人源细胞间黏附分子-1基因SYBR GreenⅠ实时荧光定量PCR检测方法的建立、验证及应用

Development,verification and application of a SYBR GreenⅠ real-time fluorescent quantitative PCR method for determination of human intercellular cell adhesion molecule-1 gene

目的建立并验证细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)基因的SYBR GreenⅠ实时荧光定量PCR检测方法,并用该方法检测不同细胞中ICAM-1的基因水平。方法根据GenBank中登录的ICAM-1基因序列设计并合成引物,建立ICAM-1基因的SYBR GreenⅠ实时荧光定量PCR法,验证方法的适用性、线性范围、灵敏性、特异性及精密性。同时采用该方法对人胚肺成纤维细胞(MRC-5)、人星形神经胶质瘤细胞(u251)、人原发性肝癌细胞(PLC/PRF/5)、人胚肾细胞(HEK293)及人脐静脉内皮细胞(HuvEc)进行检测。结果该方法的溶解曲线峰单一,无非特异性产物和引物二聚体;标准质粒在6.74×104~6.74×109 copies/μL浓度范围内与Ct值呈良好的线性关系,线性方程为y=-3.576 log x+42.982,R2=1.000;建立方法的灵敏性是普通PCR法的10倍;该方法检测不同物种细胞无交叉反应,仅能检出人源细胞ICAM-1;批内和批间CV均<1%。不同组织来源的细胞内ICAM-1基因含量不同,其中MRC-5细胞含量最高。结论成功建立了ICAM-1基因的SYBR GreenⅠ实时荧光定量PCR检测方法,且具有良好的特异性、灵敏性及精密性,可用于ICAM-1基因的定量检测。

Objective To develop and verify a SYBR GreenⅠreal-time fluorescent quantitative PCR method for determination of human intercellular cell adhesion molecule-1(ICAM-1)and apply to determination of ICAM-1 gene levels in various cells.Methods Primers were designed and synthesized according to the ICAM-1 gene sequence in GenBank,based on which a SYBR GreenⅠreal-time PCR method was developed and verified for suitability,linear range,sensitivity,specificity and precision.The ICAM-1 gene levels in human embryo lung fibroblasts(MRC-5),human astroglioma cells(u251),human primary liver cancer cells(PLC/PRF/5),human embryo lung cells(HEK293)and human umbilical vein endothelial cells(HuvEc)were determined.Results A single peak was shown on the melting curve of the developed method,and no non-specific product or primer dimers were observed.The standard plasmid at a concentration range of 6.74×104~6.74×109 copies/μL showed good linear relationship to the Ct value,while the linear equation was y=-3.576 log x+42.982,with a R2 value of 1.000.The sensitivity of the developed method was 10 times of that of common PCR.No cross reactions of various species of observed by the developed method,while only the ICAM-1 in cells from human origin was detected.Both the coefficient of variation(CVs)in intra-and inter-assays were less than 1%.The ICAM-1 gene levels in the cells from various tissues were different,which was the highest in MRC-5 cells.Conclusion The SYBR GreenⅠreal-time PCR method for determination of ICAM-1 gene was successfully developed,which showed high specificity,sensitivity and precision and might be used for the quantitative determination of ICAM-1 gene.