A型塞尼卡病毒与O型、亚洲I型、A型口蹄疫病毒多重RT-PCR检测方法的建立

Establishment of a multiplex RT-PCR method for simultaneous detection of Senecavirus A and O,Asia I,A serotypes of FMDV

为建立鉴别A型塞尼卡病毒(SVA)与O型、亚洲I型、A型口蹄疫病毒(FMDV)的快速检测方法,本研究分别设计4对特异性引物,用于扩增SVA 3D基因(157 bp)、O型FMDV VP1基因(240 bp)、亚洲I型FMDV VP1基因(460 bp)、A型FMDV VP1基因(320 bp)。经优化引物浓度、退火温度等反应条件,初步建立了同时检测SVA与O型、亚洲I型、A型FMDV的多重RT-PCR方法。利用该方法同时检测SVA及O型、亚洲I型、A型FMDV、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒2型(PCV2)、PCV3等主要猪源病毒,结果显示该方法仅对SVA及O型、亚洲I型、A型FMDV检测为阳性,其他均为阴性,特异性较强;SVA、O型、亚洲I型、A型FMDV重组质粒标准品10倍倍比稀释后利用本研究建立的多重RT-PCR方法检测,结果显示同一反应体系中,4种重组质粒标准品的检出下限均为2.5×10^2拷贝/μL,敏感性较高;批内、批间重复性试验结果一致,该方法重复性较好。利用本实验建立的方法对2019年采集自广西30份临床疑似样品进行检测,结果显示,SVA、O型FMDV的阳性检出率分别为16.67%和63.33%;未检出亚洲I型及A型FMDV;同时采用国家标准《口蹄疫诊断技术》(GB/T18935-2003)中RT-PCR方法以及文献报道的SVA套式RTPCR方法对FMDV、SVA检测,结果与本实验建立的多重RT-PCR方法检测结果的符合率为100%。本研究首次建立的多重RT-PCR检测方法具有特异性强、敏感性高、重复性好的特点,为鉴别检测SVA与O型、亚洲I型、A型FMDV提供了有效的技术方法。

To establish a rapid method for simultaneous detection of senecavirus A(SVA)and O,Asia I,A serotypes of footand-mouth disease virus(FMDV),four pairs of specific primers were designed to amplify SVA 3D gene(157 bp),FMDV VP1 gene of serotype O(240 bp),Asia I(460 bp)and A(320 bp),respectively.After optimizing the primer concentration,annealing temperature and other reaction conditions,we established a sensitive,specific and reproducible method for simultaneous detection of SVA and O,Asia I,A serotypes of FMDV.The method specifically detects SVA and O,Asia I,A serotypes of FMDV without reacting with CSFV,PRRSV,PEDV,PRV,PPV,PCV2 and PCV3.The lowest detection limits of SVA and O,Asia I,A serotypes of FMDV were all 2.5×10^2 copies/μL.Furthermore,consistent results were produced under uniform reaction conditions.We used the method to detect 30 field samples collected from Guangxi province in 2019,results showed the positive rates of SVA and O serotype of FMDV were 16.67%and 63.33%respectively,while Asia I,A serotypes of FMDV was not detected.Meanwhile,by testing the same field samples using the state standard diagnostic method for FMDV and a reported nested RT-PCR method for SVA,we found that the coincidence rate of established method and the comparative methods was 100%.The results indicated that the established multiplex RT-PCR method,which had high sensitivity,specificity and reproducibility,could be used for differential detection of SVA and O,Asia I,A serotypes of FMDV.