非洲猪瘟病毒免提取核酸荧光定量PCR检测方法的建立

Development of a direct real-time PCR assay freed from DNA extraction for detection of African swine fever virus

根据非洲猪瘟病毒(ASFV)保守的VP72基因序列,设计特异性引物和探针,经过优化反应体系中各组分及反应条件,建立了直接检测样品中ASFV的荧光定量PCR方法,并分析该方法的特异性和敏感性。结果显示,该直扩荧光定量PCR方法只对ASFV有特异性扩增,对其他几种相似疾病病原的灭活抗原,如猪瘟病毒、猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、口蹄疫病毒、印第安纳型水泡性口炎病毒、新泽西型水泡性口炎病毒、猪流行性腹泻病毒等检测均呈阴性。敏感性试验结果显示,对10倍梯度稀释的非洲猪瘟病毒灭活抗原的检测敏感性可达1×10^-6,与OIE推荐的传统的荧光PCR的检测敏感性相当。3个ASFV稀释度样品的组内和组间重复的变异系数均小于5%。该方法无须提取核酸,可在60 min内完成对样品的检测,检测快速、灵敏,结果准确,结合便携式荧光PCR仪,为野外或现场的非洲猪瘟病毒快速检测提供了一种切实可行的方法。

Specific primers and probe were designed based on the VP72 gene sequence of African swine fever virus(ASFV).A direct real-time fluorescent PCR assay freed from DNA extraction and purification was developed for rapid detection of ASFV.The specificity test results showed that the direct real-time PCR assay could specifically amplify target ASFV with the optimized concentrations of primers and probes and reaction condition,which had no cross reaction to classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine circovirus,foot and mouth disease virus,vesicular stomatitis virus(serotype Indiana and New Jersey)and porcine epidemic diarrhea virus.The sensitivity of this method reached 1 ×10^-6 dilution for the 10-fold diluted ASFV inactivated antigen,which was comparable to that of the traditional real-time fluorescent PCR method recommended by OIE for the inactivated virus.The coefficient of variation values of intra-and inter-groups repeatability of 3 different dilutions of inactivated virus were all less than 5%.Compared to the traditional real-time PCR,this method was rapid and the whole test could be completed within 60 min.Samples can be simply applied to this method without DNA extraction and purification.Due to its high specificity,sensitivity,and repeatability,the real-time PCR assay freed from DNA extraction provided a novel,rapid,and practical tool for the specific detection of ASFV in the field.