套式及免疫套式聚合酶链反应检测乙型肝炎表面抗原阴性肝病患者血清中乙型肝炎病毒DNA

NESTEDPOLYMERASECHAINREACTION(PCR)ANDMULTIPLECLONEDANTIBODYCAP┐TUREPCRFORTHEEXAMINATIONOFSERUMHEPATITISBVIRUSDNAINNEGATIVEHE┐PATITISBSURFACEANTIGENPATIENTSSUFFERINGFROMLIVERDISEASES

为了更准确、简便而又快速地了解乙型肝炎表面抗原（ＨＢｓＡｇ）阴性肝病患者的病因，建立了套式和免疫套式聚合酶链反应（ＰＣＲ）检测血清中乙型肝炎病毒（ＨＢＶ）ＤＮＡ的技术，结合丙型肝炎病毒（ＨＣＶ）感染指标的检测，对ＨＢｓＡｇ阴性肝病患者的病因进行了研究。发现套式ＰＣＲ能将单次ＰＣＲ的敏感性稳定地提高１０００倍；免疫套式ＰＣＲ可检测到０．１～０．０１ｐｇ／Ｌ水平。检测ＨＢｓＡｇ阴性肝硬化２２例（Ａ组）、ＨＢｓＡｇ阴性慢性肝炎１３例（Ｂ组）、ＨＢｓＡｇ阴性和乙型肝炎病毒核心抗体（抗－ＨＢｃ）阳性正常对照组３０例（Ｃ组）及ＨＢｓＡｇ（＋）／ＨＢｅＡｇ（－）肝硬化患者１２例（Ｄ组），分别有４５．５％、３０．８％、１３．３％和１００％患者血清中ＨＢＶＤＮＡ阳性。ＨＢＶＤＮＡ在一些抗－ＨＢｓ（＋）肝病患者和所谓健康人的血清中也存在。Ａ、Ｂ两组检出有ＨＢＶ和（或）ＨＣＶ感染患者分别占８１．８％和５３．８％。提示套式和免疫套式ＰＣＲ是简便、快速而又高度敏感的检测方法；ＨＢＶ感染可能是引起ＨＢｓＡｇ阴性慢性肝病的重要原因，且ＨＢｓＡｇ阴性肝病病因大多与病毒感染有关；应该重新认识自然感染者血清中抗－ＨＢｓ的临床意义。

Inordertofindoutrapidlythecausesoftheliverdiseasessuferedbypatientswithneg-ativehepatitisBsurfaceantigen(HBsAg),nestedpolymerasechainreaction(PCR)andmultipleclonedantibodycapturePCRtechniqueswereestablishedtoexamineserumhepatitisBvirus(HBV)DNA.ByusingbothtechniquesalongwiththeexaminationofhepatitisCvirus(HCV)infection,thecausesofchronicliverdiseaseswithnegativeHBsAgwerestudied.Itisfoundthatnested-PCRcanincreasethesensitivityofsinglePCRmorethan1000foldandmultipleclonedantibodycapture-PCRcandetectcon-centrationofHBVDNAaslowas0.1～0.01pg/L.HBVDNApositivepatientswerefoundin45.5%,30.8%,13.3%and100%respectivelyofthepatientssuferingfromlivercirhosiswithnegativeHBsAg(groupA,22cases),chronichepatitiswithnegativeHBsAg(groupB,13cases),normalsubjectswithnegativeHBsAgandpositivehepatitisBcoreantibody(HBcAb,groupC,30cases)andlivercirhosiswithpositiveHBsAgandnegativeHBeAg(groupD,12cases).HBVDNAcanbealsofoundintheserumofHBsAbpositivepatientsandsubjectssupposedtobehealthy.81.8%and53.8%ofthepatientswereinfectedwithHBVand/orHCVingroupAandgroupBrespectively.Altheseresultssuggestthatnested-PCRandmultipleclonedantibodycaputure-PCRarerapidandhighlysensitivemethodsforde-tectionofserumHBVDNA.HBVinfectionisanimportantcauseofchronicliverdiseasesinpatientswithnegativeHBsAg.ThecausesofmostoftheHBsAg-negativechronicliverdiseasesarerelatedwithinfectionofviruses.TheclinicalsignificanceofserumHBsAbinnaturalyinfectedpatientsshouldbere-considered.