猪圆环病毒2型重组腺病毒核酸扩增检测标准样品的研制

Establishment of Recombinant Adenovirus as Reference material for Porcine Circovirus Type 2 Nucleic Acid Amplification Technology(NAT)-based Assays

为研制用于猪圆环病毒2型（PCV-2）核酸扩增检测（NAT）质量控制的标准样品,本研究将猪圆环病毒2型QD/2014株ORF2连接到缺陷型腺病毒穿梭质粒pacAd5 CMV K-NpA上,构建了重组穿梭质粒CMV-PCV-2-ORF2。将其与骨架质粒pacAd5 9.2-100线性化后共转染HEK293T细胞,经胞内重组获得了内含PCV-2-ORF2的重组腺病毒pacAd5 CMV-ORF2。采用PCR和基因测序方法对重组病毒鉴定后进行大量培养、分装和冻干,经均匀性和稳定性检验后对其进行了定值。结果表明,制备的标准样品均匀、稳定,PCV-2-ORF2基因含量为8.17×10^9 GEq/mL,在HEK293细胞上的TCID_（50）为3.56×10^7/0.05 mL。本标准样品的研制成功为核酸检测标准物质制备开辟了一条新途径。

Recombinant adenovirus containing PCV-20RF2 gene, which mimics naturally DNA virus particles, can be used as candidate reference material for nucleic acid amplification technology （NAT）-based assays of PCV-2. In this paper, the com- plete ORF2 of PCV-2 stain QD/2014 was amplified and cloned to defective adenovirus shuttle plasmid pacAd5 CMV K-NpA to construct recombinant shuttle plasmid CMV-ORF2 .When the linearized CMV-PCV2-ORF2 and backbone plasmid pacAd5 9.2- 100 by Pac I were co-transfected into HEK293T ceils, the adenovirus pacAd5 PCV-2 containing ORF2 were obtained by recombination in vivo. Then the recombinant virus which was stable in propagation was verified by PCR and Sequencing. After propagation, aliquot, lyophilization, homogeneity and stability testing, the GEq/ml （gene equivalents/mL）and TCIDso of the recombinant virus were determined. The result showed the prepared candidate reference material was homogeneous and stable with a value of 8.17× 109 GEq/mL and 3.56×107/0.05 mL（TCID50）. Based on the establishment of the PCV-2 candidate reference material, a new way for preparation of reference materials for NAT-based assay was developed.