猪血凝性脑脊髓炎病毒实时荧光RT-PCR检测方法的建立与应用

Establishment and Application of a Real-time RT-PCR Assay for Detection of Porcine Hemagglutinating Encephalomyelitis Virus

为建立一种快速、准确、常规的猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)检测方法,根据PHEV N蛋白基因序列,设计1对引物和1条探针,建立了PHEV实时荧光RT-PCR检测方法。该方法特异性好,与猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)和猪圆环病毒2型(PCV2)均无交叉反应;用阳性质粒Puc57-PHEVn评估其敏感性,发现检测下限达10-6 ng/μL(224拷贝/μL)。采集233份发病猪组织样品进行临床检测,发现该方法与巢式RT-PCR的符合率达98.3%,准确筛查出巢式RT-PCR并测序阳性的样品14份,且检出率高于巢式RT-PCR。结果表明,本方法敏感、特异,可用于PHEV临床样品的检测。

In order to establish a rapid,accurate and routine method for detection of porcine hemagglutinating encephalomyelitis virus(PHEV),a pair of primers and a probe were designed based on the N gene sequence of PHEV,and a real-time RT-PCR assay was developed,which was with good specificity,and failed to crossly react with porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus(PRV),porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),classical swine fever virus(CSFV)and porcine circovirus type 2(PCV2).The sensitivity of the method was evaluated based on positive plasmid Puc57-PHEVn,and it was found that the limit detection was 10-6 ng/μL(224 copies/μL).A total of 233 swine tissues were tested by the method,and it was found that 14 positive samples were detected out,and the coincidence rate with the nested RT-PCR was 98.3%,but the detection rate was higher than that by nested RT-PCR.It was concluded that the method could be used to detect PHEV clinical samples benefited from its high sensitivity and specificity.