鸡BF1和BF2基因实时荧光定量PCR检测方法的建立

Development of a real-time fluorescent quantitative PCR assay for BF1 and BF2 genes in chickens

目的建立鸡主要组织相容性复合体(Major histocompatibility complex,MHC)BF1和BF2基因实时荧光定量PCR[Real-time fluorescent quantitative(FQ)-PCR]检测方法。方法从鸡外周血淋巴白细胞(Peripheral blood leuk-ocytes,PBLs)中提取细胞总RNA,以其为模板,根据BF1、BF2和GAPDH基因相对保守序列各设计一对引物,应用RT-PCR法扩增目的基因,分别克隆至pGM-T载体上,制备重组质粒标准品,经PCR、EcoRⅠ酶切及测序鉴定;优化反应体系及反应条件,绘制标准曲线,建立Real-time FQ-PCR检测方法,并验证其敏感性、特异性及重复性。结果重组质粒标准品经PCR、酶切及测序鉴定构建正确;建立的定量标准曲线临界循环数(Threshold cycle,Ct)与BF1、BF2和GAPDH起始质粒DNA拷贝数的对数之间线性关系良好,相关系数分别为1.00、0.98、0.99,扩增效率分别为0.8、1.1、1.1;该方法的敏感性可达101copies/μl;融解曲线分析表明扩增效率一致,特异性较好;质粒DNA标准品3次检测的CV值误差不超过0.5,均小于5%。结论已成功建立了鸡BF1和BF2基因实时荧光定量PCR检测方法,为下一步应用该法检测BF1和BF2基因的转录水平奠定了基础。

Objective To develop a real-time fluorescent quantitative PCR assay for of BF1 and BF2 genes of major histocompatibility complex （MHC） in chickens. Methods Total RNA was extracted from chicken peripheral blood leukoeytes （PBLs） and used as a template for amplification of BF1, BF2 and GAPDH genes by RT-PCR using the designed primers. The amplified genes were cloned into vector pGM-T respectively, and the constructed standard recombinant plasmids were identified by PCR, digestion with EeoR I and sequencing. The reaction system and condition were optimized, based on which a standard curve was plotted. The developed real-time FQ-PCR method was verified for sensitivity, specificity and reproducibility. Results PCR, restriction analysis and sequencing proved that the standard recombinant plasmids were constructed correctly. The threshold cycle（Ct） of standard curve showed good linear relationship to the log of original copy numbers of plasmid DNAs of BFI, BF2 and GAPDH genes, with coefficients of correlation of 1.00, 0. 98 and 0. 99, and amplification efficiencies of 0. 8, 1. 1 and 1. 1, respectively. The developed method showed a sensitivity of 101 copies / μl. Melting curve showed consistent amplification efficiencies of the genes, indicating high specificity of the method. The CV values of 3 test results of standard plasmid DNA were not more than 0. 5. Conclusion A real-time fluorescent quantitative PCR assay for of BF1 and BF2 genes in chickens was successfully developed, which laid a foundation of further application of the method to determination of transcription levels of BF1 and BF2 genes.