taqman荧光定量rt-pcr检测猪脂蛋白酯酶mrna方法的建立.doc

TaqMan荧光定量RT-PCR检测猪脂蛋白酯酶mRNA方法的建立 廉红霞1,3，卢德勋2，高 民2 （1内蒙古农业大学动物科学与医学学院，呼和浩特 010018；2内蒙古农牧业科学院，呼和浩特 010030；3河南农业大学牧医工程学院，郑州 450002） 摘要：【目的】克隆猪cDNA，作为猪LPL mRNA定量检测的标准品，建立检测方法。【方法】用RT-PCR，从猪背最长肌的总RNA中逆转录扩增LPL的cDNA，将纯化的LPL cDNA与pGM-T载体进行连接,转化宿主菌TOP10，提取重组质粒DNA，PCR鉴定并测序分析,对质粒标准进行实时荧光定量PCR 检测。纯化质粒并检测260 nm吸光度,确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品。【结果】建立了猪LPL mRNA实时定量PCR检测方法，特异性好，检测灵敏度达103拷贝，线性范围为1×103 ～1×1010拷贝，阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（R2＝0.9871）。【结论】成功克隆了LPL实时荧光PCR定量标准，且TaqMan荧光定量RT-PCR的方法可对猪背最长肌LPL mRNA的表达进行准确定量。 关键词：猪；脂蛋白酯酶；实时定量RT-PCR；TaqMan荧光探针 Establishment of Real-time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA LIAN Hong-xia1,3, LU De-xun2, GAO Min2 (1College of Animal Science and Animal Medicine, Inner Mongolia Agricultural University, Huhhot 010018; 2Inner Mongolian Academy of Agricultural and Animal Science, Huhhot 010030; 3College of Animal Science, Henan Agricultural University, Zhengzhou 450002) Abstract: 【Objective】Porcine LPL cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan fluorescence quantitative PCR assay for detection was established. 【Method】 Total RNA extracted from longissimus dorsi of porcine was reverse transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into bacterium TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analysing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for FQ-PCR. 【Result】 The method of LPL mRNA real-time PCR was well established, which detected as low as 103 copies with the linear range from 103 to 1010 copies. The standard curves showed high correlations (R2=0.9871). 【Conclusion】 A series of standards for real-time PCR analysis have been constructed successfully, and real-t