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当前位置: 首页 > 文章 > 猪IL-1β与IL-18 SYBR Green Ⅰ荧光定量PCR检测方法的建立 江苏农业学报 2011,27 (6) 289-294
Position: Home > Articles > Development of SYBR GreenⅠ-based real-time PCR assays for detection of porcine IL-1β and IL-18 Jiangsu Journal of Agricultural Sciences 2011,27 (6) 289-294

猪IL-1β与IL-18 SYBR Green Ⅰ荧光定量PCR检测方法的建立

作  者：
汪伟;倪艳秀;温立斌;吕立新;周俊明;俞正玉;茅爱华;张雪寒;李彬;郭容利;王小敏;范红结;何孔旺
单  位：
江苏省农业科学院兽医研究所;南京农业大学动物医学院
关键词：
猪;促炎症细胞因子;IL-1β;IL-18;荧光定量PCR
摘  要：
IL-1β和IL-18是重要的促炎症细胞因子,在介导机体炎症反应中起重要作用。为了建立在体外定量检测猪细胞因子IL-1β和IL-18 mRNA表达水平的SYBR GreenⅠ荧光定量PCR方法,依据GenBank中登录的猪细胞因子基因IL-1β、IL-18及管家基因β-actin核苷酸序列,设计特异性引物,经RT-PCR从3D4猪肺泡巨噬细胞系中克隆和扩增了上述3个因子核苷酸片段,构建含有各自引物扩增序列的重组质粒作为阳性模板,建立了检测IL-1β、IL-18及β-actin SYBR GREEⅠ荧光定量PCR方法。该方法起始模板数与Ct值之间线性关系好,相关系数达到0.990以上;特异性强,扩增产物形成单一的特异性熔解峰;敏感性高,初始模板的检出下限达到了1μl 1.0×102拷贝;重复性好,组内变异系数均小于4%。
译  名：
Development of SYBR GreenⅠ-based real-time PCR assays for detection of porcine IL-1β and IL-18
作  者：
WANG Wei1,2,NI Yan-xiu1,WEN Li-bin1,L Li-xin1,ZHOU Jun-ming1,YU Zheng-yu1,MAO Ai-hua1,ZHANG Xue-han1,LI Bin1,GUO Rong-li1,WANG Xiao-min1,FAN Hong-jie2,HE Kong-wang1(1.Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Key Laboratory of Veterinary Biologicals Engineering and Technology,Ministry of Agriculture,National Center for Engineering Research of Veterinary Bio-products,Nanjing 210014,China;2.College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
关键词：
porcine;pro-inflammatory cytokine;IL-1β;porcine IL-18;real-time PCR
摘  要：
The specific primers were designed and synthesized according to the nucleotide sequence of the porcine pro-inflammatory cytokines,i.e.IL-1β,IL-18 genes as well as housekeeping gene β-actin available in GenBank.IL-1β and IL-18 play important roles in mediating inflammatory response.The three fragments were amplified by RT-PCR from 3D4 porcine alveolar macrophages,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 Green Ⅰ for detection of IL-1β,IL-18 and β-actin were established.The results showed a good linear relationship between template copy number and circulation number,and the correlation coefficients(R2) of the standard curves for IL-1β,IL-18 and β-actin were over 0.990.Also,these assays were highly specific and there was single specific melting peak for every cytokine.Moreover,the assays were highly sensitive and had a detection limit of 1.0×102copies in 1 μl of initial templates.Finally,it was highly repeatable and had a coefficient of variation less than 4 percent for intra-assay.

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