猪圆环病毒2型可视化RPA快速检测方法的建立

Development of the visual RPA assay for rapid detection of porcine circovirus 2

本研究针对猪圆环病毒2型(porcine circovirus 2,PCV2)的cap基因保守序列,设计特异性RPA引物和nfo探针,建立了一种快速检测PCV2的基于侧流层析试纸条的可视化RPA方法(lateral flow strip RPA,LFS-RPA)。结果,建立的LFS-RPA方法最佳反应条件是39℃恒温扩增20 min,可在5 min内实现对检测结果的肉眼可视化判定;该方法特异性强,仅对PCV2出现阳性扩增,对本研究涉及的其他猪重要病原均无交叉反应;以含PCV2全长序列的重组质粒为模板,LFS-RPA的检出限为10 copies/μL。进一步应用建立的LFS RPA方法对30份猪临床样品进行PCV2检测,并与real-time PCR方法进行比较,结果显示两种方法的检测一致性达86.7%(26/30),其中4份C;值在36~38之间的临床样品利用LFS-RPA方法检测为阴性。上述结果表明,本研究建立的LFS-RPA方法操作简单,对仪器设备要求低,可以快速且直观的实现对检测结果的判断,可以满足现场检测的要求,对养猪场一线PCV2快速诊断和防控具有重要现实意义。

The specific RPA primers and nfo probes were designed based on the conserved sequence of cap gene of Porcine circovirus 2(PCV2),and a lateral flow strip RPA(LFS-RPA)assay based on lateral flow chromatography strips for rapid and visual detection of PCV2 was established.The results showed that the optimal reaction condition of LFS-RPA assay was performed at the constant temperature of 39℃for20 min,and the detection results were visible by naked eyes within 5min.The developed assay showed good specificity,only PCV2 was amplified and no cross-reactions to other important porcine pathogens involved in this study were observed.The limit of detection of the LFS-RPA assay was 10 copies/μL using the recombinant plasmid containing the full-length sequence of PCV2 as template.The developed PCV2 LFS-RPA assay was used to detect the viral nucleic acid of PCV2 for 30 porcine clinical samples,and the results were compared with those obtained from a real-time PCR.The detection results showed that the consistency of the two assays was 86.7%(26/30),and 4 clinical samples with C;values between 36 and 38 were negative with the LFS-RPA.The LFS-RPA assay is simple to operate,has low requirements for instruments and equipment and could quickly and intuitively realize the judgment of the detection results.The developed PCV2LFS-RPA assay meets the requirements of on-site test,which demonstrates the important practical significance for the PCV2 rapid diagnosis and prevention in the swine husbandry farms.