塞尼卡谷病毒重组酶聚合酶扩增-侧流层析试纸条检测方法的建立

Establishment of recombinase polymerase amplification-lateral flow dipstick for the detection of Seneca valley virus

为建立塞尼卡谷病毒(SVV)重组酶聚合酶扩增-侧流层析试纸条(RPA-LFD)检测方法,本研究针对SVV 3D基因设计了引物和探针,通过反应条件优化、特异性、灵敏性和重复性试验,建立了SVV RPA-LFD检测方法。特异性试验结果显示,该方法与口蹄疫病毒、猪瘟病毒、猪繁殖与呼吸道综合征病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、圆环病毒、伪狂犬病毒无交叉反应;在40℃、20 min内可检测的最低浓度为56拷贝/μL质粒标准品;通过3次重复试验检测,LFD检测线灰度值变异系数范围在4.49%~9.73%。利用该方法对120份国内临床样品进行检测,结果均为阴性。本研究首次建立了检测SVV的等温、快速RPA-LFD方法,读取结果不依赖任何仪器设备,为猪群感染SVV的现场快速诊断、流行病学研究和根除方案的制定提供帮助。

Recombinase polymerase amplification （RPA） is an isothermal and rapid amplification technique for the detection of nucleic acid developed recently. Along with lateral flow dipstick （LFD）, the result of the amplicon can be read-out directly. In this study, a RPA-LFD combined protocol was established with the primers and probes targeting seneca valley virus （SVV） 3D gene for SVV rapid detection. Under the optimized detection conditions, the assay was able to complete the detection within 20min at 40 ℃, which was specificity for SVV detection and had no cross reaction with the foot and mouth disease virus, classical swinefever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine circovirus and pseudorabies virus. The sensitive of this assay was capable to detect out of 56copies/μL of SVV. The coefficient of variation regarding the gray values of test lines was in the range of 4.49% to 9.73% assayed from seven series diluted samples （5.6×10^7 to 5.6×^101 copies/μL） with three repeat detections. A total of 120 samples were detected by this method, and the results were all negative. The established isothermal rapid amplification assay for the detection of SVV facilitates the on-site rapid diagnosis, epidemiological study and eradication of SVV among pigs0