Ⅰ亚群禽腺病毒通用PCR检测方法的建立及应用

摘要

为建立快速、特异、敏感的Ⅰ亚群禽腺病毒(FADV-Ⅰ)临床通用PCR检测方法,本研究根据GenBank登录的FADV-Ⅰ不同血清型基因组序列,比对分析后选择病毒DNA聚合酶基因设计通用检测引物,优化PCR体系各反应条件,确定该方法的敏感性与特异性,建立了检测FADV-Ⅰ通用PCR方法,并将其进行了临床应用与感染SPF鸡脏器组织病毒分布检测比较.结果显示,经优化确定引物浓度为1.0 μM,退火温度为50.4℃～61.0℃,该方法能够特异性扩增该亚群病毒494 bp的DNA聚合酶基因,对产蛋下降综合征病毒、马立克病毒、鸡痘病毒等扩增结果均为阴性,特异性强;该方法的检测限为2.8×102拷贝/μL病毒PCR产物标准品;对临床病例经PCR检测、病毒血清型鉴定与分离结果显示,该方法与病毒分离方法符合率达100％,且检测的病毒被鉴定为4、8a、8b和11血清型FADV;对病毒感染鸡脏器组织检测结果显示,该方法对其心脏、肝脏、脾脏、肺脏、肾脏、腺胃与肌肉样品均可以检测到该病毒,较文献报道方法更敏感.以上结果表明本研究建立的PCR检测方法具有通用、特异、敏感、快速、准确等特点,能够用于FADV-Ⅰ的临床检测,为防控该亚群FADV引发的疫病提供了技术保障.%To establish a rapid,specific and sensitive PCR detecting method for subgroup Ⅰ fowl adenovirus (FAdV-Ⅰ) diagnosis,a pair of primers were designed based on DNA polymerase gene of FAdV according to viral genomes submitted to GenBank,and the PCR method was constructed through the optimization of amplification conditions.The results showed that the target fragment of 494 bp was amplified with the primers.The best concentration of primers was 1.0 μM,and there were no amplification differences from 50.4 ℃ to 61.0 ℃ range of annealing temperatures.The detection limit of the viral template was 2.8 × 102 copies/μL,and the detection results of egg drop syndrome virus (EDSV),Marek's disease virus (MDV),avian poxvirus (APV) were all negative except for FAdV.The clinical diagnosis,identification of virus serotypes and virus isolation results showed that all the FAdV infected samples could be detected to be positive by the PCR method,furthermore,the viruses could be isolated from all PCR positive samples,and the infected viruses were identified as serotype 4,8a,8b and 11.The tissue virus detection of the FAdV infected SPF chicken results showed that all samples including the heart,liver,spleen,lung,kidney,glandular stomach and muscle could be detected the FAdV by this PCR method indicating the method was more sentitive than the other two reported methords.The PCR method developed in this study for detecting FAdV is a universal,specific,sensitive,rapid and accuracy diagnostic method,which provides technique support for clinical diagnosis,disease prevention and control.