九种鸡呼吸道病病原体GeXP检测方法的建立

Establishment of a GeXP assay for the differentiation of nine chicken respiratory pathogens

本研究旨在建立一种能同时检测(H5、H7、H9)亚型禽流感病毒、传染性支气管炎病毒、新城疫病毒、传染性喉气管炎病毒、鸡毒支原体、滑液囊支原体和副鸡嗜血杆菌共9种鸡呼吸道传染病病原体的GeXP高通量检测方法。根据GenBank中这9种病原体的基因保守序列,设计了10对特异性引物。用所建立的GeXP多重PCR方法对单一、混合病原体进行检测,验证多重反应体系的特异性和准确性,以含靶基因的质粒来检测多重反应体系的灵敏度,并对38份阳性样品进行检测。该法能特异性检出9种鸡呼吸道病病原体,并可在1×102 copies/μL水平同时特异地检测出9种病原体,38份阳性样品的检测结果与实际相符。本研究建立的同时鉴别9种鸡呼吸道病原体的GeXP检测方法具有高通量、特异性强、灵敏度高的特点,为同时鉴别这9种临床症状相似的疾病提供了新型检测方法。

A GeXP multiple PCR method was developed in this study for simultaneous detection of nine chicken respiratory disease pathogens in a single tube by multiplex PCR based on a GenomeLab Gene Expression（GeXP）,including Avian influenza virus of HS, H7 and H9 subtypes, Infectious bronchitis vi rus , Newcastle disease virus, Infectious laryngtracheitis virus, Mycoplasma galliselticum , Mycoplasma synoviae and Haemophillus paragallinarum. Ten pairs of specific primers were designed based on the conservative genes of the nine pathogens in GenBank. The specificity of the assay was verified by the detection of both single and mixed pathogen DNA（s） ,and the corresponding specific fragments of genes were ampli- fied. The sensitivity of the assay was evaluated by the detection of serially diluted plasmid standards, and the detection limit of GeXP was 100 copies/μL. Furthermore, the established GeXP multiple PCR method successfully detected thirty-eight positive clinical samples. In conclusion, this GeXP assay is a rapid, specific,sensitive and high-throughput detection method,which provides a new tool for the simultaneous differentiation of nine chicken respiratory diseases with similar clinical symptoms.