猪圆环病毒2型RPA恒温扩增检测方法的建立及初步应用

Establishment and Preliminary Application of the real-time RPA Method for Detection of PCV2

利用重组酶聚合酶扩增技术(RPA),以猪圆环病毒2型Cap基因的保守序列为靶序列设计并筛选出一组特异性的引物和探针,建立了快速检测猪圆环病毒2型核酸的RPA方法。试验结果表明,该方法特异性强,猪圆环病毒1型(PCV1)、猪伪狂犬病病毒(PRV)、猪细小病病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)核酸检测结果均为阴性,灵敏性高,最低可检出核酸浓度为0. 87×10-4ng/μL,简单快速,且重复性好。利用所建立的RPA方法对183份临床样品进行检测,结果与实时荧光PCR方法符合率为98. 9%。本研究为猪圆环病毒2型核酸的快速检测提供了一种新方法,尤其适合基层实验室或养猪场的快速检测。

Porcine circovirus 2 （PCV2） has been associated with porcine circovirus-associated disease （PCVAD） in pigs, caused important economic losses in commercial pig farms worldwide. In this study, a real - time reeombinase polymerase amplification （RPA） assay was developed to detect PCV2 using primers and an exo probe specific for thecap gene. The assay sensitivity and specificity was tested. The amplification results of other pathogens, including PCV1, CSFV,PRV, PPV, and PRRSV vere negative. The detection limit of RT - RPA for PCV is about0.87 × 10 ^-4ng/μL. A total ofl83clinical samples were tested by the real - time RPA assay and real - time PCR Kit. The two assays demonstrated a 98.9% diagnostic agreement. The real - time RPA assay provides a new rapid detection technology for PCV2, suitable for field quarantine and primary laboratory.