猪塞内卡病毒RT-RAA-CRISPR Cas13a检测方法的建立及应用  

Establishment and application of RT-RAA-CRISPR/Cas13a diagnostic method for porcine Senecavirus

作　　者：李晨钰 沙洲 郑辉[2] 崔进 迟田英[2] 陈峰 曹振山 张慧[2,3] 戈胜强 魏荣[2,3,4] 南福龙 古少鹏[1] 尼博 LI Chenyu;SHA Zhou;ZHENG Hui;CUI Jin;CHI Tianying;CHEN Feng;CAO Zhenshan;ZHANG Hui;GE Shengqiang;WEI Rong;NAN Fulong;GU Shaopeng;NI Bo(College of Veterinary Medicine,Shanzi Agricultural University,Jinzhong,Shanci 03080l,China;China Animal Health and Epidemiology Center,Qingdao,Shandong 266033,China;Qingdao Key Laboratory of Modern Biotechnology and Animal Epidemic Research,Qingdao,Shandong 266032,China;Key Laboratory of Animal Bio-safety Risk Waring,Prevention and Control(South China),Ministry of Agriculture and Rural Affairs,Qingdao,Shandong 266032,China;Shandong Provincial Center for Animal Disease Control and Prevention,Jinan 250100,China;College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong 266109,China)

机构地区：[1]山西农业大学动物医学学院,山西晋中030801 [2]中国动物卫生与流行病学中心,山东青岛266033 [3]青岛市现代生物工程及动物疫病研究重点实验室,山东青岛266032 [4]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032 [5]山东省动物疫病预防与控制中心,山东济南250100 [6]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医学报》2025年第2期195-203,共9页Chinese Journal of Veterinary Science

基　　金：“十四五”国家重点研发计划资助项目(2021YFDI800301-4);中国动物卫生与流行病学中心创新基金资助项目(2022001-3)。

摘　　要：建立一种基于逆转录-重组酶聚合酶等温扩增(reverse transcription-recombinase polymerase amplification, RT-RAA)及CRISPR Cas13a技术检测猪A型塞内卡病毒(Senecavirus A,SVA)的快速检测方法。根据猪SVA保守基因序列设计8对RT-RAA引物,筛选最适扩增引物、最佳反应温度、最优RT-RAA反应体系。针对优化的RT-RAA体系设计并筛选最佳CRISPR-derived RNA(crRNA),构建最优RT-RAA-CRISPR反应体系。利用6种常见猪群病原核酸验证已建立方法特异性。用数字PCR标定的SVA cRNA标准品验证已建立方法敏感性。通过重复试验验证方法的稳定性。应用已建立的方法与临床样品进行符合检验。RT-RAA及CRISPR反应体系优化结果显示,RT-RAA反应温度为37℃,扩增效果最佳;从8条crRNA中筛选出crRNA 5检测信号最强。建立的RT-RAA-CRISPR Cas13a方法特异性好,与ASFV、PRRSV、PEDV、PCV2、CSFV、PRV等常见猪群疫病均无交叉反应;方法敏感性高,对SVA最低检测限为0.86 copy/μL;对3个稀释度标准品检测均能稳定产生荧光,重复性好;能够在50 min内完成临床样品检测,对临床检毕的6份阳性样品及58份阴性样品进行符合检测,结果表明检测结果一致,符合率100%。结果表明,本研究建立了一种高特异性、高敏感性的RT-RAA-CRISPR Cas13a检测方法,有望应用于SVA的现场快速检测。The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100%agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

关 键 词：RT-RAA CRISPR/Cas13a SVA 

分 类 号：S852.65[农业科学—基础兽医学]