应用多重Digoxigenin标记探针在核酸斑点杂交检测中识别Ⅰ群禽腺病毒的感染

Identification of group Ⅰ avian adenovirus infection by nucleotide dot hybridiza-tion detection using multiple digoxigenin labeled probes

禽腺病毒(fowl adenovirus,FAdV)是鸡群中常见的免疫抑制性病毒,其中Ⅰ群FAdV有12个血清型(FAdV1-8a,8b-FAdV11),这为Ⅰ群FAdV检测带来了挑战。为开发一种可以同时检测Ⅰ群FAdV的Digoxigenin标记核酸探针或者其组合,本研究针对Ⅰ群FAdV的hexon基因保守区域设计引物,分别以12个血清型的Ⅰ群FAdV参考毒株DNA为模板合成了12种Digoxigenin标记核酸探针,并将这12种核酸探针及其组合形成的多重探针共13种探针分别与同一杂交膜上的12种不同血清型Ⅰ群FAdV参考毒株的毒株核酸进行杂交,观察各探针或其组合能够识别的最大限度。结果显示,12种血清型对应的单一核酸探针能检测和识别的病毒类型及其灵敏度差异较大,而多重探针组合PM可以同时检测出所有12种血清型毒株并体现出最高灵敏度的优势。本研究建立了应用多重Digoxigenin标记探针组合在核酸斑点杂交检测中识别Ⅰ群FAdV核酸的新方法,为解决类似病毒高通量检测提供了新的思路。

Fowl adenovirus(FAdV) is a common immunosuppressive virus in chicken flocks.Among them,group Ⅰ FAdV has 12 serotypes(FAdV1-8 a,8 b-FAdV11),which poses a challenge for the detection of group Ⅰ FAdVs.This study attempts to develop a digoxigenin-labeled probe or combination of probes that can simultaneously detect all serotypes of group Ⅰ FAdV.Primers for the conserved region of hexon gene in group Ⅰ FAdV were designed with 12 serotypes of group Ⅰ FAdV as templates,respectively,and digoxigenin-labeled nucleic acid probes were synthesized.The 12 nucleic acid probes and the multiple probes formed by the combination for a total of 13 probes were hybridized to the nucleic acids of different serotypes of group Ⅰ FAdV reference strain on the same hybrid membrane to observe the discrimination ability of each probe or multiple probes.The results showed that the types of viruses identified by 12 single nucleic acid probes corresponding to 12 serotypes was different,and the detection sensitivity of these probes was quite different.However,the multiple digoxigenin-labeled probes could simultaneously detect all 12 serotype strains,showing the advantage of sensitivity.This study established a new method for the identification of group Ⅰavian adenovirus by nucleic acid dot blot hybridization using multiple digoxigenin-labeled probes,which provides a new idea for solving high-throughput detection of similar viruses.