| 武海萍 刘云龙 童欢 马雪萍 卜莹 周国华.高灵敏焦测序结合生物发光分析仪检测甲型 H1 N1 流感病毒[J].,2014,14(35):6818-6822 |
| 高灵敏焦测序结合生物发光分析仪检测甲型 H1 N1 流感病毒 |
| Detection of Influenza A (H1 N1 ) Virus Using Highly SensitivePyrosequencing Combining Bioluminescence Analyzer |
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| DOI： |
| 中文关键词: 高灵敏焦测序 甲型 H1N1 流感病毒 耐药性突变 便携式生物发光分析仪 |
| 英文关键词: Highly sensitive pyrosequencing Influenza A (H1N1 ) virus Drug resistance mutations Portable bioluminescence
analyzer |
| 基金项目:国家自 然科学基金项目（ 31 300706）；江苏省科技支撑计划 - 社会发展项目（BE2011 607）；
第 54 批中国博士后科学基金面上资助项目（ 201 3M542575） |
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| 中文摘要: |
| 目的： 建立基于核酸序列 分析的快速、准确、低成本的 甲型 H1N1 流感病毒检测 方法。 方法： 通过优化焦测序反应体系中
ATP 硫酸化酶和荧光素酶的浓度，建立高灵敏的焦测序反应体系；将该体系应用 于低成本、小型化的便携式生物发光分析仪， 焦
测序分析流感病毒 M、 NP、HA 基因 片段的核酸序列 。 结果： 优化后的焦测序反应体系可检测低至 10 fmol 的 DNA 样本， 检测灵敏
度较传统焦测序提高了 10 倍以上。 对两例样本进行检测， 根据所测得的 M、NP、 HA 基因 特异性片段序列， 可以确认其均为甲型
H1 N1 感染； 另 外， 对 M2 蛋白阻断剂 耐药性标志位点（ S31N 突变）的测定结果显示该病毒存在 S31N 突变， 为 M2 蛋白 阻断剂 耐
药型。 结论： 高灵敏焦测序体系结合便携式生物发光分析仪成功实现了 对甲型 H1N1 流感病毒快速、准确的低成本检测。 |
| 英文摘要: |
| Objectivre:To establish a rapid, accurate and low-cost influenza A (H1 N1 ) virus detection method based on nucleic
acid sequence analysis.Methods:A highly sensitive pyrosequencing system was establised by optimizing the concentrations of ATP
sulfurylase and luciferase respectively; the nucleic acid sequences of M， NP， HA gene fragments of influenza virus were analysed with a
low-cost and portable bioluminescence analyzer by coupling the highly sensitive pyrosequencing system.Results:Compared with
conventional pyrosequencing system, the sensitivity of the proposed method increased more than 10 times, and as low as 1 0 fmol of DNA
samples can be detected. Based on the pyrosequencing results of the M, NP, HA genes, we confirmed the infection of influenza A
(H1 N1 ) virus in the two samples. The sequencing results of the gene coding M2 protein inhibitor drug resistance marker (S31N mutation)
proved the presence of S31 N mutation, indicating that the virus is M2 protein inhibitor-resistant.Conclusion:A rapid, accurate and
low-cost detection method of influenza A (H1N1 ) virus was established by combining the highly sensitive pyrosequencing system with a
portable bioluminescence analyzer. |
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