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绵羊肺炎支原体RPA检测技术的建立

林裕胜 江锦秀 张靖鹏 游伟 胡奇林

林裕胜, 江锦秀, 张靖鹏, 游伟, 胡奇林. 绵羊肺炎支原体RPA检测技术的建立[J]. 福建农业学报, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006
引用本文: 林裕胜, 江锦秀, 张靖鹏, 游伟, 胡奇林. 绵羊肺炎支原体RPA检测技术的建立[J]. 福建农业学报, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006
LIN Yu-sheng, JIANG Jin-xiu, ZHANG Jing-peng, YOU Wei, HU Qi-lin. Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae[J]. Fujian Journal of Agricultural Sciences, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006
Citation: LIN Yu-sheng, JIANG Jin-xiu, ZHANG Jing-peng, YOU Wei, HU Qi-lin. Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae[J]. Fujian Journal of Agricultural Sciences, 2019, 34(4): 416-421. doi: 10.19303/j.issn.1008-0384.2019.04.006

绵羊肺炎支原体RPA检测技术的建立

doi: 10.19303/j.issn.1008-0384.2019.04.006
基金项目: 

国家重点研发计划项目 2016YFD0500906

福建省科技计划项目——省属公益类科研院所基本科研专项 2018R1101014-17

福建省农业科学院科技创新项目 PC2018-6

福建省财政专项——福建省农业科学院科技创新团队建设项目 STIT2017-1-5

福建省农业科学院青年创新团队项目 STIT2017-3-10

详细信息
    作者简介:

    林裕胜(1988-), 男, 硕士, 助理研究员, 主要从事动物传染病研究

    通讯作者:

    胡奇林(1963-), 男, 研究员, 主要从事动物传染病学和免疫学研究(E-mail:hql562713@163.com)

  • 中图分类号: S852.62

Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae

  • 摘要:   目的  建立一种快速简便检测绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)的方法。  方法  根据Mo膜蛋白P80基因序列,利用Oligo 7软件设计并筛选出特异性扩增引物,通过条件优化建立检测Mo的RPA方法。  结果  该方法可特异性扩增Mo,对其他羊常见病原无特异性扩增,对Mo核酸的检测灵敏度为70 fg·μL-1,与常规PCR敏感性一致。批内和批间结果显示Mo阳性样品均能扩增条带,而阴性对照均无扩增,表明重复性好。对186份临床样品应用建立的RPA方法和常规PCR方法同时进行检测,并对其中40份肺脏样品进行支原体的分离鉴定,结果显示分离鉴定出的13份Mo阳性样品及常规PCR法检测出的95份阳性样品经RPA检测,结果均为阳性。  结论  建立的Mo RPA方法特异性强、重复性好,可作为Mo的快速检测和流行病学调查的一项候选技术。
  • 图  1  Mo P80基因的RPA扩增结果

    注:M为1 000标注; 1为阴性对照; 2为Y98 P80基因。

    Figure  1.  Amplification of P80 gene in Mo by RPA

    Note:M: DNA Marker DL1 000; 1: Negative control; 2:Y98 P80 gene.

    图  2  Mo RPA特异性试验结果

    注:M为1 000标注; 1~3为绵羊肺炎支原体Y98、FJ-CL01和FJ-ND株模板; 4~16为分别为Mcc、M.arginini、M.bovis、M.agalactiae、Mh、ORFV、Mccp、Mmc、Mmm LC、AL、Ec、SA和Pm RPA扩增模板; 17为空白对照。

    Figure  2.  Specificity test on Mo by RPA

    Note:M:DNA Marker DL1000; 1-3: The templates of Mo strain Y98, strain FJ-CL01 and strain FJ-ND; 4-16: The templates of RPA were Mcc, M.arginini, M.bovis, M.agalactiae, Mh, ORFV, Mccp, Mmc, Mmm LC, AL, Ec, SA and Pm respectively; 17: Blank control.

    图  3  RPA (A)和PCR (B)的感性试验

    注:M为1 000标注; 1~9为绵羊肺炎支原体Y98株DNA模板7 ng·μL-1、700 pg·μL-1、70 pg·μL-1、7 pg·μL-1、700 fg·μL-1、70 fg·μL-1、7 fg·μL-1、700 ag·μL-1、70 ag·μL-1

    Figure  3.  Sensitivity test on RPA(A) and PCR(B) methods

    Note: M: DNA Marker DL1000; 1-9: Template DNAs of Mo strain Y98 in copies from 7 ng·μL-1、700 pg·μL-1、70 pg·μL-1、7 pg·μL-1、700 fg·μL-1、70 fg·μL-1、7 fg·μL-1、700 ag·μL-1、70 ag·μL-1.

    表  1  RPA、常规PCR和病原分离鉴定法检测临床样品结果比较

    Table  1.   Mo detection, isolation, and identification on clinical specimens by RPA and conventional PCR

    检测方法
    Detecting method number
    样品数
    No. of samples
    阳性样品数
    No. of positive
    阴性样品数
    No. of negative
    阳性率
    Positive rate/%
    RPA 186 95 91 51.1
    常规PCR
    Conventional PCR
    186 95 91 51.1
    支原体分离鉴定法
    Isolation and identification for Mo
    40 13 27 32.5
    下载: 导出CSV
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  • 收稿日期:  2018-12-27
  • 修回日期:  2019-01-14
  • 刊出日期:  2019-04-28

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