关键词: 猪, 脂蛋白酯酶, 实时定量RT-PCR, TaqMan荧光探针

Abstract: bstract: Objective : Cloning porcine LPL cDNA , using it as the standard for real－time quantifying LPL mRNA and establishing TaqMan FQ-RT-PCR assay for detection of it. Methods : Total RNA extracted from longissimus dorsi of porcine was reverse transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed to 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 was diluted to series standard concentrations of LPL αre combined plasmid for FQ-PCR. Results : 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-time TaqMan-Fluorescence Quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in longissimus dorsi of porcine.

Key words: porcine, Lipoprotein Lipase, FQ-PCR, TaqMan Fluorogenic Probe