减蛋综合征病毒环介导等温扩增检测方法的建立

根据GenBank登录的减蛋综合征病毒(EDSV)六邻体基因的保守区,设计了3对环介导等温基因扩增(LAMP)引物,优化反应体系,并对其灵敏性、特异性进行验证.结果表明:LAMP优化后的反应体系为外引物浓度0.1μm01·L-1,内引物浓度0.8 μmol·L-1,环引物浓度0.4μmol·L-1,Mg2+浓度8.0 ...

Establishment of loop-mediated isothermal amplification method for detecting egg drop syndrome virus

In order to develop an efficient,rapid and highly specific loop-mediated isothermal gene amplification(LAMP) assay to detect egg drop syndrome virus(EDSV),three pairs of primers were designed according to hexon conservative region of EDSV.The LAMP was performed using Bst DNA polymerase large fragment under the effect of isothermal amplification.The LAMP reaction system was optimized and the sensitivity and specificity were tested.The results showed that a batter amplification of LAMP was abtained with the reaction system containing 0.1 μmol·L-1 outer primer,0.8 μmol·L-1 imner primer,0.4 μmol·L-1 ring primer,8 mmol·L-1mg2+,0.8 mmol·L-1 dNTP,0.5 mmol·L-1 betaine,8 U Bst DNA polymerase,reaction temperature was 65 ℃.The products amplified by LAMP from EDSV were distinctively ladder-like band in agarose gel electrophoresis,and green fluorescence should be seen in colorable reaction.The sensitivity of LAMP assay developed in this study was 10 times higher than that of PCR.It had no cross reaction with NDV,IBDV,IBV and CELO.The LAMP assay was more simple and convenient,which could be used economically and effectively in both laboratorial and pen-side testing.