新城疫疫苗毒与野毒的区别诊断及其多功能检测软件的开发

Differentiation of Newcastle disease virus vaccine strains from NDV field strains and development of multifunctional software for differentiation

运用生物信息学软件对GenBank中收录的30株新城疫病毒(NDV)全基因组序列间的差异和它们对应的F基因片段22～420核苷酸序列间的差异进行了分析。发现这些差异高度相关(r=0.937),该片段可作为区分NDV野毒和疫苗毒的指纹序列。据此设计了1对扩增用简并引物(其中一条用于PCR产物的直接测序),建立了RT-PCR-测序技术,并测得2个参数:NDV标准疫苗指纹序列库和指纹序列的变异参数。在此基础上,开发出多功能自动分析软件,通过对该指纹序列的分析,不仅可以区分NDV疫苗毒和野毒,还能同步测定这些病毒的毒力和基因型。该技术对NDV标准毒株的测定结果与已知信息完全吻合,且只需3 d即可获得结果。对不同禽类(鹅、鸽、鸵鸟、鸡)中分离的NDV的测定结果表明,59%为残留的疫苗毒。

The nucleic acid sequence of fragment 22-420 of Newcastle disease virus（NDV） fusion protein gene could be used as the fingerprint sequence to differentiate the field strains from the vaccine strains according to the statistic analysis of the sequence divergence correlation between the genome and the fragment of 30 NDV strains by bioinformatics softwares（r=0. 937）. The degenerate primers were synthesized to amplify this fragment, with the established RT-PCR sequencing method. Two parameters,namely, the fingerprint sequence database and variations of the standard NDV live vaccines,were determined. Based on these results,we developed a multifunctional automatic analysis software which can differentiate the live vaccines and field strains,and predict their virulence genotypes simultaneously. This new diagnosis method was useful in the analysis of the standard strains of NDV and could obtain results within three days. Field samples of NDV isolated from different species of poultry,i, e. goose, pigeon,ostrich and chicken, were also tested with this new method. Approximately 59% of the isolated strains of NDV represented the live vaccines.