非洲猪瘟病毒LFD-RPA快速检测方法的建立

Development of LFD-RPA assay for rapid detection of African swine fever virus

本研究根据非洲猪瘟病毒(ASFV)B646L基因保守序列设计nfo RPA特异性引物和探针,建立了ASFV核酸LFD-RPA快速检测法。结果表明,该方法特异性强,与猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、口蹄疫病毒(FMDV)、猪A型塞内卡病毒(SVA)等病原核酸无交叉反应;最低可以检测到1.57×10^2copies/μL重组质粒pUC-B646L;检测时间短,其中RPA扩增20 min,LFD检测10~20 min。应用该法及OIE推荐的荧光PCR法对24个模拟样品进行检测,检测结果一致。对50份已知的临床样品核酸进行检测,阳性符合率为73.3%。上述结果表明,本研究建立的LFD-RPA快速检测方法操作简便,为ASFV现场快速检测提供了技术支持。

In this study,a LFD-RPA assay was developed to detect African swine fever virus(ASFV)using primers and probes specific for the conserved region of B646 L gene.The results showed that ASFV was specifically detected by the assay without cross-reactions with other nucleic acids of PCV-2,CSFV,PRRSV,FMDV or SVA.A minimum of 1.57×10^2 copies/μL recombinant plasmid pUC-B646 L could be detected in this test.The detection process was rapid,just including RPA reaction in 20 min and LFD in 10-20 min.The simulated samples were tested in parallel by the LFD-RPA and real-time PCR recommended by OIE,and the results were consistent between these two assays.Detection of nucleic acids in 50 known clinical samples,and the positive coincidence rate is 73.3%.The LFD-RPA rapid detection method established in this study provides technical support for the rapid on-site detection of ASFV.