中国兽医杂志2026，Vol.62Issue(9)：55-63,9.DOI:10.20157/j.cnki.zgsyzz.2026.sf.062
ASFV和PRRSV双重RAA侧流层析试纸条检测方法的建立及应用
Establishment and Application of a Dual RAA Lateral Flow Chromatographic Test Strip Detection Method for ASFV and PRRSV
摘要
Abstract
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.关键词
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Key words
African swine fever virus(ASFV)/porcine reproductive and respiratory syndrome virus(PRRSV)/recombinase-assisted amplification(RAA)/test strip/identification分类
农业科技引用本文复制引用
岳怀宁,任静,孙泰然,郭恒宽,李潭清,袁晨,宋勤叶..ASFV和PRRSV双重RAA侧流层析试纸条检测方法的建立及应用[J].中国兽医杂志,2026,62(9):55-63,9.基金项目
河北省重点研发计划项目(21326613D) （21326613D）
2022年河北省省属高等学校基本科研业务费研究项目(KY2022042) （KY2022042）
