Abstract
目的 采用自行研制的微量核酸释放剂(micro-nucleic acid releasing reagent, MNRR),建立微量血清直接进行PCR扩增的一步法实时荧光PCR检测拉米夫定治疗后YMDD变异的情况,探讨乙肝病毒YMDD变异与肝脏功能损伤程度的关系.方法设计包含YIDD和YVDD变异区和无变异参照的三条下游引物,与同一上游引物和探针建立3管荧光PCR扩增体系,变异Ct值与参照Ct值之差在3.3之内判断为阳性变异.建立将血清标本直接加到含有MNRR的扩增管进行核酸处理和PCR扩增的一步法检测.将0.2 ml 的PCR扩增管按3管/份标本排放到核酸提取仪,每管加入3.5 μl 的MNRR和3.5μl待测血清,用带虑芯吸头的加样器轻轻吹打混匀,加30 μl无菌石蜡油覆盖,于80℃静置8 min和10℃静置1 min,直接加入PCR扩增液,封盖后移至实时荧光PCR仪进行扩增.对154例采用拉米夫定治疗1年后的乙肝患者YIDD变异发生率进行分析,并研究其与肝脏功能损伤程度之间的关系.结果拉米夫定治疗1年后YIDD变异率为15.9%;YVDD变异率为9.6%,YIDD和YVDD共生变异率为4.4%.发生YIDD/YVDD共生变异的患者其血清HBV DNA 含量显著高于单一发生YIDD变异的患者(P<0.05),发生YMDD共生变异的乙肝患者其肝脏功能损伤的程度明显较单一发生YIDD变异者严重.结论拉米夫定治疗发生共生变异的患者其血清HBV DNA含量显著高于单一发生YIDD或YVDD变异的患者;发生YMDD共生变异的乙肝患者其肝功能损伤的程度明显重于无变异或单一发生YMDD变异的患者。
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