病毒样颗粒内标在荧光定量RT-PCR检测高致病性猪繁殖与呼吸综合征病毒方法中的应用

为建立对高致病性猪繁殖与呼吸综合征病毒（H-PRRSV）快速准确的检测方法，本试验构建兽医临床用病毒样颗粒内标表达载体，制备检测H-PRRSV的病毒样颗粒内标。在H-PRRSV Nsp2基因保守区域设计了1对特异性引物和1个TaqMan荧光探针，通过对反应体系和扩增条件的优化，建立了检测H-PRRSV的荧光定量RT-PCR检测方法；将MS2噬菌体的成熟酶蛋白和衣壳蛋白基因序列定向插入pET-32a载体的相应位置，在载体下游插入人工合成包含引物和探针的内标基因序列，采用双酶切和PCR鉴定阳性质粒，将阳性质粒转化大肠杆菌BL21工程菌，IPTG诱导表达，制备了病毒样颗粒内标。试验结果表明，所建立的方法特异性强，灵敏度高，对8份已知病毒的检测结果显示特异性为100%，检测滴度为1 TCID₅₀/mL病毒液，对42份临床样本的检测结果与农业部备案试剂盒检测结果完全一致，体现了较好的实用性。

Application of Armored Virus as Internal Control in the Detection of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus with Real-time RT-PCR

To establish a rapid and accurate detection method of highly pathogenic porcine reproductive and respiratory syndrome virus(H-PRRSV), we constructed an expression vector of armored virus for veterinary clinic detection and prepared an internal control for the detection. A pair of specific primers and a TaqMan probe were designed based on Nsp2 gene in the conserved region, the detection method for H-PRRSV was established by optimization of the reaction system and amplification parameter. Mature enzyme protein and capsid protein gene sequence of MS2 phage were inserted into pET-32a vector, and internal control gene of artificial synthesis including primer and probe sequence was cloned into the downstream vector,it was identified by double enzyme digestion and PCR, the recombinant vector was transformed into Escherichia coli BL21, IPTG induced the expression of recombinant vector to prepare armored virus. The result showed that the detection method for H-PRRSV by Real-time RT-PCR was established through the optimization of reaction system and amplification conditions, the proposed method was specific and sensitive, the detection result of 8 known specific viruses was accordant with the fact and the method could detect 1 TCID₅₀/mL H-PRRSV.In the detection of 42 clinical samples,the result was fully consistent with that of the kit designated by ministry of agriculture, the method showed good practicality.