RealTime HBV Quantification kit Input HBV (Copies/mL) Result Replicates Positive reaction % 270 10 10 100 135 4 4 100 90 7 7 100 67.5 10 10 100 33.75 9 9 100

Evaluation of RealTime HBV Quantification Kit 147 Observed HBV DNA, Log10 copies/ml 8.00 6.00 4.00 2.00 y=1.0008x+0.369 R 2 =0.9933 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 Input HBV DNA, Log10 copies/ml Fig. 2. Correlation of test results between BBI control target value and Abbott RealTime HBV Quantification kit. Table 2. Carry-over tests of Abbott RealTime HBV Quantification kit Log 10 HBV concentration (copies/ml) Abbott RealTime HBV 10.00 8.00 6.00 4.00 2.00 y=1.0357x-0.7751 R 2 =0.9609 0.00 0.00 2.00 4.00 6.00 8.00 10.00 Log10 HBV concentration (copies/ml) BioSewoom Real-Q HBV Quantification Fig. 3. Quantitative comparison of Abbott RealTime HBV with Real-Q HBV Quantification kit. Specimen Results of day 1 (copies/ml) Results of day 2 (copies/ml) 6. 검체간오염 (Carry-over) 평가 1 Negative Negative 2 88,251,410 90,571,031 3 Negative Negative 4 123,554,860 86,190,454 5 Negative Negative 6 105,460,281 72,717,937 7 Negative Negative 8 80,464,680 76,413,781 9 Negative Negative 10 70,053,646 9,402,591 를보였고, 나머지 6검체에대해서는목표값의 3.2배범위에들었다. 목표값이 160 copies/ml 미만인 4개의검체를제외한나머지 6검체에서의상관성은 r 2 =0.9933이었다 (Fig. 2). Biosewoom Real-Q HBV Quantification kit에서 1,000 copies/ ml 이하이었던 12검체중 6검체는 Abbott RealTime HBV Quantification kit에서음성결과를보였다. 이들을제외하고 58개검체에대해상관성을평가한결과는기울기 1.0357, r 2 은 0.9609이었으며 (Fig. 3), Abbott RealTime HBV Quantification kit에서음성을보인결과들의정량값을 50 copies/ml 로대치하여상관성을평가한경우에는기울기 1.0571, r 2 은 0.9624로매우좋은상관성을보였다. 5. 교차반응평가 HBsAg 음성이면서 HCV RNA 양성인혈청 20검체로교차반응을평가한결과모든검체에서 HBV DNA가증폭되지않아 HCV RNA에의한교차반응은없는것으로판단하였다. 강양성 5검체와음성혈청 5개검체를교대로배치하여 2일간검사한결과양성검체와음성검체사이의검체간오염 (carryover) 은없는것으로나타났다 (Table 2). 고찰만성 B형간염은국내만성간질환의주된원인으로치료를시행하지않을경우대부분에서간경변증및간암등의합병증을유발하게된다. 현재만성 B형간염의치료는 interferon- 의주사나라미부딘 (lamivudine) 혹은아데포비어 (adefovir) 등의경구투여요법이흔히시행되고있다. 이러한치료약제의사용에있어혈청내 HBV의정량적측정은환자예후를예측하고치료반응을평가하여치료의시작과종료를결정하는매우중요한판단근거를제공하고있다. 문제가되고있는라미부딘의내성은치료 1년이후 14-32% 에서나타나며 [15-17] 치료 5년후에는 60-70% 에서나타나는데 [18, 19] 이는치료기간이긴경우, 치료전혈청내 HBV DNA 수치가높은경우, 그리고치료시작후남아있는바이러스의양이많은경우더높은빈도로나타난다고알려져있다 [3, 19]. 또한아데포비어내성은치료시작후 2년째에 20% 가넘는데, 이전에라미부딘내성으로단독요법을시행하는경우내성률이더높다 [20, 21]. 따라서이러한약제들로치료할경우치료의경과를예측하고약제내성변이주출현으로인한바이러스재활성 (viral breakthrough) 의조기발견을위해서 HBV 정량검사는필수검사가되었다. 2-10 log copies/ml의넓은측정범위를가지고있는 HBV DNA 정량검사법은실시간으로형광신호를관찰하기때문에

148 김명희 차충환 안동희외 2 인 증폭후전기영동을하는과정이필요없어검사보고시간이빠르고검사시발생할수있는 carryover 위험성을최소화할수있다 [22]. 또한본연구에서평가한 Abbott RealTime HBV assay는 HBV 유전체중 S 유전자의기시부위를목표 (target) 로하기때문에 core/precore 변이, HBsAg escape 변이, YMDD 변이및기타약제내성변이등에영향을받지않고모두검출할수있도록고안되었다. 또한 COBAS Amplicor HBV 시약처럼 core region을 target으로하는경우는유전자형 F의경우민감도가떨어지는것으로알려져있는반면 Abbott Real- Time HBV assay는모든유전자형에대해동일한민감도를보이는것으로알려져있다 [23, 24]. Abbott RealTime HBV Quantification Kit의정밀도는본연구에서 7.8 10 3, 1 10 6 copies/ml 두농도에대해검사차례내정밀도의변이계수의경우 3.56-4.71% 이었으며검사일간정밀도의변이계수의경우 3.03-4.98% 이었다. 이는이전에황등 [14] 에의해연구되었던 BioSewoom Real-Q HBV Quantitation kit의경우본검사와유사한농도를보이는구간에서검사차례내정밀도의변이계수 8.7-11.9%, 검사일간정밀도의변이계수는 13.2-21.4% 이었고, Weiss 등 [24] 이실시한 Roche COBAS TaqMan HBV Test의경우검사차례내정밀도의변이계수는 7.9-11.6%, 검사일간정밀도의변이계수는 13.0-18.6% 이었던것보다더좋은정밀도를보이는것이다. 직선성은 53-10 9 copies/ml 범위의경우선형회귀모델 (y=a+b 1 X) 에서 b 1 = 0.999로직선성이유지되었는데, 이는 51-3.1 10 9 copies/ml 에서직선성을보인다는제조사의주장을인정할만한결과로판단된다. 또한 33.75 copies/ml까지양성으로검출하였으므로제조사에서제시한값인 51 copies/ml을검출한계로인정할수있었다. 이는 Real-Q HBV Quantitation kit (56 copies/ ml) 이나 COBAS TaqMan HBV Test (34 copies/ml) 와는비슷한수준이라할수있다 [14, 24]. HCV양성검체와교차반응은관찰되지않았으며, HBV 강양성및음성검체를교대로검사한경우에도교차오염이발생하지않았다. 본연구에사용된검사키트와유사하게 HBV 유전체의 S 유전자를 target으로하는 Biosewoom Real-Q HBV Quantitation kit와의상관성평가에서는기울기 1.0357, r 2 =0.9609 로좋은상관성을보였다. 그러나 Biosewoom Real-Q HBV Quantitation kit에서는 1,000 copies/ml 이하로검출되었는데 Abbott RealTime HBV Quantification kit에서는음성으로나온검체가 6검체가있었다. 이번실험에서는검체의부족으로추가연구를시행하지못해어느결과가참인지를확인할수없었다. 낮은농도에대한 Abbott RealTime HBV Quantification kit 검출능은검출한계와낮은농도의 BBI panel 등으로평가하였는데, 두시약간검출한계의차이는없는것으로판단되었다. 또한 BBI panel과의상관성검사에서는 160 copies/ m 미만으로표기된검체중한검체만을검출하지못하였으나이외의모든대조물질에서좋은상관성을보여주었다. 본연구는 Biosewoom Real-Q HBV Quantitation kit의결과를기준으로 Abbott RealTime HBV Quantification kit와의성능을비교한것이므로비교한두키트중어느쪽이우월하다는결론을내릴수는없다. 그러나 1,000 copies/ml 이하에서는키트에따라검체내의어떤다른요인에의해위음성혹은위양성결과를보일수있다는점을고려하여해석할필요는있다고여겨진다. 결론적으로본연구를통하여평가된 Abbott RealTime HBV Quantification kit 는정밀도, 검출한계, 직선성, 상관성, 교차반응, 교차오염등의성능에있어다른실시간정량적중합효소연쇄반응 (RQ-PCR) 제품에비해손색이없다고판단되며, HBV DNA 정량을이용한만성 B형간염치료의추적관찰에유용하게사용될수있을것으로생각된다. 요약배경 : B형간염바이러스 DNA 정량검사는만성 B형간염환자에대한항바이러스제치료의시작과추적관찰을위해필요하다. 본연구에서는 Abbott RealTime HBV Quantification kit (Abbott Laboratories, USA) 의성능을평가하였다. 방법 : Abbott RealTime HBV Quantification kit의정밀도, 직선성, 검출민감도, 교차반응성및검체간교차오염여부를평가하였다. 서울아산병원에서라미부딘치료를받고있는만성간염환자 64명의혈청을이용하여 Real-Q HBV Quantification kit (BioSewoom Inc., Korea) 와의상관성을분석하였고, BBI panel (BBI diagnostics, USA) 을이용하여목표값과의일치도를비교하였다. 결과 : 평가시약의정밀도는검사차례내정밀도변이계수 3.56-4.71%, 검사일간정밀도변이계수는 3.03-4.98% 이었다. 53-10 9 copies/ml에서직선성이유지되었으며검출한계는 51 copies/ml임을검증할수있었다. C형간염바이러스양성검체에의한교차반응은없었으며, HBV 강양성검체와음성검체를교대로검사하여조사한결과도교차오염은없었다. Real-Q HBV Quantification kit와는우수한상관관계를보였으며 (r 2 =0.9609), BBI panel에대한검사에서목표값과유사하여좋은상관성을보였다 (r 2 =0.9933).

Evaluation of RealTime HBV Quantification Kit 149 결론 : Abbott RealTime HBV Quantification kit는성능이매우우수하므로국내에서도 HBV 감염의항바이러스제치료시작과모니터링에많이활용될것으로판단된다. 참고문헌 1. Di Bisceglie A, Lai CL, Gane E, Chen YC, Thongsawat S, Wang Y, et al. Telbivudine globe trial: maximal early HBV suppression is predictive of optimal two-year efficacy in nucleoside-treated hepatitis B patients. Hepatology 2006;44(S1):230-1. 2. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, et al. Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years. Gastroenterology 2006;131:1743-51. 3. Yuen MF, Sablon E, Hui CK, Yuan HJ, Decraemer H, Lai CL. Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001;34:785-91. 4. Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 2006;295:65-73. 5. Kim SH, Ki CS, Park HK, Cho HJ, Kim JW, Park SS, et al. Annual report on external quality assessment in diagnostic genetics in Korea (2006). J Lab Med Qual Assur 2007;29:153-74. ( 김선희, 기창석, 박현경, 조현정, 김종원, 박성섭등. 진단유전학분과신빙도조사결과보고 (2006). 임상검사와정도관리 2007;29:153-74.) 6. Konnick EQ, Erali M, Ashwood ER, Hillyard DR. Evaluation of the COBAS amplicor HBV monitor assay and comparison with the ultrasensitive HBV hybrid capture 2 assay for quantification of hepatitis B virus DNA. J Clin Microbiol 2005;43:596-603. 7. Pawlotsky JM, Bastie A, Hezode C, Lonjon I, Darthuy F, Remire J, et al. Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays. J Virol Methods 2000;85:11-21. 8. Lee HC, Suh DJ, Ryu SH, Kim H, Shin JW, Lim YS, et al. Quantitative polymerase chain reaction assay for serum hepatitis B virus DNA as a predictive factor for post-treatment relapse after lamivudine induced hepatitis B e antigen loss or seroconversion. Gut 2003; 52:1779-83. 9. Lok AS and McMahon BJ. Chronic hepatitis B. Hepatology 2007;45: 507-39. 10. Mackay IM. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004;10:190-212. 11. Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5 ----3 exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991;88:7276-80. 12. Lee LG, Connell CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 1993;21: 3761-6. 13. Valasek MA and Repa JJ. The power of real-time PCR. Adv Physiol Educ 2005;29:151-9. 14. Hwang SH, Cha CH, Kim YL, Kwon OJ, Oh HB. Performance evaluation of Real-Q HBV Quantification Kit for HBV DNA by real-time PCR. Korean J Lab Med 2006;26:442-8. ( 황상현, 차충환, 김유리, 권오중, 오흥범. 실시간정량적중합효소연쇄반응을이용한 Real-Q HBV Quantification Kit의성능평가. 대한진단검사의학회지 2006;26:442-8.) 15. Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z, et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256-63. 16. Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998;339:61-8. 17. Schalm SW, Heathcote J, Cianciara J, Farrell G, Sherman M, Willems B, et al. Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: a randomised trial. Gut 2000;46:562-8. 18. Chang TT, Lai CL, Chien RN, Guan R, Lim SG, Lee CM, et al. Four years of lamivudine treatment in Chinese patients with chronic hepatitis B. J Gastroenterol Hepatol 2004;19:1276-82. 19. Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, et al. Longterm safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology 2003;125:1714-22. 20. Fung SK, Chae HB, Fontana RJ, Conjeevaram H, Marrero J, Oberhelman K, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol 2006;44:283-90. 21. Lee YS, Suh DJ, Lim YS, Jung SW, Kim KM, Lee HC, et al. Increased risk of adefovir resistance in patients with lamivudine-resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy. Hepatology 2006;43:1385-91. 22. Klein D. Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 2002;8:257-60. 23. Laperche S, Thibault V, Bouchardeau F, Alain S, Castelain S, Gassin

150 김명희 차충환 안동희외 2 인 M, et al. Expertise of laboratories in viral load quantification, genotyping, and precore mutant determination for hepatitis B virus in a multicenter study. J Clin Microbiol 2006;44:3600-7. 24. Weiss J, Wu H, Farrenkopf B, Schultz T, Song G, Shah S, et al. Real time TaqMan PCR detection and quantitation of HBV genotypes A-G with the use of an internal quantitation standard. J Clin Virol 2004;30:86-93.