To develop a rapid and practical method to differentiate wild-type strains of classical swine fever virus (CSFV) and the attenuated C-strain, a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was established with a set of primers based on NS5B gene sequence. The viral cDNA generated by reverse transcription was amplified with Bst DNA polymerase at a constant temperature of 62 ℃, and the products could be visualized under the UV light with SYBR Green I dye. The RT-LAMP was able to detect different genotypes of wild-type CSFV strains, but not for the C-strain, bovine viral diarrhea virus or other swine viruses. The detection limit of the RT-LAMP assay was 2.5 TCID_(50) of CSFV, comparable to the sensitivity of the real-time RT-PCR. The agreement rate between RT-LAMP and the real-time RT-PCR or the primer-probe energy transfer real-time PCR was 100 % or 98.4 % in detecting 126 samples. Thus, the assay is a rapid, sensitive, simple and practical method for the detection of wild-type CSFV in the field.%本研究旨在建立一种可视化检测猪瘟病毒(CSFV)野毒株的反转录.环介导等温扩增方法(RT-LAMP).根据CSFV的NS5B基因序列设计一套RT-LAMP引物,以样品的cDNA为模板,利用Bsf DNA聚合酶,在62℃恒温条件下进行扩增,扩增产物中加入sYBR Green I染料直接或在紫外光下观察判定扩增结果.该方法可检测出不同基因型的CSFV厂野毒株,其检出极限为2.5 TCID_(50)的CSFV,与实时荧光定量RT.PCR方法的敏感性相当;特异性试验表明,该方法对猪瘟免化弱毒疫苗株(HCLV)、牛病毒性腹泻病毒以及其它常见猪源病毒均无扩增反应;通过对126份不同样品进行检测比较,该方法与实时荧光定量RT-LAMP检测方法的符合率达100%.与引物.探针能量转移PCR方法的符合率为98.4%.该方法无需特殊仪器,是一种适用于基层的快速、简便的CSFV野毒鉴别检测方法.
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机译：Development of an One-step Reverse Transcription Loop-mediated lsothermal Amplification Method for Rapid Detection of Bovine Viral Diarrhea Virus牛病毒性腹泻病毒一步 RT-LAMP快速检测方法的建立
