【目的】建立鸡传染性贫血病毒(CAV)的环介导等温扩增(LAMP)技术,以便简单、快速、特异地检测CAV,同时搭建LAMP技术平台。【方法】选取GenBank中69株CAV序列,选择保守序列区域,使用在线设计软件Pirmer Explorer V4软件对引物进行设计并...
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【目的】建立鸡传染性贫血病毒(CAV)的环介导等温扩增(LAMP)技术,以便简单、快速、特异地检测CAV,同时搭建LAMP技术平台。【方法】选取GenBank中69株CAV序列,选择保守序列区域,使用在线设计软件Pirmer Explorer V4软件对引物进行设计并筛选,以CAV阳性质粒为模板用3对LAMP引物进行等温扩增,扩增产物分别进行电泳、SupergreenI荧光染色以及酶切鉴定,正确后对该方法的反应体系及反应条件进行优化。【结果】根据引物设计优化结果,最终选定的引物组选取了215bp～395bp的181bp序列作为扩增片段;扩增产物电泳呈LAMP特有的拖带,能被PstI和BgⅢ有效酶切,SupergreenI染色后肉眼可见特有的绿色荧光,阴性产物呈红棕色;经优化后25μL体系为:dNTPs(10mmol/L)0.3μl、FIP/BIP(100umol/L)0.3μL、F3/B3(10umol/L)10μmol/L、LF/LB(10umol/L)10μmol/L、Betaine(5mol/L)5.0μL、MgCl2(25mmol/L)2.5μL、BstDNAPolymerase1μl、10×ThermoPolbuffer2.5μl、模板DNA2μl,超纯水补至25μL;反应条件为:65℃温浴1h,80℃灭活5min。【结论】成功建立了CAV的LAMP检测方法,为基层实验室的快速诊断及CIA防控提供了技术支撑,同时为其它病原的LAMP方法的建立奠定了基础。
Border disease virus belonging to the genus of pestivirus causes the border disease of small ru minants. The disease has been found worldwide, however, no any reports of BDV infection was ava ilable;and it was commonl...
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Border disease virus belonging to the genus of pestivirus causes the border disease of small ru minants. The disease has been found worldwide, however, no any reports of BDV infection was ava ilable;and it was commonly considered that there were no the virus infections before in China. We have detected border disease viruses in the serum and tissue samples of the goats and sheep being clinical signs in east China by RT-PCR. Four BDV strains with no cytopathologic effects (CPE) w
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