Hepatitis B Virus Quantitation 진단검사의학과 정유진 2004.5.13.

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Presentation transcript:

Hepatitis B Virus Quantitation 진단검사의학과 정유진

Serology Antigens HBsAg, HBeAg Antibodies IgM anti-HBc, Total anti-HBc, anti-HBe, anti-HBs Molecular Methods Signal Amplication Hybrid Capture (Digene) bDNA (Bayer) Target Amplification PCR Markers of HBV Infection

HBV DNA Quantitation Clinical Use HBV 간염의 follow-up 치료 약제의 개발과 치료경과 추적 약제 내성 검사방법 Bayer bDNA Roche COBAS AMPLICOR ™ Digene Hybrid Capture ® II HBV DNA

배경 본원 시행 : DIGENE 의 Hybrid Capture ® II HBV DNA HBV 치료약 개발을 위한 임상시험 : 본원이 Central Laboratory 역할 ( 용역연구 ) → LG 연구소 : YMDD Genotype, HBV Quntitation COBAS AMPLICOR ™ 의 HBV MONITOR ™

HBV Genome pre-S1 pre-S2 ORF-Surface HBsAg (envelope) ORF-Pol ORF- pre-C ORF-X L(-) S(+) DNA polymerase HBcAg (nucleocapsid) HBeAg HBV genome, partial ds DNA open reading frame “ORF” (unknown) Core / 1 gaps in fixed position bps long

COBAS AMPLICOR ™ System Routine Diagnostic PCR

HBV MONITOR ™ TEST Specimen Preparation Polymerase Chain Raction (PCR) Hybridization of the amplifed products to probes specific for the targets Dectection of the probe-bound amplified products

Serum 100 µL 50 µL Lysis 1 Mix, Incubate O C Lysis µL Specimen Preparation Centrifuge 15 min, Remove Supernatant Lysis 2 Buffer With QS DNA 50 µL 100 µL Mix, Incubate O C Centrifuge 15 min, 50µL A-Tube With Working Master Mix 50µL

HBV MONITOR Working MASTER MIX Mg 2+ dCTP dGTP dUTP dATP Taq Polymerase AmpErase ® Biotinylated Primers HBV-104UB HBV-104D Target HBV DNA HBV QS

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES HBVRUO PCRITS CE 05/16/00 page 20 of 35 Pertains to SAP# Preliminary Document. Not approved by Quality Management Denaturation & Hybridization Denaturation Denatured Amplicon Strands Biotin Magnetic Microparticles Capture Probe D-cup A-Tube Wash Wheel

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES HBVRUO PCRITS CE 05/16/00 page 21 of 35 Pertains to SAP# Preliminary Document. Not approved by Quality Management Detection Reaction (conjugate&substrate addition) Avidin-horseradish Peroxidase Conjugate forming complex with Biotin Tetramethylbenzidine (TMB) Substrate Hydrogen Peroxide Magnetic Microparticles Capture Probe

비 교

재료 대상 : ~ 명의 환자 21 검체 Amplicor Monitor: 높은 수치인 경우 희석 재검이 필요 ? 100 x, 1000 x, x

비교 결과

DiGene vs. Monitor

Amplicor Monitor 희석 결과 ( 단위 :10000 copies/mL)

결론 Study 진행 : x 로. HBV MONITOR ™ 는 Detection Range 가 낮은 범위에서 더 정확함. 그러나, 일반 검사용으로 사용 시 HBV MONITOR ™ 는 높은 결과치의 경우 검 체 희석 재검이 필요함.