用RT-PCR对新城疫病毒分离株毒力的快速鉴定

Rapid differentiation of the virulence of Newcastle disease virus isolates by RT-PCR

根据新城疫病毒NDV强、弱毒株在F蛋白裂解位点氨基酸组成的序列上的差异,参照有关文献,设计了三对引物A+B,A+C,A+D。引物A+B能从Lasoa,B1,I系和强毒F48E8的含毒尿囊液中扩增出362bp的核酸片段,引物A+C仅能从强毒F48E8的含毒尿囊液中扩增出254bp的片段,引物A+D仅能从Lasota,B1,I系的含毒尿囊液扩增出254bp片段。三对引物均不能从IB,IBD,AI的含毒尿囊液和正常鸡胚尿液中扩增出特异片段。从发病鸡群中分离到3株NDV毒株中均被A+C引物扩出,它们的鸡胚平均死亡时间MDT分别为51.8h、53.2h、52.6h,1日龄雏鸡脑内接种指数ICPI为1.65、1.63、1.60,证明是NDV强毒。利用RT-PCR对NDV人工感染鸡最早可于攻毒后第二天从病鸡气管中检测到NDV,其次为泄殖腔和脾、肾。利用RT-PCR对NDV毒力的鉴定与传统生物学试验对强弱毒的测定结果完全吻合。但前者可在24小时内直接对组织匀浆进行诊断,具有快速简便和敏感的特点。

On the basis of difference incomposions and se-quce of amino acid at the cleavagesite of F oprotein of virulent and non-virulent strains of Newcastl disease viruses(NDV)three primer pairs A+B,A+C and A+D were designed according to relevant references. A fragment of 362bp was amplified by the primers a+b from the allantoic fluid containing Lasota,B1 I or virulent F48E8.A fragment of 254bp was amplified by the primers A+Conly from allantoic fluid containing virulent F48E8 while afragment of 254bp was amplified by the primers A+D from allantoic fluid of Laso-ta B1 and I.But no specfic fragment was amplifie from both normal all antoic fluid and allantoic fluid containing infectious bronchitis virus or infectious bursa disease virus or avian influenza virus with any of these three primer pairs.A frag-ment of 254bp was amplified by the primers A+C from three isolated from infectious chicken groups.The mean death time of embryos inoculated with these three isolates were51.8 53.2 and 52.8 hours respectively and the intracerebral pathogenicity indes ICPI were 1.65 1.63 and 1.60 re-spectively this proved they were virulent. NDV could be de-tected by RT-PCR in the tracheas of the artificially infected chicken next day then in the cloaca spleen and kidney.The results of indentifing the virulenceofNDVbyRT-PCRaccorded with those by convenfionally biological methods. But the former can diagnose ND directly with tissue homogenate in twenty-four hours and have senstive simple and rapid characters