PCR检测鸭瘟病毒的研究

Detection of duck plague virus by polymerase chain reaction

根据鸭瘟病毒DNA聚合酶基因序列 ,设计、合成了 1对引物 ,以 1株鸭瘟病毒疫苗株DNA为模板 ,进行PCR扩增 ,扩增出预期 5 6 3bp的目的DNA片段。将扩增出的DNA片段克隆到pMD18 T载体 ,经Amp/IPTG/X gal平板筛选 ,HindⅢ、XbaⅠ双酶切鉴定 ,获得阳性重组质粒。对重组质粒进行序列测定 ,与参考序列比较 ,二者同源性为 99.3%。小鹅瘟病毒、鸭肝炎病毒、鹅副黏病毒PCR扩增均为阴性。用此方法检测人工感染和自然感染鸭瘟的组织 (脑、肝、脾 ) ,均能检测到鸭瘟病毒DNA。PCR检测鸭瘟病毒具有高度的特异性、敏感性 。

According to the DNA polymerase gene data of du ck plague virus (DPV) , a set of primers was designed and used for a polymerase ch ain reaction (PCR) with the vaccine DPV strain. A specific 563 bp DNA product w as amplified , which was cloned into pMD18-T vector ,and the positive recombina nt clone was selected by the Amp/IPTG/X-gal agar plate and characterized by HindⅢ and XbaⅠenzyme digestion . The r ecombinant plasmid was sequenced and compared with published sequence. The homog eneity was 99.3%. The DPV specific DNA product was also amplified from the infec ted brains,livers and spleens from 4 experimentally infected ducks and 2 outbrea k samples of DPV, but not from the goose parvovirus and other avian virus . As little as 0.22 pg of DPV DNA was detected by the PCR. The result suggested that the PCR is a specific and sensitive method which can be used for detecti on and diagnosis of latent and subclinical DPV infections.