平行等位基因特异序列检测技术对低比率乙型肝炎病毒耐药突变位点的检测

Detection of minority drug-resistant mutations in patients infected with hepatitis B virus by parallel allele-specific sequencing

目的评价应用平行等位基因特异性序列检测技术(parallel allele-specific sequencing,PASS),对乙型肝炎病毒(HBV)进行耐药突变位点检测效果。方法应用PASS方法对17例未经抗病毒治疗和50例经抗病毒治疗失败的乙肝患者血浆进行耐药突变检测,其中50例乙肝抗病毒治疗失败的患者血浆同时采用测序法进行耐药分析。结果 17例未接受抗病毒治疗的患者样本中有8例检测到低比率的耐药突变位点(0.04%～16.89%),9例未发现有耐药突变。50例抗病毒治疗失败患者标本有7例PASS法和测序都未检测到耐药突变,16例PASS法检测到低比率耐药突变(0.03%～5.95%),测序法未检测到任何突变位点,27例PASS法和测序法都检测到了耐药突变(36%～100%)。应用PASS法进行连锁分析表明当多个耐药突变位点以高比率在同一患者体内检测到时,它们多以连锁的关系存在于同一病毒基因组。结论 PASS方法对检测HBV感染个体中耐药病毒群具有高度的灵敏性和特异性,并能对多个耐药突变位点进行连锁性分析。

Objective To detect hepatitis B virus （HBV） anti-viral drug-resistant mutations by parallel allele-specific sequencing （PASS）.Methods Seventeen baseline patients and fifty patients who failed antiviral treatment were analyzed using both PASS and clonal sequencing methods.Results In seventeen baseline patients,minor drug-resistant mutations （0.04% ～16.89%） were detected in eight patients by PASS assay.In all fifty treatment-failure patients,drug-resistant mutations were not detected by either method in seven patients.Minor drug-resistant mutations （0.03%～ 5.95%） were detected by PASS assay,but not by PCR product population sequencing in sixteen patients.Major drug-resistant mutations （36%～100%） were detected by both methods in twenty-seven patients.In addition,PASS detected minority drug-resistant mutations,while population sequencing only detected majority drug-resistant mutations.Linkage analysis by PASS also showed that linked drug-resistant mutations in the same viral genome were detected in majority of the viruses in patients in whom multiple drug-resistant mutations were persent.Conclusions PASS assay can detect minority drug-resistant mutations in HBV-infected individuals with high sensitivity and specificity and can be used for linkage analysis of multiple drug-resistant mutations.