摘要
用ＥＤＳ７６病毒标准ＡＶ１２７株和分离株（Ｂ９６株）感染鸭胚，提取病毒核酸，分别经ＥｃｏＲｌ和Ｐｓｔｌ双酶切，获得图形和大小相似的酶切片段。分别进行ＥｃｏＲｌ和Ｐｓｔｌ单酶切，也得到相似结果。病毒ＤＮＡ经ＥｃｏＲｌ和Ｐｓｔｌ双酶切后，与ＰＵＣ１９质粒载体重组，并转化到ＥｃｏｌｉＪＭ１０１中，筛选出一个插入片段（Ｇ片段）约为２７ｋｂ的重组质粒。用Ｄｉｇｏｘｉｇｅｍｉｎ标记ＥＤＳ７６ＤＮＡＧ片段（ＥｃｏＲｌ和Ｐｓｔｌ双酶切片段）及含该片段的重组质粒分别制备探针，对ＥＤＳ７６病毒ＤＮＡ进行斑点杂交，两种探针均为阳性，而对照组ＮＤＶ、ＩＢＶ、ＩＬＴＶ、ＩＢＤＶ正常鸭胚尿囊液的核酸为阴性。且后一种探针的敏感性高于前者，它的ＤＮＡ检出限量为４ｐｇ水平。结果表明，两种探针具有高度的特异性、敏感性。
After a standard EDS76 strain AV 127 virus or a field isolate strain B96 virus was inoculated in duck embryos,viral DNA was extracted from the duck embryo allamoic fluids and digested with endonuclease EcoRI and PstI,The produced fragments were very similar in restriction patterns and size of fragments,An EDS76 virus DNAG fragment with 27kb was obtained,The fragment was recombined with PUC 19 plasmid lineared with the same restriction enzyme,then it was trasformed into E coli JM 101,The recombinant which has an inserted fragment with 27kb was obtained,The recombinant and the EDS76 virus DNAG fragment were labelled with digoxigenin as DNA probes for EDS76 virus,Dotblot bybridiztion with the two probes showed positive result for EDS76 virus DNA,but negative for the nucleic acid samples obtained from NDV,IBV,ILTV,IBDV and duck embryo allantoic fuild,The sensitivity of the recombinant plasmid PUC19 probe,DNAdetection limit being 4pg was superior to that of the EDS76 virus DNAG fragmentprobe,It was demonstrated that two probes were of high specificity and sensitivity.
关键词
减蛋综合症
病毒
核酸探针
鸡病
Egg Drop Syndrome1976 virus digoxigenin DNA probes
