鸭乙型肝炎病毒核酸荧光定量PCR方法的建立及应用

Establishment and application of the method for detecting DHBV DNA with fluorescence quantitative PCR

目的:建立鸭乙型肝炎病毒核酸(DHBV DNA)荧光定量的方法。方法:根据DHBV DNA S基因区的序列设计扩增所需3条引物,通过偶联反应用AmpliSensor荧光信号标记半巢式引物,经过前期不对称扩增、半巢式扩增及在线检测建立DHBV DNA阳性标准品QPCR的标准曲线,并将70份鸭血清分别用荧光定量PCR方法和地高辛标记的DHBV DNA探针斑点杂交的方法检测,比较结果的相关性。结果:DHBV DNA阳性标准品经过30次循环后,扩增产物经2％琼脂糖凝胶电泳检测,可以看出有180bp、70bp两个片段,与设计的片段大小相符,扩增产物的浓度与标准品的起始浓度成正比,扩增指数的数值大小与该循环时已合成的扩增产物的量成正比,起始浓度的大小与要合成的一定扩增产物的量所需的循环次数成反比。用荧定量PCR方法和斑点杂交的方法检测血清中DHBV DNA的结果有良好的相关性(r=0.97,P<0.01,v=58)。结论:荧光定量PCR方法可作为检测DHBV DNA含量的一个重要手段。

Objective: To establish the method for detecting duck hepatitis B virus (DHBV) DNA using fluorescence quantitative PCR. Methods: Three primers derived from DHBV DNA S gene were designed . The semi - nested primer was labelled by AmpliSen-sor using coupling reagent.The standard curve of the positive standards of DHBV DNA was established after asymmetric preamplifi-cation ,semi - nested amplification and on - line detetion. 70 samples of duck serum were tested by fluorescent quantitative DHBV DNA PCR method and dot blot hybridization assay using digoxigenin - labelled DHBV DNA probe. The correlation of results between the two methods was analysed. Results : After 30 cycles , amplification products showed two bands about 180bp and 70bp by 2% a-garose gel electrophoresis. The concentration of amplification products was in direct proportion to the initial concentration of the positive standards. The magnitude of detection index was in direct proportion to the amount of amplification products accumulating at the current cycle. The initial concentration of the positive standards was inversely proportional to the number of cycles needed for the amount of amplification products.The correlation coefficient of results was 0. 97 (P<0. 01) between fluorescent quantitative PCR method and dot blot hybridization assay. Conclusion : Fluorescence quantitative PCR can be used as a method to detect DHBV DNA.