Abstract: 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.

Key words: armored virus; highly pathogenic porcine reproductive and respiratory syndrome virus(H-PRRSV); internal control; MS2 phage; pET-32a vector