非洲猪瘟病毒实时荧光RPA快速检测方法的建立

Establishment of a Real-time Recombinase Polymerase Amplification Assay for Rapid Detection of African Swine Fever Virus

非洲猪瘟是一种跨境动物疫病,对世界养猪业危害严重。目前,该病的流行范围越来越广。为防止该病传入我国,当前急需建立快速、简便、可靠的检测方法。本研究通过分析非洲猪瘟病毒B646L基因保守区域,设计并合成了特异性引物和exo探针,建立了检测非洲猪瘟病毒的实时荧光RPA方法。利用该方法可在20 min内完成检测过程,最低可检测到16拷贝/反应;与猪的其他常见病原核酸无交叉反应;与荧光PCR方法具有相似的灵敏度和特异性;利用该方法对100份国内临床样品进行检测,结果均为阴性。本研究所建立的非洲猪瘟病毒实时荧光RPA检测方法具有简单、快速、敏感性高、特异性强的优点,在临床鉴别诊断、检验检疫和病原监测等方面具有广泛的应用价值。

African swine fever(ASF)is a transboundary swine disease,causing huge economic losses for swineindustry in the world.At present,the prevalence of the disease has been spreading widely.In order to prevent thedisease from being introduced into China,it is urgent to establish a fast,simple and reliable method.In this study,the specific primers and exo probes were designed and synthesized by analyzing the conserved region of B646L gene ofAfrican swine fever virus(ASFV).A real-time fluorescence RPA method for detection of ASFV was established.Theminimal detection limit of the novel real-time RPA assay was16copies per reaction,and the detection could be finishedin20minutes.Cross reactivity with other swine associated viruses was not observed.The sensitivity and specificity ofthe assay was comparable to that of the real-time PCR.100domestic samples were detected by this method,and theresults were negative.The real-time RPA assay developed in this study was of great significance of simple,fast,highsensitivity and strong specificity,with wide application value in clinical differential diagnosis,laboratory quarantineand pathogen surveillance.