应用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和序列上的差异 ,参照有关文献 ,设计了 3对引物 (A+B,A+C,A+D)。引物 A+B能从 L a Sota、B1 、 系和强毒 F4 8E8的含毒尿囊液中扩增出 36 2 bp的核酸片段 ,引物 A+C仅能从强毒 F4 8E8的含毒尿囊液中扩增出 2 5 4 bp的片段 ,引物 A+D仅能从 L a Sota、B1 、 系的含毒尿囊液中扩增出 2 5 4 bp片段。 3对引物均不能从 IB、IBD、AI的含毒尿囊液和正常鸡胚尿囊液中扩增出特异片段。从发病鸡群中分离的 3株 NDV毒株均被 A+C引物扩出 ,它们的鸡胚平均死亡时间 (MDT)分别为 5 1.8、5 3.2、5 2 .6 h,1日龄雏鸡脑内接种指数 (ICPI)为 1.6 5、1.6 3、1.6 0 ,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 2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毒力的鉴定结果与传统生物学试验的测定结果完全吻合 ,但前者可在 2 4 h内直接对组织匀浆进行诊断 ,具有快速。

On the basis of difference in composions and sequence of amino acid at the cleavage site of F protein of virulent and non-virulent strains of Newcastle disease viruses(NDV) three primer pairs A+B,A+C and A+D were designed according to relevant references.A fragment of 362 bp was amplified by the primers A+B from the allantoic fluid containing LaSota,B_1,Ⅰ or virulent F_(48)E_(8).A fragment of 254 bp was amplified by the primers A+C only from allantoic fluid containing virulent F_(48)E_(8),while a fragment of 254 bp was amplified by the primers A+D from allantoic fluid of LaSota,B_1 and Ⅰ.But no specfic fragment was amplified from both the normal allantoic 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 fragment of 254 bp was amplified by the primers A+C from three isolates from infectious chicken groups.The mean death time of embryos inoculated with these three isolates were 51.8,53.2 and 52.8 hours respectively,and the intracerebral pathogenicity index(ICPI) were 1.65,1.63 and 1.60 respectively.It was proved that they were virulent.NDV could be detected by RT-PCR in the tracheas of the artificially infected chicken next day,then in the cloaca,spleen and kidney.The virulence of NDV identified by RT-PCR accorded with those by conventionally biological methods.Furthermore the former can diagnose ND directly with tissue homogenate within twenty-four hours and have sensitive,simple and rapid characters.