甲型H1N1流感病毒实时荧光定量PCR标准阳性模板的构建  

Real-time fluorescent quantitative PCR positive standard template preparation of influenza virus H1N1

作　　者：方盛藩 郑夔[1] 李小波[1] 苏锦坤[1] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心卫生检疫实验室,广东广州510700

出　　处：《中国卫生检验杂志》2015年第1期1-3,共3页Chinese Journal of Health Laboratory Technology

基　　金：国家质检总局科研项目(2010IK222);国家质检总局科技计划项目(2013IK223)

摘　　要：目的制备甲型H1N1流感病毒核酸检测标准阳性模板,将其应用于日常甲型流感病毒荧光定量PCR(RTPCR)检测中。方法根据甲型H1N1流感病毒基因序列设计出M、HA、NA片段的全基因扩增引物,采用RT-PCR方法扩增以上3个基因片段,将3个目标片段分别连接到PGM-T载体上构建重组质粒,筛选出分别含M、HA、NA基因阳性质粒,将以上阳性质粒两两连接,最后制备成含M、HA、NA基因的标准阳性模板。结果此模板在-20℃保存3个月对荧光定量PCR结果无显著影响,具有较好的稳定性,Ct值最大变异系数为1.30%。使用该模板建立的定量范围为1×1010copy/ml^1×1019copy/ml,标准曲线方程为:y=-2.7232x+40.99。检测方法表明该标准阳性模板具有良好的特异性。结论本次制备成的标准阳性模板适用于各甲型H1N1流感病毒检测实验室的质量控制,可用于临床诊断。Objective To prepare the nucleic acid detection standard positive templates of influenza virus H1N1,and make it applied to the daily fluorescence quantitative PCR detection of influenza virus. Methods According to the gene sequences of influenza virus H1N1,designed the whole genome amplification primer of M,HA and NA pieces. Amplifying the three segments above by real- time polymerase chain reaction（ RT- PCR）,the three target segments were respectively connected to the PGM- T carrier to compare recombinant plasmid. Screened out positive plasmids containing M,HA and NA genes respectively,connected two of them respectively,and prepared the final positive standard template containing M,HA and NA genes. Results Keeping in the template- 20 ℃ for 3 months,this positive standard template had no significant effect on fluorescence quantitative PCR results,and showed its good stability,the maximum variation coefficient of Ct value was 1. 30%. Using this template the established quantitative range was 1 × 10^10 copy / ml ～ 1 × 10^19 copy / ml,standard curve equation was： y =- 2. 7232 x ＋ 40. 99. Dissolution curves show that the method had good specificity. Conclusion The prepared standard positive template was suitable for the quality control of the influenza virus H1N1 detection laboratory,and can be used for clinical diagnosis.

关 键 词：甲型H1N1流感病毒 标准模板 荧光定量聚合酶链式反应 

分 类 号：R511.7[医药卫生—内科学]