Mari J, Bonami J R, Lightner D V
Department of Veterinary Science, University of Arizona, Tucson 85721, USA.
Dis Aquat Organ. 1998 May 14;33(1):11-7. doi: 10.3354/dao033011.
The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.
从纯化的桃拉综合征病毒（TSV）中提取的单链RNA基因组被转录成双链平端cDNA，并用于构建pUC 18或pBluescript II KS载体的cDNA文库。在筛选文库后选择的12个重组质粒进行限制性酶切消化，以确定插入片段大小和限制性图谱。其中两个，pP15和pQ1，被选用于探针构建。插入片段分别为1500和1300个碱基对（bp），用DIG-11dUTP标记，相应的探针分别命名为P15和Q1。在Northern印迹和斑点印迹上，使用不同的变性方法，这两种探针与提取的RNA-TSV基因组、TSV和感染TSV的对虾匀浆特异性杂交。与测试的其他对虾病毒[传染性皮下和造血组织坏死病毒（IHHNV）和肝胰腺细小病毒（HPV）]未获得阳性杂交信号。通过对患TSV病对虾的组织切片进行原位杂交，证实了这两种探针的特异性。
