ASFV和PRRSV双重RAA侧流层析试纸条检测方法的建立及应用OA
Establishment and Application of a Dual RAA Lateral Flow Chromatographic Test Strip Detection Method for ASFV and PRRSV
为了建立一种双重重组酶辅助扩增(RAA)侧流层析试纸条检测方法,以满足临床快速检测非洲猪瘟病毒(ASFV)和猪繁殖与呼吸综合征病毒(PRRSV)的需求.本试验选取ASFV B646L基因(编码p72蛋白)和PRRSV ORF7基因[编码核衣壳(N)蛋白]的保守区域作为特异性引物和探针的目标序列,分别设计引物和探针,筛选最佳的引物和探针组合,确定RAA反应的最佳扩增体系和扩增条件,验证该方法的特异性和敏感性,并比较该方法与三重实时荧光定量聚合酶链式反应(qPCR)方法对临床样品的检测.结果显示,ASFV引物和探针组合选择RAA-ASFV-F3/-R3/-P3,PRRSV引物探针组合选择RAA-PRRSV-F3/-R3/-P3进行后续试验;筛选的PRRSV(RAA-PRRSV-F3/-R3/-P3)和ASFV(RAA-ASFV-F3/-R3/-P3)的引物和探针序列均具有高度保守性和特异性,建立的双重RAA方法的扩增体系为25 μL,引物(RAA-ASFV-F3/-R3)各0.75 μL,探针(RAA-ASFV-P3)0.2 μL,引物(RAA-PRRSV-F3/-R3)各1.0 μL,探针(RAA-PRRSV-P3)0.3 μL,于38℃经20 min即可完成反应.该方法与猪瘟病毒、伪狂犬病病毒、猪圆环病毒2型和猪流行性腹泻病毒的核酸无交叉反应,对ASFV B646L基因的最低检测限为4.000拷贝/反应,对PRRSV ORF7基因的最低检测限为6.375拷贝/反应.临床样品检测结果显示,建立的双重RAA侧流层析试纸条与三重qPCR方法检测结果一致,其阴性和阳性符合率均为100％,并且可以同时检测ASFV和PRRSV.本试验建立的双重RAA侧流层析试纸条检测方法为临床ASFV和PRRSV的快速检测和诊断提供技术手段.
To establish a dual recombinase-aided amplification(RAA)lateral flow chromatographic test strip detection method for rapid clinical detection of African swine fever virus(ASFV)and porcine reproductive and respiratory syndrome virus(PRRSV),this study selected the conserved regions of the ASFV B646L gene(encoding p72 protein)and PRRSV ORF7 gene[encoding nucleocapsid(N)protein]as target sequences for the design of specific primers and probes.Optimal primer-probe combinations were screened to determine the best amplification system and reaction conditions.The specificity and sensitivity of the method were evaluated,and its performance was compared with a triple real-time fluorescence quantitative polymerase chain reaction(qPCR)assay using clinical samples.The results showed that the optimal combinations were RAA-ASFV-F3/-R3/-P3 for ASFV and RAA-PRRSV-F3/-R3/-P3 for PRRSV.Both PRRSV(RAA-PRRSV-F3/-R3/-P3)and ASFV(RAA-ASFV-F3/-R3/-P3)primer-probe sets exhibited high conservation and specificity.The established dual RAA amplification system(25 μL total volume)included 0.75 μL of each ASFV primer(RAA-ASFV-F3/-R3),0.2 μL of probe(RAA-ASFV-P3),1.0 μL of each PRRSV primer(RAA-PRRSV-F3/-R3),and 0.3 μL of probe(RAA-PRRSV-P3).The reaction was completed at 38℃ within 20 min.The assay showed no cross-reactivity with classical swine fever virus,pseudorabies virus,porcine circovirus type 2,or porcine epidemic diarrhea virus.The limits of detection were 4.000 copies/reaction for the ASFV B646L gene and 6.375 copies/reaction for the PRRSV ORF7 gene.When tested with clinical samples,the dual RAA-lateral flow chromatographic test strip method showed 100％concordance with the triple qPCR assay for both positive and negative results,effectively allowing simultaneous detection of ASFV and PRRSV.In conclusion,the dual RAA-lateral flow chromatographic test strip assay established in this study provides a rapid,accurate,and reliable tool for the on-site detection and diagnosis of ASFV and PRRSV in clinical settings.
岳怀宁;任静;孙泰然;郭恒宽;李潭清;袁晨;宋勤叶
河北农业大学动物医学院,河北 保定 071000||中国兽医药品监察所,北京 海淀 100081河北农业大学动物医学院,河北 保定 071000保定市动物疫病预防控制中心,河北 保定 071000饶阳县动物卫生监督所,河北 衡水 053000河北农业大学动物医学院,河北 保定 071000河北农业大学动物医学院,河北 保定 071000河北农业大学动物医学院,河北 保定 071000
农业科技
非洲猪瘟病毒(ASFV)猪繁殖与呼吸综合征病毒(PRRSV)重组酶辅助扩增(RAA)试纸条鉴别
African swine fever virus(ASFV)porcine reproductive and respiratory syndrome virus(PRRSV)recombinase-assisted amplification(RAA)test stripidentification
《中国兽医杂志》 2026 (9)
55-63,9
河北省重点研发计划项目(21326613D)2022年河北省省属高等学校基本科研业务费研究项目(KY2022042)
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