肉食兽细小病毒核酸探针的制备及其初步应用研究

Preparation and Preliminary Use of Nucleic Acid Probes for Carnivore Parvoviruses

将提纯的貂肠炎病毒(MEV)中间复制型DNA(RF-DNA),以HinaⅡ酶切,回收0.7kbC片段并克隆至质粒pBR322中,构成重组质粒pBM,经转化大肠杆菌RRⅠ并扩增后,提纯pBM,酶切电泳回收克隆的C片段,以[α-32P]dATP标记C片段和pBM,用光生物素标记pBM,分别制成放射性同位素和光生物素核酸探针。采用打点杂交技术检测了5种肉食兽细小病毒的细胞培养物和非细小病毒科5种病毒的细胞培养物,同时检测了貂和犬的粪便样品33份。结果表明,32P标记的C片段和pBM探针与光生物素际记的pBM探针均具有相同的杂交特异性,与5种肉食兽细小病毒及感染细小病毒的貂、犬粪样呈杂交阳性反应,与其他科病毒及健貂、犬粪样呈阴性。同位素32P和光生物素标记探针分别检出1 pg和10 pg貂肠炎病毒RF-DNA,敏感性比血凝试验分别离100倍和10倍。

Double-stranded replicative form (RF) DMA of mink enteritis virus(MEV) was extracted and purified. A 0.7 kb DNA fragment C obtained by Hind I digestion of MEV RF DNA was cloned into a plasmid vector pBR322. The recombinant pBM was purified from transformed Escherichia coll strain RRI, and fragment C was recovered from pBM. pBM and fragment C were labelled with [α-32P] dATP, and pBM with photobiotin. By dothybridization, 5 members of carnivore parvoviruses and 5 virus strains of non-parvoviridae in infected cell cultures and 33 fecal samples from healthy or non-infected minks and dogs were examined. Photobiotin-labelled pBM probe and 32P-radiolabelled pBM and fragment C probes had the same specificity. All the carnivore parvovirus infected cell cultures and fecal samples showed positive, and those from healthy minks and dogs were negative. 32P-radio-labelled probes and photobiotin-labelled probe detected 1 pg and 10 pg MEV RF DNA respectively, i.e.100 times and 10 times more sensitive than the standard hemagglutination test.