实验动物科学 ›› 2024, Vol. 41 ›› Issue (5): 36-41.DOI: 10.3969/j.issn.1006-6179.2024.05.007

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三种小鼠肠道病毒多重 PCR 检测方法的建立和初步应用

  

  1. ( 1. 中国人民解放军东部战区总医院第三派驻门诊部,南京 210002) ( 2. 中国人民解放军东部战区总医院医疗保障中心
    实验动物室,南京 210002) ( 3. 南京农业大学农业部动物生理生化重点开放实验室,南京 210095)
  • 收稿日期:2024-04-12 出版日期:2024-10-28 发布日期:2024-10-28
  • 通讯作者: 恽时锋( 1965—) ,男,主任医师,研究方向为实验动物与比较医学,E-mail:yunshifeng1@ 163. com。
  • 作者简介:赵迎峰( 1981—) ,男,主治医师,硕士,研究方向为病原体检测,E-mail:zhaoyf316@ 126. com。
  • 基金资助:
    2023 年科技创新研究计划项目( 2023JCYJZD077)

Establishment and Preliminary Application of a Multiplex PCR Method for
Detecting Three Types of Enterovirus in Mice

  1. ( 1. the Third Outpatient Department, Chinese PLA General Hospital of Eastern Theater Command, Nanjing 210002,China)
    ( 2. Department of Laboratory Animal, Medical Security,Center Chinese PLA General Hospital of Eastern Theater Command,
    Nanjing 210002,China) ( 3. Key Open Laboratory of Animal Physiology and Biochemistry,
    Department of Agriculture, Nanjing Agricultural University, Nanjing 210095,China)
  • Received:2024-04-12 Online:2024-10-28 Published:2024-10-28

摘要:

目的 建立一种同时检测小鼠细小病毒(MVM) 、肝炎病毒(MHV)和诺如病毒(MNV)的多重 PCR 检测方法。
方法 分别根据 MVM、MHV 和 MNV 基因保守区应用软件设计特异性引物,构建在同一反应体系中可同时检测
MVM、MHV 和 MNV 的多重 PCR 检测方法,验证其特异性、敏感性及重复性,并进行初步检测应用。 结果 通过优
化 PCR 扩增条件成功建立多重 PCR 检测方法,特异性实验结果表明,该方法可同时扩增 MVM、MHV 和 MNV 的目
的片段,片段大小与预期相符,未出现非特异性片段;敏感性测试显示,MVM、MHV 和 MNV 三种病毒的最低检测下
限为 10 2 、10 4 、10 2 copies/ μL;重复性实验显示该方法在不同时间、不同场所应用的重复性稳定。 应用该方法对 287
份小鼠结肠匀浆样品进行检测,与单重 PCR 检测结果完全一致。 结论 所建立的 MVM、MHV 和 MNV 三种病毒多
重 PCR 检测方法具有特异性高、敏感性强和重复性优的特点,适用于三种病原体感染的临床样品检测和大量样品
的流行病学调查。

关键词:

Abstract:

Objective To establish a multiplex PCR method for the detection of minute virus of mice
(MVM) , mouse hepatitis virus (MHV) and murine norovirus (MNV) . Method Specific primers were
designed according to the application software of MVM, MHV and MNV gene conserved regions. By
optimizing PCR amplification conditions, a multiplex PCR assay was established to detect MVM, MHV
and MNV simultaneously. The specificity, sensitivity and repeatability of the assay were verified, and the
preliminary clinical application of the assay was carried out. Result The multi-PCR method was
successfully established by optimizing the PCR amplification conditions. The specificity test result showed
that the method could simultaneously amplify the target fragments of MVM, MHV and MNV, and the

fragment size was consistent with the expectation, without the occurrence of non-specific fragments.

Sensitivity test result showed that the lower limits of detection of MVM, MHV and MNV were 10  , 10 
and 10  copies/ μL, respectively. Repeatability test shows that the proposed method has good
repeatability. The method was used to detect 287 clinical samples, and the coincidence rate of detection
result with single PCR result reached 100%. Conclusion The established multiplex PCR method for detection of MVM, MHV and MNV viruses has the characteristics of high specificity, high sensitivity and excellent repeatability, which is suitable for clinical sample detection of the infection of the three pathogens and epidemiological investigation of a large number of samples.

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