Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.
J Virol Methods. 2012 Jul;183(1):80-5. doi: 10.1016/j.jviromet.2012.03.022. Epub 2012 Mar 30.
Reverse transcription-real time polymerase chain reaction (real time RT-PCR) assay with Universal Probe Library (UPL) probes has been developed for the detection and genotyping of Chilean infectious pancreatic necrosis virus (IPNV) isolates from infected cell culture. Partial nucleotide sequences (1175 bp) of the VP2 coding region from a selection of 7 Chilean IPNV isolates showed that they clustered into two main groups strongly correlated with Genogroups 1 and 5 proposed by Blake et al. (2001), corresponding to types West Buxton (WB) and Spajarup (Sp), respectively. Based on the VP2 gene sequences of those 7 Chilean isolates and different reference IPNV strains, 2 sets of candidate primer/UPL probes (# 8 and # 117) were designed and evaluated with a total of 32 field isolates isolated from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and Pacific salmon (Oncorhynchus kisutch) farms from 2006 to 2010 in Chile. The UPL probes clearly differentiated the same two major Genogroups that those recognized by sequencing analysis. Among the Chilean isolates examined, 18 yielded amplification with UPL probe # 8, and 14 with probe # 117, respectively corresponding to types Sp and WB, as demonstrated by typing by sequencing. Based on the findings reported below, it has been demonstrated that the combined real time RT-PCR protocol with UPLs approach was efficient in discriminating distinct Genogroups of IPNV cultured in fish cell lines and, therefore, recommended its use for detection and typing of IPN viruses. The study also confirmed the existence of two IPNV type strains in Chilean salmonid aquaculture.
建立了一种基于通用探针库（UPL）探针的逆转录实时聚合酶链反应（real time RT-PCR）检测方法，用于检测和基因分型感染细胞培养物中的智利传染性胰脏坏死病毒（IPNV）分离株。对 7 株智利 IPNV 分离株的 VP2 编码区部分核苷酸序列（1175bp）进行分析，结果表明它们聚为两个主要组群，与 Blake 等人（2001 年）提出的 1 型和 5 型基因群高度相关，分别对应 West Buxton（WB）和 Spajarup（Sp）型。根据这 7 株智利分离株的 VP2 基因序列和不同的参考 IPNV 株，设计了 2 组候选引物/UPL 探针（#8 和#117），并对 2006 年至 2010 年期间智利大西洋鲑（Salmo salar）、虹鳟（Oncorhynchus mykiss）和太平洋鲑（Oncorhynchus kisutch）养殖场分离的 32 株田间分离株进行了评估。UPL 探针可清晰地区分与测序分析结果一致的两个主要基因群。在所检测的智利分离株中，分别有 18 株和 14 株与 UPL 探针#8 和#117 扩增，这与测序分型结果显示的 Sp 和 WB 型相对应。根据下面的研究结果，证明了该联合实时 RT-PCR 协议与 UPL 方法可有效区分鱼类细胞系中培养的不同 IPNV 基因群，因此推荐用于 IPN 病毒的检测和分型。该研究还证实了智利鲑鱼养殖业中存在两种 IPNV 型株。
