同时鉴别6种鸡病毒性呼吸道病GeXP检测方法的建立

Development of a GeXP Assay for Simultaneous Differentiation of Six Chicken Respiratory Viruses

为了建立一种能够同时鉴别H5、H7、H9亚型禽流感、新城疫、传染性支气管炎和传染性喉气管炎6种鸡病毒性呼吸道传染病病原体的GeXP检测方法,本研究根据这些病原体各自的基因保守序列,设计合成了7对特异性引物,优化反应条件,用单一病毒、混合病毒样品来验证所建立的GeXP方法的特异性和准确性,以不同拷贝数的克隆质粒来检测GeXP方法的灵敏度。检测34份临床阳性样品,进一步验证该法的可靠性。结果显示,在7对引物同时存在的GeXP反应体系中,可特异性地扩增出相关6种病毒目的片段,并可在102拷贝/μL水平同时特异地检测出鸡6种病毒性呼吸道传染病,GeXP方法检测34份临床阳性样品结果与病毒分离一致。本研究建立的GeXP方法不仅可高通量检测6种病原体,而且具有特异性强和灵敏度高的特点,适用于临床样品混合感染的快速鉴别诊断,对这6种发病特征及临床症状相似的鸡病毒性呼吸道病的有效防控有很高的实际应用价值。

A GeXP based multiplex PCR assay was developed to simultaneously detect six different chicken respiratory viruses including HS, HT, H9 subtypes of avian influenza virus（AIV）, new castle disease virus （NDV）, infectious bronchitis virus（IBV） and infectious laryngotracheitis virus（ILTV）. According to theconserved sequences of genes of each pathogen, se tion conditions w gle and mixed inf ere optimized. The specificity an ections of virus. The sensitivity dilutions of cloned plasmids. To further evaluate by GeXP. The corresponding specific fragments o yen pairs of specific primers were designed, and the r d accuracy of GeXP were examined using samples of was evaluated bY performing the assay on serial 10- the reliability, thirty-four clinical samples were dete f genes were amplified. The detection limit of GeXP eac- sin- fold cted was102 copies/μL when all of 7 pre-mixed plasmids containing target genes of six chicken respiratory viruses were present. In the detection of thirty-four clinical samples, the results of GeXP were accorded with the viral isolation completely. In conclusion, this GeXP assay is a rapid, specific, sensitive and high-through- put method for the detection of chicken respiratory virus infections. It can be applied in rapid differential diagnosis for clinical samples, and also provide an effective tool to prevent and control chicken respiratory diseases with similar clinical symptoms