荧光定量RT-PCR检测鸡α干扰素mRNA方法的建立

Establishment of a real-time RT-PCR for detecting the transcriptional level of interferon alpha mRNA in chicken

根据鸡IFN-α基因序列设计引物,建立了定量检测鸡IFN-α基因表达水平的荧光定量RT-PCR（RRT-PCR）方法,并对新城疫病毒（NDV）感染的鸡法氏囊、胸腺中IFN-αmRNA表达水平进行了检测.结果表明,该方法对鸡IFN-αmRNA的扩增效率为103.99%,线性范围为10-2～10-9,相关系数为0.996,最低能检出73拷贝/反应;熔解曲线分析RRT-PCR产物在（88.6±0.2）℃出现单特异峰;特异性评价结果显示,本方法只扩增鸡IFN-αmRNA,不与常见禽源病原微生物发生交叉反应.对NDV感染鸡的法氏囊、胸腺中IFN-αmRNA表达水平的定量检测结果表明,IFN-αmRNA在感染后36,48 h显著升高.本研究建立的检测鸡IFN-αmRNA表达水平的RRT-PCR方法具有敏感性高、稳定性好和特异性强的特点,为鸡IFN-αmRNA的精确定量提供了新方法.

Interferon alpha （IFN-α） employed an important role of virus resistance and immunomodulatory in host. In the present study, a real-time reverse-transcription PCR （RRT-PCR） based on SYBR Greenlfor quantitative analysis of chicken IFN-α mRNA was developed and the expression levels of IFN-α mRNA in the bursals and thymus in SPF chicken infected with Newcastle disease virus （NDV） were investigated by using this method. The results showed ： the amplification efficiency of this assay was 103.99% , and the linear range was 10^-2 ～ 10^-9 with good correlation coefficient of 0. 996, as well as the detection limit reached 73 copies per reaction. The melting curve presented a single peak at （88.6 ± 0.2） ℃. The RRT-PCR only can amplify the specific fragments of the chicken IFN-α mRNA without cross-reactive with the common pathogenic microorganisms of chickens. The expression level of IFN-α mRNA in infected chicken bursa and thymus at 36 h and 48 h post-infection increased significantly. Our results showed that the RRT-PCR developed in this study was of good sensitivity, stability and specificity thus rendering it precise for quantitative detection of IFN-α mRNA.