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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 a positive template for the detection of influenza A (H1N1) virus nucleic acid and to apply it in routine quantitative PCR (RTPCR) detection of influenza A virus. Methods According to the sequence of the influenza A (H1N1) virus, the whole genome amplification primers of M, HA and NA fragments were designed. The above three gene fragments were amplified by RT-PCR, and the three target fragments were respectively linked to the PGM-T vector Recombinant plasmids were constructed and positive plasmids containing M, HA and NA genes were screened out. The above positive plasmids were ligated to each other. Finally, a standard positive template containing M, HA and NA genes was prepared. Results The template stored at -20 ℃ for 3 months had no significant effect on the results of real-time PCR, and had a good stability. The maximum coefficient of variation of Ct was 1.30%. The quantitative range established using this template was 1 × 10 10 copies / ml to 1 × 10 19 copies / ml. The standard curve equation was: y = -2.7232 × + 40.99. The detection method shows that the standard positive template has good specificity. Conclusion The prepared standard positive template is suitable for the quality control of each influenza A H1N1 influenza virus testing laboratory and can be used for clinical diagnosis.