期刊文献+

三种小鼠肠道病毒多重PCR检测方法的建立和初步应用

Establishment and Preliminary Application of a Multiplex PCR Method for Detecting Three Types of Enterovirus in Mice
下载PDF
导出
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。 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^(2),10^(4) and 10^(2) 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.
作者 赵迎峰 马畅 刘彪 尤金炜 张旭亮 恽时锋 ZHAO Yingfeng;MA Chang;LIU Biao;YOU Jinwei;ZHANG Xuliang;YUN Shifeng(the Third Outpatient Department,Chinese PLA General Hospital of Eastern Theater Command,Nanjing 210002,China;Department of Laboratory Animal,Medical Security,Center Chinese PLA General Hospital of Eastern Theater Command,Nanjing 210002,China;Key Open Laboratory of Animal Physiology and Biochemistry,Department of Agriculture,Nanjing Agricultural University,Nanjing 210095,China)
出处 《实验动物科学》 2024年第5期36-41,共6页 Laboratory Animal Science
基金 2023年科技创新研究计划项目(2023JCYJZD077)。
关键词 小鼠细小病毒 小鼠肝炎病毒 小鼠诺如病毒 多重PCR检测方法 minute virus of mice mouse hepatitis virus mouse norovirus multiplex PCR
  • 引文网络
  • 相关文献

参考文献7

二级参考文献45

共引文献49

;
使用帮助 返回顶部