非洲猪瘟病毒微滴式数字PCR检测方法的建立及临床应用

Establishment and Clinical Application of a Droplet Digital PCR Assay for African Swine Fever Virus

为建立非洲猪瘟病毒微滴式数字PCR方法,以非洲猪瘟病毒P72蛋白编码基因为靶基因设计引物和探针,利用正交试验方法优化反应体系及扩增程序,建立了非洲猪瘟病毒微滴式数字PCR方法,并对其敏感性、特异性及稳定性进行评估。结果显示:建立的方法最低检出限为0.58 copies/μL,与荧光PCR方法相比更敏感;本方法与布鲁氏菌、猪伪狂犬病病毒、猪圆环病毒2型和猪瘟病毒无交叉反应;对10倍稀释的标准物质检测10次,变异系数为8.44%,重复性好。利用建立的方法对100份临床样品进行检测,结果均为阴性,与荧光PCR检测结果一致;对5份实验室间比对样品进行检测,检测出阳性样品4份,阴性样品1份,与实验室间比对给定的样品盘结果一致。结果表明,本研究建立的微滴式数字PCR方法的敏感性、特异性和稳定性符合要求,适于非洲猪瘟病毒临床检测。

In order to establish a droplet digital PCR(ddPCR)assay for African swine fever virus(ASFV),primers and probes were designed taking the gene encoding P72 protein as a target gene,the reaction system and amplification program were optimized using orthogonal experimental design,and a ddPCR assay was established,followed by evaluation on its sensitivity,specificity and repeatability.The results revealed that the detection limit of established method could reach 0.58 copies/μL,which was more sensitive than fluorescent PCR;it could not produce any crossreaction with Brucella,porcine pseudorabies virus(PRV),porcine circovirus type 2(PCV-2)and classical swine fever virus(CSFV);its coefficient of variation(CV)was 8.44%when ten-fold diluted standard materials were tested for 10 times,indicating a good repeatability.100 clinical samples were detected by the established method,all their results were negative and consistent with those by fluorescent PCR;for 5 inter-laboratory comparison samples,4 positive samples and 1 negative sample were detected,which were consistent with the given sample panel for interlaboratory comparison.In conclusion,the established ddPCR,with good sensitivity,specificity and repeatability,was appropriate to detect ASFV in practice.