文章摘要
利用RT-LAMP可视化检测技术检测猪瘟病毒及特异性鉴别猪瘟兔化弱毒疫苗
Visual detection of classical swine fever virus and identification of HCLV vaccine strain by reverse transcription loop-mediated Isothermal amplification assay
投稿时间:2014-06-10  修订日期:2014-07-20
DOI:
中文关键词: 猪瘟病毒,RT-LAMP,检测
英文关键词: Classical swine fever(CSF)  RT-LAMP  Detection
基金项目:广西科技重大专项(1222003-2-4)、广西特聘专家专项(2011B020)和国家百千万人才工程人选专项(945200603)共同资助。
作者单位E-mail
邓显文 广西兽医研究所 dengxianwen@126.com 
谢芝勋 广西兽医研究所  
谢志勤 广西兽医研究所  
刘加波 广西兽医研究所  
庞耀珊 广西兽医研究所  
谢丽基 广西兽医研究所  
范晴 广西兽医研究所  
罗思思 广西兽医研究所  
黄莉 广西兽医研究所  
黄娇玲 广西兽医研究所  
曾婷婷 广西兽医研究所  
黄秀琴 广西兽医研究所  
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中文摘要:
      【目的】为建立可视化检测猪瘟病毒(CSF)及特异性鉴别猪瘟兔化弱毒疫苗病毒(HCLV)的逆转录环介导等温扩增(RT-LAMP)方法。【方法】根据基因库中CSF非结构蛋白NS5B基因的保守序列,利用在线软件Primer Explorer V4 Software,设计了二套分别针对CSF所有毒株及HCLV特异性引物,并优化各反应条件。通过特异性、敏感性试验评价建立的RT-LAMP可视化检测方法,同时用实时浊度仪进行实时测量。【结果】该方法可通过肉眼观察钙黄绿素(Calcein)颜色变化直接判定结果;对CSF的检测结果为阳性(肉眼观察可见翠绿色),对其它常见病原体的检测结果均为阴性(肉眼观察可见桔红色)并能特异性鉴别HCLV,该方法的敏感性高于常规RT-PCR方法;同时用实时浊度仪进行实时测量,结果相一致。【结论】所建立的RT-LAMP方法简便、灵敏、特异,可用于快速检测CSF及特异性鉴别HCLV。
英文摘要:
      【Objective】 To establish a rapid method for visual detection of classical swine fever virus (CSFV) and identification of hog cholera lapinized virus ( HCLV) vaccine strain by reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. 【Method】According to the sequence of a gene library of non-structural protein NS5B gene of CSF using Primer Explorer V4 software, two degenerate primer sets specific to CSF of all strains,HCLV were designed,and the reaction conditions were optimized.Visual detection of RT-LAMP was method established by evafuation of specificity and sensitivity of the assay as well as real-time measurement of turbidity. 【Results】 This method can be directly judged by the naked eyes through calcein color change results; detection of CSF is positive (the naked eyes visible emerald green), detection of other common pathogens of pig was negative (the naked eyes visible orange), and to specific discriminating HCLV, more sensitive than conventional RT-PCR method. At the same time the results are consistent with real-time measurement of turbidity. 【Conclusion】 RT-LAMP method is simple, sensitive, specific and can be used for rapid detection of CSFV, specific identification of HCLV.
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