PCR在HBV核酸定量检测中的应用

Explore the Application of Quantitative PCR in the HBV Detect

目的:探索一种简便定量分析系统,通过检测HBV携带者血清中的HBV X区DNA,fRNA和trRNA拷贝数,为提高隐匿性感染期低复制状态HBV检测效果提供可能。方法:从血清中提取HBV DNA和RNA,对Core区、X区DNA进行PCR扩增,使用半巢式PCR法对fRNA、trRNA在同一离心管中进行反转录并扩增,选取大小相近的质粒作为竞争模板对其进行定量。并对拉米夫定干预治疗14周前后的患者血清中HBV XDNA、Core DNA、fRNA和trRNA拷贝数进行检测与比较。所有检测结果均通过southern杂交进行验证。结果:建立了针对DNA的定量分析系统及针对RNA的RT-PCR定量分析系统,并且阐明了阿米夫定治疗前后HBV DNA和X区RNA结构和数量的变化。治疗前治疗后DNA和RNA的拷贝数均下降。Core DNA下降显著,为103-104倍,而XDNA拷贝数下降102倍。而fRNA和trRNA仅有小幅下降,为10倍左右。结论:可以通过竞争性PCR方法对血清中HBV DNA和RNA进行定量检测,以期为HBV病毒的诊断提供更充足依据。

Objective： To build a feasible quantitative analysis system to improve the possibility of detection of the HBV at the occult infection according to the copy numbers of HBV XDNA, fRNA and trRNA in the serum. Methods： HBV DNA and RNA were extracted from serum samples. HBV Core DNA, X DNA were amplified by polymerase chain reaction （PCR）, and the heminested PCR were used in the reverse transcriptase-polymerase chain reaction （RT-PCR） system for fRNA and trRNA. They were marked by the plasmid sized around them as competitive templates. The copy numbers of HBV XDNA, Core DNA fRNA and trRNA in the serum treated by lamivudine for 14 weeks were compared with before. Results were verified by the southern hybridization. Results： The DNA quantitative system and RNA RT-PCR quantitative system were established. The exchange of the HBV DNA and RNA in number and structure were clarified. The copy number of DNA and RNA decreased after lamivudine treatment. Core DNA decreased dramatically by 10^3-10^4 times, and X DNA decreased 10^2 times. While fRNA and trRNA decreased around 10 times. Conclution： HBV DNA and RNA can be detected by the competitive PCR and it is likely to be useful to the RBV virus examination.