摘要
本研究根据伪狂犬病病毒(PRV)gp50的基因序列设计并合成引物,以我国闽A株细胞培养毒为模板,筛选最佳反应条件,建立了PRV的PCR检测方法。应用该方法对双城、Shope、Bartha等毒株的细胞培养物进行基因扩增都获得了分子量为321bp的特异性目的DNA片段,其检出敏感度为2×10^(-5)个TCID_(50)。用该方法检测人工感染仔猪的扁桃体、三叉神经节、鼻拭子、肺、脑,家兔的白细胞,小鼠的脑组织,其结果都呈现特异性阳性反应,检测狂犬病病毒,腺病毒、细小病毒皆为阴性结果。但是,应用该引物扩增马立克氏病病毒(MDV)基因,可发现一条大小约为27bp的DNA片段,表明PRV与MDV可能具有源性。本研究还建立了两种模板制备方法,即:通过煮沸裂解由细胞培养物制备DNA模板的简法,通过EDTA-胰蛋白酶消化→煮沸裂解→酚:仿:醇抽提由病料制备DNA模板的新法,为在临床上应用PRV PCR方法论断该病提供了可能。
One set of primers was designed and synthesized for amplification of the 321bp regionly-ing in the gene encoding the glycoprotein gp50 of pseudorabies virus (PRV). The positive template was prepared with the Chinese F-A strain of PRV in cell culture samples. The best procedure of polymerase chain reaction (PCR) was selected and determined. We applied this technique to specifically amplify the 321 bp DNA fragment of the PRV strains including Shuangcheng,Shope and Bartha in cultured samples By this technique,the tissue samples of experimentally infected animals including tonsils ,trigeminal gangliones,nasal scabs,lungs, cerebra of swine,white blood cells f rabbit,and the cerebra of mouse were fested for this specific fragment. The sensitvity of PRV PCR reached to 2 × 10-5TCID50. The negative results were achieved from rabies virus,adenovirus and par-vovirus. Howerer,the approximately 270 bp DNA fragment was amplified from Marek's disease virus (MDV). It indicated that homol-ogy between PRV and MDV may exist.Two methods of preparing templates were established in this study. They in eluded that (l)the cell culture samples were split by boiling and (2) the tissue samples were digested by EDTA-trypsin, disrupted by boiling and extracted by phenol : chloroform : isoamyl alco hoi. It would provide the possibility that the PCR be used to diagnose the pseudorabies in clinic.
