基因扩增产物微孔杂交法检测新城疫病毒致病毒株

Detction of the Amplified Products of Virulent Newcastle Disease Virus-RNAwith Hybridization Technique in Microwells

目的建立一种快速、简便、敏感、实用的方法以检测新城疫病毒致病性毒株。方法在F蛋白裂解位点的两侧设计引物 ,引物1的5'端用生物素标记 ;按Aus/32强毒株F基因裂解区域设计24个碱基的寡核苷酸探针 ,并对之进行5'端地高辛标记。生物素化的双链扩增产物被固相的链霉亲和素捕获后经变性成为生物素化单链。然后微孔中加入地高辛标记的探针进行杂交 ,并用抗 -地高辛 -碱性磷酸酶显色。结果所建立的方法快速、简便、特异 ,敏感性比琼脂糖电泳法高约3～4倍 ,批内CV为9.88 % ,批间CV为18.31%。结论所建立的方法优化PCR条件后可作为新城疫病毒感染诊断。

Objective To establish a simple,rapid,sensitive and practical method to detect velogenic and mesogenic strains of Newcastle diaease virus.Methods A pair of 20-mer oligonucleotides,flanking the cleavage site,were used as primers,one of which was labeled at 5'-terminal with biotin.A 24-mer oligonucleotide probe labeled with digoxigenin at 5'-terminal was designed to complement the cleavage region of F gene of the Aus/32 virulent strain of NDV.After RT-PCR,the amplified products were added into the steptavidin-coated microwells,and the biotinlylated strands were captured,followed by denaturation,and non-biotinlylated strands were removed.Hybridization was performed by adding the specific probe labeled witn digoxigenin.Finally anti-digoxigenin-alkaline phosphatase conjugates were added.After washing,the substrate pNPP was added into the wells and measured on microplate reader.Results The established assay with features of simplifity,rapidity,specificity had an intrarun CV of 9.88% and an interrun CV of 18.31%,it could detect the amplified products of 0.17%,which is about 3～4 times more sensitive than that of agarose gel electrophoresis and ethidium bromide staining.Conclusion The method could be widely used as a routine assay for NDV-RNA of mesogenic and velogenic strains during diagnosis and quarantine after optimization of the parameters of PCR.