基于酶促重组酶扩增的非洲猪瘟病毒检测方法

A Detection Method of African Swine Fever Virus based on Enzymatic Recombinase Amplification

建立基于酶促重组酶扩增(enzymatic recombinase amplification,ERA)技术的非洲猪瘟病毒(African swine fever virus,ASFV)核酸快速检测方法。针对ASFV B646L基因保守序列,设计特异性的ERA探针和引物,经过反应条件的优化,建立等温条件下检测ASFV DNA的ERA方法。结果显示:建立的ERA检测ASFV方法特异性强与其它病原无交叉反应;CV均小于10%,具有良好的重复性;对阳性样品拷贝数的检测下限为85 copies·μL^(-1);与世界动物卫生组织(WOAH)非洲猪瘟qPCR诊断方法对比,Kappa值为0.961,具有高度一致性。本研究建立的基于ERA检测ASFV方法可以用于ASFV的现场快速检测。

This experiment was conducted to establish a rapid nucleic acid detection method for African swine fever virus(ASFV)based on enzymatic recombinase amplification(ERA).We designed ERA probes and primers specific for the conserved sequence of ASFV B646L gene,and optimized the reaction conditions in order to establish an ERA method for the detection of ASFV DNA under isothermal conditions.The ERA method for ASFV demonstrated high specificity and no cross-reaction with other pathogens;the CV was less than 10%,indicative of good reproducibility.The lowest detection limit for the ERA method is 85 copies·μL^(-1);comparison with the World Organization for Animal Health(WOAH)recommended qPCR diagnostic method for African swine fever(ASF)demonstrated a Kappa value of 0.961,suggestive of high identity with African swine fever qPCR diagnostic method.ERA-based method for the detection of ASFV can be used for the rapid detection of ASFV in the field.