鸡新城疫病毒荧光双链引物实时荧光PCR检测方法的研究

The Real-time PCR Detection of Newcastle Disease Virus of Chickens by Fluorescent Double-stranded Primers

根据鸡新城疫F糖蛋白的保守基因序列,设计一对正引物。根据正引物设计一对与之相互补的负引物。正引物的5’端标记FAM,负引物的3’标记淬灭剂DABCYL。反应体系中进行引物杂交后,由于正、负引物完全互补,FAM产生的荧光被标记在负引物上的DAB-CYL淬灭掉。然后利用杂交后生成的荧光双引物对鸡新城疫及AIVI、LTVI、BV四种病毒和MG进行了实时荧光PCR检测。结果表明,只有以鸡新城疫标准毒株的克隆质粒为模板的管中能够检测到荧光信号,其他管中没有信号产生。该方法特异性强,灵敏度高,重复性好,在鸡新城疫临床诊断和出入境检疫中有着广阔的应用前景。

A positive primers were designed based on the mRNA of the F-Glycoprotein of the Newcastle Disease virus. At the same time , the negative primers which were designed based on positive primers can completely combine with positive primers. FAM was labelled on the 5 ＇ end of the positive primers and DABCYL was labelled on the 3＇ end of the negative primers . When positive primers were hybridized with negative primers , the fluorescence generated by FAM was quenched by DABCYL. Then the fluorescent double-stranded primers were formed, and they can be used to detectd Newcastle Disease virus and other 4 species virus including of AIV, ILTV, IBV and MG. The experiment showed that no signal was detected for the others except Newcastle Disease virus. The assay gives better specificity, good reproducibility and high sensitivity to the NDV,so it is prospective in the clinical diagnosis and quarantine on the export and import in detecting the NDV.