| 张强,唐青,刘卫滨,李浩,梁国栋.狂犬病毒TaqMan PCR检测方法的建立[J].Chinese journal of Epidemiology,2006,27(10):889-893 |
| 狂犬病毒TaqMan PCR检测方法的建立 |
| Establishment of TaqMan PCR detection method for rabies virus |
| Received:December 02, 2005 |
| DOI： |
| KeyWord: 狂犬病毒 TaqManPCR检测 定量 |
| English Key Word: Rabies virus TaqMan PCR Quantification |
| FundProject: |
| Author Name | Affiliation | E-mail | | ZHANG Qiang | Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention,Beijing 100052,China | | | TANG Qing | Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention,Beijing 100052,China | qtang04@sina.com | | LIU Weibin | Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention,Beijing 100052,China | | | LI Hao | Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention,Beijing 100052,China | | | LIANG Guodong | Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention,Beijing 100052,China | |
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| Abstract: |
| 目的建立检测狂犬病毒(RV)核蛋白(N)基因片段的TaqMan PCR检测方法。方法针对RV N基因保守区域设计特异性引物与探针;优化检测体系中引物与探针的浓度;以体外转录的RV完整的N基因RNA作为定量分析模型;考核检测体系灵敏性、特异性、稳定性;初步用于RV的检测。结果引物和探针具有良好的特异性,使用浓度分别为0．6μmol/L和0．2μmol／L,可检测到2700个RNA拷贝数,较传统RT-PCR检测方法敏感性提高了10倍;同一样品Ct值重复检测5次,变异系数均＜5%,表明检测体系具有较好的稳定性;通过所建立的检测体系,绘制了以模板拷贝数为分析指标的RV定量检测标准曲线,对实验室内保存毒株的检测结果显示TaqMan PCR检测比普通PCR检测更快捷、敏感。结论所建立的RV TaqMan PCR可以用于RV的实验室检测,并且比普通PCR检测方法更灵敏、特异。 |
| English Abstract: |
| Objective To establish a molecular diagnostic method for rabies virus(RV) based on TaqMan PCR. Methods Base on the rabies virus nucleoprotein gene sequences published in GenBank,RV specific primers and probe were designed by Primer Premier 5.0. The primers and probe were optimized and the sensitivity,specificity,and reproducibility of the system were tested. Quantitative standard curve of RV TaqMan PCR was established. Some RV samples were detected using this system. Results The optimized primers and probe were 0.6μmol/L and 0.2μmol/L. Reproducibility test showed that coefficient variables were all less than 5 % in 4 different system. Quantification standard curve based on the genomic copy was drawn. RV detection using the established method proved that TaqMan PCR was more sensitive and easier performed than traditional RT-PCR. Conclusion TaqMan PCR for RV detection had been established, which was more sensitive and specific than the general RT-PCR. |
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