| 季节性流感病毒H1N1实时荧光定量PCR检测方法的建立 |
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| 引用本文： | 许黎黎,鲍琳琳,秦川.季节性流感病毒H1N1实时荧光定量PCR检测方法的建立[J].中国比较医学杂志,2011,21(7):62-66. |
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| 作者姓名： | 许黎黎 鲍琳琳 秦川 |
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| 作者单位： | 中国医学科学院医学实验动物研究所,卫生部人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,北京,100021 |
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| 基金项目： | 科技重大专项--艾滋病和病毒性肝炎等重大传染病防治:2009ZX10004-016，2009ZX10004-402，2009ZX10004-503;卫生公益性行业科研专项项目:200802036;中央级公益性科研院所基本科研业务费:DWS201112 |
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| 摘 要： | 目的建立一种快速定量检测季节性流感病毒H1N1核酸的实时荧光定量PCR检测方法及试剂盒。方法选择季节性流感病毒H1N1的保守基因NP基因作为检测靶目标,应用Clustal W软件进行序列同源性比对分析,筛选出季节性流感病毒H1N1特异性的保守序列作为引物候选区域,然后应用Primer Express及PrimerPremier 5.0软件包对候选引物进行进一步配对及筛选,得到最优特异性检测引物。同时,由病毒全长cDNA扩增出NP基因,琼脂糖凝胶电泳检测NP基因的扩增情况并对目的条带进行切胶回收及纯化,对回收后的NP全长基因进行核酸浓度测定,并换算成拷贝数,作为定量标准品。结果应用ABI公司的Power SYBR Green PCR MasterMix及StepOne实时荧光定量PCR仪,该检测系统灵敏度可达102 copies/μL,不同梯度标准品间线性关系(R2)达0.999,斜率为-0.3433,扩增效率为95.572%,所有标准品均在83.2℃出现尖且窄的特异性熔解峰。结论利用该检测系统可以快速定量检测季节性流感病毒H1N1,灵敏度高,可用作基础及临床实验室对季节性流感病毒H1N1感染的辅助诊断方法和临床效果的监测手段,对实验操作者要求相对较低,具有实际的应用价值。
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| 关 键 词： | 季节性流感病毒H1N1 荧光定量PCR 病毒载量 |
A Real-time PCR Detection System for Seasonal Influenza H1N1 Virus |
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| XU Li-li,BAO Lin-lin,QIN Chuan.A Real-time PCR Detection System for Seasonal Influenza H1N1 Virus[J].Chinese Journal of Comparative Medicine,2011,21(7):62-66. |
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| Authors: | XU Li-li BAO Lin-lin QIN Chuan |
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| Institution: | XU Li-li,BAO Lin-lin,QIN Chuan(Key Labortaory of Human Diseases Comparative Medicine,Ministry of Health,Institute of Laboratory Animal Science,Key Laboratory of Human Diseases Animal Model,State Administration of Traditional Chinese Medicine,Beijing 100021,China) |
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| Abstract: | Objective To develop a real-time PCR detection method and system for seasonal influenza H1N1 virus.Methods NP gene of seasonal influenza H1N1 virus was selected as the detection object.Sequences of different subtypes of influenza viruses were alignmented by using Clustal W software,and the most unique and conserved regions of seasonal influenza H1N1 virus were recruited as the candidate sequences for specific primers.Primer Express and Primer Premier 5.0 softwares were used to filter the optimizing pair of ... |
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| Keywords: | Seasonal influenza H1N1 virus Real-time PCR Viral load |
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