RT-PCR鉴别鸡新城疫病毒强弱毒株检测方法的建立

Establishment of RT-PCR Method for Identification of Strong and Weak Virulence NDV Strains in Chickens

通过比对分析NCBI数据库中新城疫病毒(NDV)不同毒株V蛋白基因序列,设计2对引物,并优化试验条件,建立一种快速鉴别NDV强弱毒株的RT-PCR诊断方法。结果显示,用2对引物对NDV强毒可扩增出703 bp和456 bp的特异性片段,而对NDV弱毒只能扩增出703 bp的特异性片段;特异性试验结果表明该方法对IBDV、IBV和ILTV的检测均为阴性;灵敏性试验结果显示,该方法对NDV强弱毒株最小RNA检出量为均为0.01 ng/μL,敏感性高于当前通用检测引物。所建方法具有高灵敏性、强特异性、准确快速等优点,可用于临床新城疫强弱毒的快速检测及新城疫的监测。

In order to meet the need of rapid identification and diagnosis of strong and weak virulence Newcastle disease virus(NDV)strains in clinical practice,it is convenient to carry out the research on the difference of pathogenicity of different NDV strains.In this study,by comparing and analyzing the V protein gene sequences of different NDV strains in the NCBI database,two pairs of primers were designed and the test conditions were optimized to establish a rapid RT-PCR diagnostic method for the identification of strong and weak virulence NDV strains.The results showed that the specific fragments of 703 bp and 456 bp could be amplified by using two pairs of primers to the strong virulence NDV strain,while the specific fragments of 703 bp could be amplified to the weak virulence NDV strain,the results of specificity test showed that the method was negative for IBDV,IBV and ILTV.The results of sensitivity test showed that the minimum RNA detection amount of this method against the strong and weak virulence NDV strains was 0.01 ng/μL,and the sensitivity of this method was higher than that of the current common detection primers.The method established in this study has the advantages of high sensitivity,strong specificity,accuracy and rapidity,and can be used for the rapid diagnosis of the strong and weak virulence NDV strains and the monitoring research of Newcastle disease.