Laboratório de Virologia, FEPAGRO Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Caixa Postal 47, Eldorado do Sul, 92990-000 RS, Brazil.
Res Vet Sci. 2010 Jun;88(3):436-40. doi: 10.1016/j.rvsc.2009.10.006. Epub 2009 Nov 14.
Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93-99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10(5.55) TCID(50)/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.
多引物滚环扩增（MPRCA）被用于扩增来自患有断奶后多系统衰弱综合征（PMWS）迹象的猪组织中的猪圆环病毒 2 型（PCV2）基因组。从扩增的 PCV2 基因组中克隆了两个原核质粒并进行了测序。除了 ORF2 区编码衣壳蛋白的一个沉默取代外，它们几乎完全相同（1767nt）。此外，它们与来自其他国家的 PCV2 分离株具有高度核苷酸序列相似性（93-99%）。为了研究 MPRCA 扩增的 PCV2 基因组是否可用于产生感染性病毒，将克隆的基因组从质粒中分离出来，重新环化并用于 PK-15 细胞的转染。该过程导致产生了高达 10（5.55）TCID（50）/mL 的感染性病毒。因此，可以得出结论，MPRCA 是一种有用的工具，可用于扩增 PCV2 基因组，以用于测序和病毒分离策略，其特别有用的一点是，它允许从扩增的基因组中直接构建 PCV2 感染性克隆。然而，与用于诊断目的的 PCR 相比，其灵敏度较低。
