用热启动聚合酶链反应检测血清中乙型肝炎病毒DNA技术的建立和应用

建立了热启动聚合酶链反应（ＰＣＲ）检测乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）的技术。ＰＣＲ所有反应成分被２次加样。先加Ａ液（含ｄＮＴＰｓ、一对引物和ＭｇＣｌ＿２）与一粒石蜡珠。７０℃加热后冷却至室温，使蜡珠先融化后凝固形成蜡盖封住Ａ液，然后在向其上加入Ｂ液（含耐热性ＤＮＡ聚合酶、ＨＢＶＤＮＡ模板和ＫＣｌ）并开始循环扩增。当反应管内第一次变性温度升至６０℃以上时，中隔蜡层融化，蜡上浮形成防蒸发屏障，Ａ、Ｂ两液则由于热分子运动而混匀，从而保证引物与靶基因在较高温度下严格退火以减少错导非特异性扩增和引物聚体形成。提高了ＰＣＲ的特异性和灵敏性，应用这种技术对７６例肝炎血清进行了检测，结果ＨＢＶＤＮＡ检出率为６８％，其中ＨＢｓＡｇ（＋）、ＨＢｅＡｇ（＋）、抗－ＨＢｃ（＋）血清检出率为１００％；ＨＢｓＡｇ（＋）、抗－ＨＢｅ（＋）、抗－ＨＢｃ（＋）血清检出率为７０％；ＨＢｓＡｇ（＋）、抗－ＨＢＣ（＋）血清检出率为８９％；抗ＨＢＣ（＋）血清检出率为对％；标记全阴性血清检出率为３３％。

Detection of Hepatitis B Virus DNA in Serum by Hot Start Polymerase Chain Reaction

The technique of hot start polymerase chain reaction used to detect HBV DNA in sera was established.The reactants were divided into two groups, the first containing primers, dNTPs and MgCl_2which were loaded at bottom of the tube with a wax bead,the other test sample,Taq DNA polymerase and KCl which were added on the solidified wax after the first is heated to make the wex melted and than cooled to solidify it to form a cover that could separte the two.In such a way,the complete mixture of all agents was delayed until the temperature rose to an extent in which the primer annealing to nontarget sequences was prevented.By the technique,76 sera were tested and HBV DNA was detected in 68%. Among them,HBV DNA was pick up in 100% which were positive to HBsAg, HBeAg and anti-HBc;in 70% positive to HBsAg,anti-HBe and anti-HBc;in 89% positive to HBsAg and anti-HBc:in 31% positive to anti-HBc and in 33% negative to all HBV serum markers.