鸽白细胞介素8基因实时荧光定量PCR检测方法的建立

Development of a Quantitative Real-time PCR Method for Detection of Pigeon IL-8 Gene

为建立鸽白细胞介素8(interleukin-8,IL-8)基因实时荧光定量PCR检测方法,本研究根据GenBank上公布的鸽IL-8基因序列,在保守区域设计1对特异性引物,以鸽子淋巴细胞提取的核酸为模板扩增鸽IL-8基因部分片段并克隆到pMD18-T载体上。提取重组质粒通过系列制备标准品,建立鸽IL-8基因SYBR GreenⅠ染料法实时荧光定量PCR标准曲线,并进行特异性、敏感性和重复性试验。结果显示,实时荧光定量PCR熔解曲线呈单一熔解峰,试验线性相关系数为1.0,IL-8基因的扩增效率为103%。敏感性结果显示最低可检测到16个拷贝数;用该方法检测其他鸽白细胞介素细胞因子(IL-1β、IL-6、IL-18)和双蒸水的结果均为阴性。批间、批内变异系数均≤1.52%。因此,本研究建立的实时荧光定量PCR检测方法可用于鸽IL-8mRNA的检测,为病毒感染宿主细胞后细胞因子表达的定量分析奠定基础。

To develop aquantitative Real-time PCR method for detection of pigeon interleukin-8（IL-8）,apair of specific primers was designed based on the conserved region of IL-8gene sequence published on GenBank.A fragment of IL-8gene was amplified from the pigeon lymphocytes template and cloned into pMD18-T vector.Plasmid DNA was extracted from the bacteria and was serially diluted to serve as a standard.A standard curve for the SYBR GreenⅠof quantitative Real-time PCR was established and the specificity,sensitivity and reproducibility of this assay were investigated.The results showed that the quantitative Real-time PCR melting curve only had one single melting peak.The assay was linear with R2 values was 1.0;The reaction efficiency for the pigeon IL-8gene was 103%.The detection limit of this assay was 16 copies per reaction.Other interleukin including IL-1β,IL-6and IL-18 and double distilled water control were tested by this assay and the results were all negative.The CV values of intra-and inter-assay were less than1.52%.The established quantitative Real-time PCR assay of this study was suitable for the detection of pigeon IL-8.It would provide basis for analyzing cytokine expression quantitatively after the host cell was infected by the virus.