应用生物素标记的NDV－cDNA探针检测感染尿囊液中NDV－RNA的初步研究

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＮＤＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体──Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐｃＤＮＡ片段经光敏生物素标记后,即成ＮＤＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＮＤＶ强毒株和疫苗毒株的基因组ＲＮＡ,而不与ＩＢＤｖ－ｄｓＲＮＡ、ＡＩＢｖ－ｓｓＲＮＡ、ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ－ｄｓＤＮＡ,ＦＰＶ－ｄｓＲＮＡ及ＡＩＬＶ－ｄｓＤＮＡ发生交叉杂交反应。试验结果表明：尽管该探钎含有编码Ｆｏ蛋白酶切位点序列的碱基顺序,但它还是不能把ＮＤＶ的强、弱毒株区分开。这说明ＮＤＶ强、弱毒株比区域内的碱基存在着相当大的同源性。不过,此探针对ＮＤＶ来说具有特异性,这就为ＮＤＶ的诊断技术开创了基因水平检测的新途径。

DETECTION OF NEWCASTLE DISEASE VIRUS RNA IN INFECTED ALLANTOIC FLUID WITH PHOTOBIOTIN LABELED cDNA PROBE

The photobiotin labeled cDNA probe,which was made up with 359 bp sequence,lying in the gene encoding the fusion glycoprotein F for a virulent strain,amplifiedby polymerase chain reaction(PCR)from NDV-cDNA gene library, was studied fordetecting NDV-RNA in infected allantic fluids.Preliminary results show that the probecould specially detect the RNA from a virulent strain and two vaccine strains of NDVusing dot-blot hybridization on nitrocellulose membrane. Control experiments revealedthat it could not hybridize with dsRNA from infectious bursal disease virus(IBDV) of poultry,ssRNA from avian infectious bronchitis virus(AIBV) dsDNA from egg dropsyndrome 1976 (EDS-76),dsDNA from Marek's disease virus (MDy),dsDNA fromfowl pox virus(FPV),and dsDNA from avian infectious laryngotracheitis virus(AILV). AIthough the 359 bp fragment probe spans the sequence encoding the proteolytic clea-vage site of the Fo protein,it could not differentiate the genome RNA of virulentstrain from the one of avirulent strain.It is probable that there is base-homology atleast in the region between virulent and avirulent strains. As the same reason, theprobe is very specific for NDV-RNA.