| 张佳瑞 巩丽 朱少君 韩秀娟 姚丽 王姝妹 张伟 李艳红.PCR 在HBV核酸定量检测中的应用[J].,2014,14(28):5424-5426 |
| PCR 在HBV核酸定量检测中的应用 |
| Explore the Application of Quantitative PCR in the HBV Detect |
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| DOI： |
| 中文关键词: 竞争性PCR 拉米夫定 fRNA trRNA |
| 英文关键词: Competitive PCR Lamivudine fRNA trRNA |
| 基金项目:国家自然科学基金项目（30672013；81372226）；陕西省科技统筹创新工程（2011KTCL03-11） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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 103-104
times, and X DNA decreased 102 times. While fRNA and trRNA decreased around 10 times.Conclusion:HBV DNA and RNA can be detected
by the competitive PCR and it is likely to be useful to the HBV virus examination. |
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