鸡宿主限制因子SAMHD1实时荧光定量PCR检测方法的建立

为建立一种快速检测chSAMHD1表达量的方法,本研究以SPF鸡外周血单个核细胞总cDNA为模板,构建了包含chSAMHD1全长CDS的重组质粒pMD-chSAMHD1。跨chSAMHD1内含子设计1对荧光定量PCR特异性引物,以构建的重组质粒作为标准品,建立chSAMHD1实时荧光定量PCR检测方法,并对其敏感性、特异性及重复性进行验证,将其应用于1日龄SPF鸡组织chSAMHD1表达量的检测。结果显示:用建立的实时荧光定量PCR方法对5×10^8~5×10^4拷贝/μL的标准质粒进行检测,所得标准曲线呈现良好的线性关系;最低检测限为50拷贝/μL,灵敏度是普通PCR的100倍;特异性良好,仅能检测到鸡源细胞中SAMHD1基因;重复性好,组内和组间变异系数均小于2%;应用该方法检测1日龄正常SPF鸡心脏、肝脏、脾脏、肺脏、肾脏、脑、神经、胸腺、法氏囊、十二指肠、腿肌、腺胃等组织的chSAMHD1基因拷贝数,结果显示chSAMHD1具有广泛的组织分布性。本研究为chSAMHD1功能研究提供了一种准确、有效的检测手段。

Establishment of Real-time Fluorescent Quantitative PCR for Detection of Chicken Host Restriction Factor SAMHD1

In this study,in order to establish a method for the rapid detection of expression of chSAMHD1 gene,the recombinant plasmid pMD-chSAMHD1 containing full-length CDS of chSAMHD1 was constructed with the total cDNA of peripheral blood mononuclear cells of SPF chicken as the template.One pair of specific primers across the chSAMHD1 introns was designed,and the constructed recombinant plasmid was used as the standard substance to establish a real-time fluorescent quantitative PCR method for the detection of chSAMHD1 gene.The sensitivity,specificity and repeatability of the real-time PCR method were verified,and it was applied to the detection of chSAMHD1 expression in 1-day-old SPF chicken tissues.The results showed that the standard curve showed a good linear relationship when the established real-time PCR was used to detect 5×10^8 copies/μL to 5×10^4 copies/μL standard plasmid.The detection limit of this method was 50 copies/μL,which was 100 times more sensitive than the traditional PCR.And there was no cross reaction with the SAMHD1 genes of other species.With good repeatability,the intra-group and inter-group coefficients of variation were both less than 2%.This method was used to detect the copies of chSAMHD1 gene in the heart,liver,spleen,lung,kidney,brain,nerve,thymus,bursa of Fabricius,duodenum,leg muscles and glandular stomach of 1-day-old SPF chickens.The results showed that the chSAMHD1 had a wide tissue distribution.This study provides an accurate and effective detection method for the functional study of chSAMHD1.