摘要
本研究根据GenBank上公布的鸡白细胞介素1β(IL-1β)、白细胞介素18(IL-18)、肿瘤坏死因子α(TNF-α)和3-磷酸甘油醛脱氢酶(GAPDH)基因序列,在保守区域各设计合成1对特异性引物,以鸡胚成纤维细胞的cDNA为模板构建重组阳性质粒。采用梯度稀释重组标准品质粒DNA,构建SYBR GreenⅠ染料法荧光定量PCR标准曲线。结果显示,各基因的熔解曲线均呈单一熔解峰,IL-1β、IL-18、TNF-α和GAPDH的扩增效率分别是101.2%、95.6%、100.1%和98.2%,相关系数分别为0.9996、0.9998、0.9957和0.9989。敏感性试验结果表明,该检测方法在35个Ct值内可检测到100个模板拷贝数;重复性试验结果表明,组内变异系数均小于1.4%。本研究建立的实时荧光定量PCR检测方法可用于鸡IL-1β、IL-18和TNF-αmRNA的检测,为导致免疫抑制性疾病病毒感染宿主细胞后细胞因子表达的定量分析奠定基础。
In order to develop a SYBR GreenⅠ Real-time PCR assay for detection of chicken IL-1β,IL-18 and tumor necrosis factor-α （TNF-α） genes, four specific primer pairs were designed according to the chicken＇s IL-1β,IL-18, TNF-α and glyceraldehyde-3-phosphate dehydrogenase （GAPDH） gene sequences in GenBank. The four fragments were amplified by RT-PCR from chicken embryo fibroblasts, cloned and sequenced. The recombinant plasmids containing the target gene were constructed and used as the Real-time PCR standard templates. Real-time PCR assays based on SYBR GreenⅠfor detection of chicken IL-1β,IL-18, TNF-α and GAPDH were established. The results showed that each gene＇s melting curve also had a single peak,each gene＇s amplification efficiency was 101.2%, 95.6%, 100.1% and 98.2%, R2 was 0.9996,0.9998,0.9957 and 0.9989. Moreover,the assays were highly sensitive,the detection limit of 100 copies in 35 Ct and each gene＇s coefficient of variation less than 1.4 percent for intra-assay. This reliable Real-time PCR assay might be used for decting chicken＇s IL-1β,IL-18 and TNF-α mRNA expressing and provided the basis for quantitative analysis of cytokine expression in host cell after virus infect which cause immunosuppressive diseases.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第9期90-95,共6页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31160512)
广西科技项目(桂科重1222003-2-4
桂科攻10100014-5)
广西特聘专家专项(2011B020)
