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A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus
基于SYBR Green荧光染料的实时逆转录聚合酶链式反应(RT-PCR)试验检测新生 H7N9 病毒   

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Abstract

Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Molecular diagnostic methods are thus urgently needed in public health laboratories. We developed a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) to detect the novel H7N9 virus.

Materials and Reagents

  1. Respiratory specimens (throat-swabs, sputum and tracheal aspirate) from patients with influenza-like illnesses
  2. Madin-Darby canine kidney (MDCK) cells (Shanghai Institutes for Biological Sciences)
  3. Eagle's Minimum Essential Medium (EMEM) (Life Technologies, Gibco®, catalog number: 11095-080 )
  4. Fetal bovine serum (Life Technologies, Gibco®, catalog number: 10099 )
  5. Viral transportation medium (Yocon Bio-technology, catalog number: MT0301-1 )
  6. QIAsymphony Virus/Bacteria Mini Kit (QIAGEN, catalog number: 931036 )
  7. Nuclease-free water (Life Technologies, catalog number: 10977 )
  8. SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Life Technologies, catalog number: 11736 )
  9. The primer sequences used for detection of influenza A H7N9 virus

    Primera
    Sequence (5’-3’)b
    PCR amplicon (bp)
    H7F
    H7R
    N9F
    N9R
    TGAAAATGGVTGGGAAGGYY
    TGCCGATTGRGTGCTYTTRT
    ACAGTGTACAAYAGCARRGT
    GTTTCGRGCCCAYGTRTTAA
    103

    165
    a F: Forward; R: Reverse
    b V: A/C/G; Y: C/T; R: A/G

Equipment

  1. T-25 cell culture flask (Corning, catalog number: 430168 )
  2. A biosafety 3 laboratory (BSL-3) (required for virus isolation)
  3. QIAsymphony SP instrument (QIAGEN, catalog number: 9001297 )
  4. ABI Prism 7900HT Sequence Detection System (Life Technologies, Applied Biosystems®, catalog number: 4329001 )

Software

  1. Sequence Detection System Software (v2.3) (Life Technologies, Applied Biosystems®)

Procedure

  1. Virus isolation
    1. MDCK cells were cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% heat-inactivated fetal bovine serum (FBS) in a T-25 cell culture flask and incubated at 37 °C under a humidified atmosphere with 5% CO2.
    2. Disperse the respiratory specimens (throat-swabs, sputum and tracheal aspirate) maintained in the viral transportation medium by vortex for 1 min. Sputum was pre-diluted with an equal volume of PBS solution prior to a vortex procedure.
    3. When the MDCK cells grew to 85% confluence, 0.5 ml of the respiratory specimens positive for H7N9 virus infection was inoculated into cells.
    4. Cytopathic effects were observed every day by microscopy.
    5. When 90% of MDCK cells experienced cytopathic effects (Figure 1), the cell culture supernatants were harvested by centrifuge at 2,000 rpm for 10 min and stored at -70 °C until use.


      Figure 1. Rounding of infected cells, fusion with adjacent cells to form a syncytia and ultimate lysis, as compared with non-infected cells. A: Non-infected cells; B: Cells with cytopathic effects.

    6. 200 μl of cell culture supernatants was subjected to RNA extraction. Purified viral nucleic acid was used as assay control.
  2. RNA extraction
    1. Viral RNA was extracted from 200 μl of cell culture supernatants or respiratory specimens using QIAsymphony Virus/Bacteria Mini Kit combined with the QIAsymphony SP instrument, following the manufacturer’s instructions.
    2. The extracted RNA was eluted in 60 μl of nuclease-free water. RNA integrity was analyzed by agarose gel electrophoresis and then stored at -70 °C until use.
  3. SYBR green-based real time RT-PCR assay
    1. The one-step real time quantitative RT-PCR assays were performed to amplify the HA and NA genes of H7N9 virus respectively using SuperScript III Platinum SYBR Green One-Step qRT-PCR kit. The assay was run in duplicate or triplicate for each unknown sample.
    2. The assay was carried out in a 10 μl reaction mixture containing:
      5 μl of 2x SYBR Green Reaction Mix
      0.8 μM of each primer
      0.2 μl of ROX Reference Dye (500 nM)
      0.2 μl of SuperScript III RT/Platinum Taq Mix
      1 μl of purified RNA
      x μl of nuclease-free water
    3. The optimized thermal cycling conditions were as follows:
      1. A reverse transcription step at 50 °C for 10 min
      2. An initial denaturation step at 95 °C for 5 min
      3. 40 cycles of PCR amplification at 95 °C for 15 sec, 60 °C for 20 sec, and 72 °C for 30 sec, followed by a melting curve analysis program according to the instrument documentation.
    4. Finally, data were collected and results were analyzed with the use of Sequence Detection System Software v2.3. The Tm values of H7 and N9 specific amplicons were 80.77 and 81.20, respectively.

Acknowledgments

This experimental protocol was partly adapted from the previously published paper: Zhu et al. (2013). This protocol was supported in part by the Jiangsu Province Health Development Project with Science and Education (ZX201109), the Jiangsu Province Key Medical Talent Foundation (JKRC2011002, RC2011191), and the Science and Technology Pillar program of Jiangsu Province (BE2011796). We thank Dr. Shu Yuelong, China National Influenza Center, Chinese Center for Disease Control and Prevention, Beijing, China, for providing the entire gene sequence data of the first three H7N9 virus isolates used for primer design.

Reference

  1. Zhu, Z., Fan, H., Qi, X., Qi, Y., Shi, Z., Wang, H., Cui, L. and Zhou, M. (2013). Development and evaluation of a SYBR Green-based Real Time RT-PCR assay for detection of the emerging avian influenza A (H7N9) birus. PLoS One 8(11): e80028.

简介

最近,一种新型禽流感A型(H7N9)病毒在中国出现,并且与许多人类感染和致命病例有关。 因此,公共卫生实验室迫切需要分子诊断方法。 我们开发了基于SYBR绿色的一步实时逆转录PCR(RT-PCR),以检测新型H7N9病毒。

材料和试剂

  1. 来自流感样疾病患者的呼吸道标本(喉拭子,痰和气管吸出物)
  2. Madin-Darby犬肾(MDCK)细胞(上海生物科学研究所)
  3. Eagle的最低必需培养基(EMEM)(Life Technologies,Gibco ,目录号:11095-080)
  4. 胎牛血清(Life Technologies,Gibco ,目录号:10099)
  5. 病毒运输培养基(Yocon Bio-technology,目录号:MT0301-1)
  6. QIAsymphony Virus/Bacteria Mini Kit(QIAGEN,目录号:931036)
  7. 无核酸酶水(Life Technologies,目录号:10977)
  8. SuperScript III Platinum SYBR Green一步法qRT-PCR试剂盒(Life Technologies,目录号:11736)
  9. 用于检测甲型流感H7N9病毒的引物序列

    底漆 a
    序列(5'-3') b
    PCR扩增子(bp)
    H7F
    H7R
    N9F
    N9R
    TGAAAATGGVTGGGAAGGYY
    TGCCGATTGRGTGCTYTTRT
    ACAGTGTACAAYAGCARRGT
    GTTTCGRGCCCAYGTRTTAA
    103

    165
    F:Forward; R:反向
    b V:A/C/G; Y:C/T; R:A/G

设备

  1. T-25细胞培养瓶(Corning,目录号:430168)
  2. 生物安全3实验室(BSL-3)(病毒分离所需)
  3. QIAsymphony SP仪器(QIAGEN,目录号:9001297)
  4. ABI Prism 7900HT序列检测系统(Life Technologies,Applied Biosystems ,目录号:4329001)

软件

  1. 序列检测系统软件(v2.3)(Life Technologies,Applied Biosystems )

程序

  1. 病毒隔离
    1. MDCK细胞在Eagle's Minimum Essential Medium(EMEM) 在T-25细胞中含有10%热灭活的胎牛血清(FBS) 并在37℃,在潮湿气氛下温育 5%CO <2>。
    2. 分散呼吸道标本(咽喉拭子, 痰和气管吸出物)维持在病毒运输中 介质通过涡旋1分钟。 痰用等体积预稀释 的PBS溶液,然后进行涡旋程序
    3. 当MDCK 细胞生长至85%汇合,0.5ml呼吸标本 将H7N9病毒感染的阳性接种到细胞中
    4. 每天通过显微镜观察细胞病变效应
    5. 当90%的MDCK细胞经历细胞病变效应(图1)时, 通过在2,000rpm离心收获细胞培养物上清液 10分钟,储存于-70℃直至使用

      图1.舍入 感染的细胞,与相邻细胞融合以形成合胞体 最终裂解,与未感染的细胞相比。 A:未感染 细胞; B:具有细胞病变效应的细胞
    6. 200微升细胞 培养上清液进行RNA提取。 纯化病毒 核酸用作测定对照。
  2. RNA提取
    1. 从200μl的细胞培养上清液或提取病毒RNA 呼吸标本使用QIAsymphony Virus/Bacteria Mini Kit组合   与QIAsymphony SP仪器,遵循制造商 指令。
    2. 提取的RNA用60μl洗脱 无核酸酶水。 通过琼脂糖凝胶分析RNA完整性 电泳,然后储存在-70℃直至使用。
  3. 基于SYBR绿色的实时RT-PCR测定
    1. 进行一步实时定量RT-PCR测定 使用SuperScript分别扩增H7N9病毒的HA和NA基因   III铂金SYBR Green一步法qRT-PCR试剂盒。 进行测定 每个未知样品重复或重复三次。
    2. 该测定在含有:
      的10μl反应混合物中进行 5μl2x SYBR Green Reaction Mix
      0.8μM的每条引物
      0.2μlROX参比染料(500nM)
      0.2μl的SuperScript III RT/Platinum 混合物
      1μl纯化的RNA xμl不含核酸酶的水
    3. 优化的热循环条件如下:
      1. 在50℃下10分钟的逆转录步骤
      2. 在95℃5分钟的初始变性步骤
      3. 95个循环的PCR扩增15秒,60℃20秒, 和72℃30秒,然后进行解链曲线分析程序 根据仪器文档。
    4. 最后,数据 并使用Sequence分析结果 检测系统软件v2.3。 H7和N9特异性的T m1值 扩增子分别为80.77和81.20。

致谢

该实验方案部分改变自先前发表的论文:Zhu等人(2013)。 该方案由江苏省科技教育健康发展项目(ZX201109),江苏省重点医学人才基金(JKRC2011002,RC2011191),江苏省科技支柱计划(BE2011796)部分支持。 我们感谢中国北京疾病预防控制中心中国国家流感中心Shu Yuelong教授提供用于引物设计的前三个H7N9病毒分离株的全部基因序列数据。

参考

  1. Zhu,Z.,Fan,H.,Qi,X.,Qi,Y.,Shi,Z.,Wang,H.,Cui,L.and Zhou,M.(2013)。 基于SYBR Green的实时RT-PCR测定法的开发和评估,用于检测新出现的 禽流感A(H7N9)birus。 PLoS One 8(11):e80028。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, Z. and Cui, L. (2014). A SYBR Green-based Real Time RT-PCR Assay for Detection of the Emerging H7N9 Virus. Bio-protocol 4(12): e1150. DOI: 10.21769/BioProtoc.1150.
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