Ⅰ群禽腺病毒SYBR GreenⅠ荧光PCR检测方法的建立

Development of a SYBR GreenⅠ real-time PCR assay for the detection of aviadenovirus groupⅠ

根据GenBank中Ⅰ群禽腺病毒六邻体基因的保守序列设计了1对引物，建立了检测Ⅰ群禽腺病毒12个血清型的SYBR GreenⅠ荧光PCR方法。用Ⅰ群禽腺病毒阳性样品进行了特异性、敏感性和重复性试验。结果显示。该方法可检测到每个反应相当于1×10^2个拷贝的标准品DNA，与鸡减蛋综合征病毒、禽呼肠孤病毒、传染性腔上囊炎病毒和传染性支气管炎病毒等非Ⅰ群禽腺病毒不发生交叉反应，具有良好的重复性。对保存的3份Ⅰ群禽腺病毒的细胞培养物进行荧光定量PCR检测，结果都为阳性，检测的病毒含量为1×10^6～1×10^8拷贝／μL。表明．建立的实时荧光定量PCR具有特异、敏感、快速、定量、重复性好等优点，可用于临床上Ⅰ群禽腺病毒感染的检测。

A pair of primers were designed and synthesized according to the conserved hexon gene sequences of aviadenovirus group Ⅰ available in GenBank,and then reaction parameters were optimized to develop a SYBR Green Ⅰ real time PCR. Sensitivity analysis showed that the developed SYBR Green Ⅰ real time PCR could detect 1 × 10^2 template copies of plasmid DNA in one reaction. The assay exhibited specificity as all negative controls and other avian pathogens,such as egg drop syndrome virus,avian reovirus,infectious bursal disease virus and infectious bronchitis virus showed negative results. The real-time PCR results of 3 cell culture samples showed that the concentrations of the samples DNA were 1×10^6 to 1 × 10^8 copies/μL. As a result of the sensitivity and specificity of the assay with a rapid and simple procedure,the real time PCR will be useful for the clinical diagnosis of aviadenovirus group Ⅰ infection.