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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 explore a simple and convenient quantitative analysis system to detect the HBV DNA, fRNA and trRNA copy number in the serum of HBV carriers, so as to improve the detection effect of HBV in low replication state during occult infection. Methods: HBV DNA and RNA were extracted from serum and DNA of Core region and X region were amplified by PCR. Semi-nested PCR was used to reverse transcript and amplify fRNA and trRNA in the same centrifuge tube. The plasmid was used as a competition template to quantify it. The levels of HBV XDNA, Core DNA, fRNA and trRNA in serum of patients before and after lamivudine intervention were detected and compared. All test results were verified by Southern blotting. Results: Quantitative DNA analysis system and RNA-specific RT-PCR quantitative analysis system were established, and the changes of the structure and quantity of HBV DNA and X region before and after amifudine treatment were clarified. Pretreatment DNA and RNA copy number decreased after treatment. Core DNA decreased significantly, 103-104 times, while the XDNA copy number decreased 102 times. The fRNA and trRNA decreased only slightly, about 10 times. Conclusion: HBV DNA and RNA in serum can be quantitatively detected by competitive PCR in order to provide a more adequate basis for the diagnosis of HBV.