碱性磷酸酶直接标记核酸探针检测血清中鸭乙型肝炎病毒DNA方法的建立及应用

目的 :建立碱性磷酸酶直接 (AlkPhosDirec)标记核酸探针检测血清中鸭乙型肝炎病毒核酸 (DHBVDNA)方法。方法 :应用AlkPhosDirecTM] 将碱性磷酸酶直接标记纯化的DHBVDNA全基因制备探针 ,与目标核酸杂交后加入CDP -Star化学发光试剂 ,用胶片放射自显影检测DHBVDNA。同时检测了探针的灵敏度和特异性。比较了AlkPhosDirec标记DHBVDNA探针与地高辛标记的DHBVDNA探针检测鸭血清标本 10 0份结果。并用AlkPhosDirec标记探针检测血清中鸭乙型肝炎病毒核酸的方法考核了活性肽类物质保尔佳 (Polyerga)及其 8种单体体内抗鸭乙型肝炎病毒作用。 结果 :探针灵敏度为10 pg ,无非特异性的结果出现 ,与地高辛探针比较 ,用AlkPhosDirect标记探针检测方法的敏感性为 10 0 % ,特异性为 10 0 % ,符合率为 10 0 % ;抗鸭乙型肝炎病毒作用动物实验表明 ,保尔佳 0 0 1号单体 (CMS0 0 1)能使血清中DHBVDNA明显降低 (P<0 .0 5 )。结论 :AlkPhosDirec标记DHBVDNA探针检测血清中鸭乙型肝炎病毒核酸方法灵敏、特异 ,与地高辛标记的DHBVDNA探针检测结果完全相符合 ,可作为药物抗鸭乙型肝炎病毒作用动物实验疗效评价的手段。

Establishment and application of the method for detecting DHBV DNA in serum using DHBV DNA probe labeled directly by alkaline phosphatase

Objective:To establish the sensitive and specific technique for detecting DHBV DNA in serum using DHBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direct probe). Methods: The probe that purified DHBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP were used in the hybridization assay.DHBV DNA was detected by autoradiography on the film.The test compared the chemiluminescent dot blot hybridization assay for 100 samples with digoxigenin-labeled DHBV DNA probe detective method .The antiviral effects of polypeptide Polyerga and 8 kinds of monomers on duck hepatitis B virus in vivo were observed by AlkPhos Direct probe detective method. Results: The sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least.The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe detection.The monomer CMS001 could significantly reduce the serum level of DHBV DNA( P<0.05).Conclusion: The method detecting DHBV DNA in serum by DHBV DNA AlkPhos Direct probe is sensitive and specific.The results between two methods with AlkPhos Direct and digoxigenin-labeled HBV DNA probe are coincident completely.The method might be useful for the evaluation on anti-DHBV effects of drugs.