小反刍兽疫病毒RT-RAA-CRISPR/Cas12a可视化快速检测方法的建立  

Establishment of a visual method for rapid detection of peste des petits ruminants virus based on RT-RAA-CRISPR/Cas12a

作　　者：黄超华 阮周曦 路平[2] 吴江 曹琛福 林彦星 花群义 HUANG Chaohua;RUAN Zhouxi;LU Ping;WU Jiang;CAO Chenfu;LIN Yanxing;HUA Qunyi(Animal and Plant Inspection and Quarantine Center,Shenzhen Customs,Shenzhen,Guangdong 518045,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 370200,China)

机构地区：[1]深圳海关动植物检验检疫技术中心,广东深圳518045 [2]中国动物卫生与流行病学中心,山东青岛370200

出　　处：《中国兽医学报》2023年第10期2030-2034,共5页Chinese Journal of Veterinary Science

基　　金：海关总署科技资助项目(2021HK169)。

摘　　要：为建立小反刍兽疫病毒(peste des petits ruminants virus,PPRV)可视化快速检测方法,本研究根据PPRV N基因的保守序列设计、合成和筛选出反转录重组聚合酶介导恒温扩增(RT-RAA)特异性引物和CRISPR/Cas12a特异性crRNA。结合侧向流试纸,本研究建立了一种PPRV RT-RAA-CRISPR/Cas12a可视化快速检测方法,并对该方法的性能进行了评估。结果显示,该方法特异性良好,与蓝舌病毒(BTV)、鹿流行性出血热病毒(EHDV)、口蹄疫病毒(FMDV)、羊痘病毒(CaPV)、猪水泡病病毒(SVDV)、伪狂犬病毒(PRV)等病毒核酸无交叉反应;敏感性高,最低可检出4.30×10^(1)拷贝/μL的PPRV核酸;应用该方法及国家标准推荐的RT-qPCR方法对60个模拟样品进行平行检测,结果显示Kappa值为0.918,表明两者具有高度一致性。综上表明,本研究建立的PPRV RT-RAA-CRISPR/Cas12a可视化快速检测方法特异性好、敏感性高,且操作简单、无需特殊仪器,可应用于野外或基层农场的PPRV快速筛查检测工作。In order to establish a rapid visualization method for detecting peste des petits ruminants virus(PPRV),the RT-RAA amplified primers and crRNA synthesized primers were designed,synthesized and screened according to the conservative sequence of PPRV N gene.Based on RTRAA,Casl2a trans-cleavage reaction and lateral flow dipstick,a rapid visualization method for PPRV detection was developed and validated.Specificity assay showed this method only detected PPRV with no detection for BTV,EHDV,FMDV,CaPV,SVDV and PRV.Sensitivity test results showed that the detection limit of this method for PPRV RNA reached 4.30×10^(1)copies/μL.Parallel detection of the simulated samples by this method and the standard RT-qPCR demonstrated a high consistency with the Kappa value of o.918.In conclusion,the established visualized method was specific,sensitive,simple,and fast,which provide a reliable tool for rapid screening and detection of PPRV in field or basic farm.

关 键 词：小反刍兽疫病毒 RT-RAA CRISPR/Cas12a 

分 类 号：S855.3[农业科学—临床兽医学]