猪谷氧还蛋白I(GLRX1)基因实时荧光定量PCR检测方法的建立

DESIGN AND VALIDATION OF REAL TIME FLUORESENCE QUANTITATIVE RT-PCR ASSAY FOR DETECTION OF PORCINE GLUTAREDOXIN

本研究针对猪GLRX1设计特异性引物,将PCR扩增片段分别连接至T-easy载体上构建重组质粒,经筛选、鉴定纯化后,10倍梯度稀释作为质控样品,用于实时荧光定量PCR中GLRX1标准曲线的构建,并进行反应的灵敏性、特异性和重复性试验。结果显示标准曲线线性关系R2值均在0.99以上;特异性结果表明只能检测到猪GLRX1扩增曲线;组内和组间变异系数均小于5%。本研究初步建立了检测猪GLRX1基因的SYBR Green荧光定量RT-PCR的方法,为后续对猪传染性疾病与猪GLRX1之间相互关系的研究提供了一种特异、灵敏的检测方法。

The objective of the study is to establish a method for detecting porcine GLRX1 by SYBR Green I relative fluorescence quantitative RT-PCR, Special primers based on porcine GLRX1 were designed. Then these amplified fragments were cloned into T-easy. Using the recombinant plasmid as standard products, a real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR） was performed to construct the standard curves of porcine GLRX1 and detect the sensitivity, specificity and repeatability. The results showed a precise linear relationship with a correlation coefficient ofR2 〉 0.99. The amplification curve showing a single peak could only been detected for porcine GLRX1. The variation coefficient was less than 0.5% by within and between the groups of repeatability tests. The developed real-time PCR assay was highly specific, sensitive and reproducible, and could be an available tool for monitoring the relationship between pig infectious diseases and porcine GLRX1.