狂犬病诊断基因芯片的建立和检测应用的初步研究

Application of diagnostic DNA microarray for detecion of rabies virus

根据已发表的狂犬病病毒(RV)的序列,设计合成能扩增高度保守核(N)蛋白片断的一对引物。通过生物素标记PCR技术,将核(N)蛋白基因片断作为探针点在硝酸素纤维膜上,制作成疾病诊断基因芯片。取160份可疑动物的血液,提取核酸作模板进行PCR扩增,将其产物与诊断基因芯片进行特异性逆向点杂交检测;并用蔗糖密度梯度离心和凝胶层析纯化狂犬病病毒作抗原,建立检测RV抗体的间接ELISA。把以上两种方法应用于临床,结果基因芯片的检测率比ELISA方法和(RT)PCR要高15%左右,表明建立的狂犬病诊断基因芯片具有更高的灵敏度和特异性。

Fragments of Canine coronavirus protein N gene were amplified by reverse transcription-polymerase chain reaction （RT-PCR） using primers designed for the highly conserved regions. The DNA fragments were labeled with biotin and spotted onto colloxylin to form microarrays, Polynucleotide, The gene chip were tested by hybridization of PCR products from 160 the Canine blood. The results showed that the microarray technique could rapidly identify and specifically distinguish the Rabies virus. The sensitivity of this method was 15 % higher than that of the indirect EnZyme-linked immunosorbent assay （ELISA）.