快速检测乙型肝炎病毒前核心区基因变异株方法的建立与初步应用

A RAPID METHOD FOR DETECTION OF HEPATITIS B VIRUS PRE-CORE VARIANTS AND ITS PRELIMINARY APPLICATION

用聚合酶链反应（ＰＯＲ）选择性地扩增血清标本中乙型肝炎病毒（ＨＢＶ）基因前Ｃ区（Ｐｒｅ－Ｃ）后，以三种寡核苷酸探针Ｍ０（野毒株基因序列）、Ｍ１（ｎｔ．１８９８位点突变株基因序列）、Ｍ２（ｎｔ．１９９８位及ｎｔ．１９０１位上双点突变株基因序列）在极其严谨的条件下分别杂交，检测ＥＢＶＰｒｅ－Ｃ的最常见变异，结果本法具有根高的特异性。应用上述方法检测了３１份乙肝病人的血清标本，结果７份ＨＢｅＡｇ阳性标本中，有４份ＰＣＲ阳性，均与Ｍ０探针杂交，未出现变异；２４份抗－ＨＢｅ阳性标本中，有１０份ＰＣＲ阳性，其中８份表现为Ｍ０伴Ｍ１和（或）Ｍ２阳性，一份为野毒株，另一份与三种探针均不杂交。

We describe here a rapid method to screen for hepatitis B virus(HBV)variants containing a TAG stop codon in the distal Pre-Core region.The entire Pre-Core region was amplified by the polymerase chain reaction (POR)and hybridized under stringent conditions with oligonucleotide probes of Mo(wild-type),M_1(one point mutation at nt.1898),M_2(two point mutations at nt.1898 and nt.1901),Results indicate that this method is highly specific that can identify even a single mismatch.Thirty one serum samples from viral hepatitis B were analysed using the established method,of the 7 HBeAg positive sera,4 samples were POR positive,in which all hybridize with Mo only.Among the M anti-HBe postive sera,10samples were PCR postive,which 80%(8/10) hybridized with M_1 or M_2,or both,only 1 sample failed to hybridize with any of the there probes.This Oligonucleotide bybridization assay is therefore a rapid and reliable method which can be Cmployed in clinical diagnosis or molecular epidemiological survey of HBV Pre-Core variants.