NADC30-like谱系猪繁殖与呼吸综合征病毒荧光定量PCR检测方法的建立及应用

Establishment and application of a real-time PCR assay for the detection of porcine reproductive and respiratory syndrome virus NADC30-like strain

为定量检测NADC30-like谱系猪繁殖与呼吸综合征病毒(NADC30-like PRRSV),本研究针对NADC30 PRRSV株的ORF6基因保守区域设计1对特异性引物,通过优化反应条件,建立了一种灵敏度高、耗时短、操作简便且可量化的SYBR Green I荧光定量PCR检测方法。建立的标准曲线结果显示:Ct值与质粒标准品浓度之间存在良好的线性关系,相关系数(R~2)为0.995。该方法特异性强,除对NADC30-like PRRSV有特异性扩增外,对猪圆环病毒2型(PCV2)、猪塞尼卡病毒(SVA)、牛病毒性腹泻病毒(BVDV)、猪细小病毒(PPV)、经典型猪繁殖与呼吸综合征病毒(C-PRRSV)和NADC34-like PRRSV等病原均无扩增。对质粒标准品的检测下限可达4.62×10~1拷贝/μL,敏感性为常规PCR方法的100倍。组内变异系数与组间变异系数均小于1%,重复性较好。利用该方法与常规PCR方法同时检测53份血清样品,该方法的阳性检出率为11.3%(6/53),高于普通PCR的阳性检出率(9.4%,5/53),二者的总符合率为89.11%。本研究建立的SYBR Green I荧光定量PCR方法可以用于NADC30-like PRRSV的快速检测,为PRRS的防控提供技术支持。

In order to establish a new detection method for NADC30-like strains of porcine reproductive and respiratory syndrome virus(PRRSV),a quantitative SYBR Green I real-time PCR assay was developed with a pair of specific primers targeting to the conservative region of ORF6 gene of PRRSV.The result showed that a strong linear relationship was obtained between the Ct values and the log10 number of the corresponding positive standard plasmids,and the correlation coefficient was 0.995(R^(2)=0.995).This assay showed a high specificity,because there was no cross-reaction with PCV2,SVA,BVDV,PPV,C-PRRSV,and NADC34-like PRRSV.The sensitivity tests showed that the minimum detection limit of this method was 46.2 copies/μL,which was 100 times more sensitive than the conventional PCR.The intra-and inter-assay coefficient of variation of this assay was less than 1%.Fifty-three serum samples were collected and tested for PRRSV by using this assay and conventional PCR.This assay detected a positive rate of PRRSV of 11.3%(6/53),which was significantly higher than that of the conventional PCR(9.4%,5/53),and the coincidence rate of these two methods was 89.11%.This SYBR Green I real-time PCR assay can be used for the rapid detection of NADC30-like PRRRSV.Meanwhile,it might provide a technical support for the prevention and control of PRRS.