绵羊SP-A基因实时荧光定量RT-PCR检测方法的建立及应用  

Development and application of a RT-qPCR for detection of ovine SP-A gene

作　　者：谢秀兰[1,2] 黎帅[3] 刘溪源 杨慧君[5] 李颖康[2] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [2]宁夏农林科学院动物科学研究所,宁夏银川750002 [3]宁夏大学新华学院,宁夏银川750021 [4]银川市畜牧技术推广服务中心,宁夏银川750001 [5]宁夏大学农学院,宁夏银川750021

出　　处：《中国兽医科学》2016年第6期781-785,共5页Chinese Veterinary Science

基　　金：中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室开放基金资助(SKLVEB2012KFKT004);宁夏自然科学基金项目(NZ13127)

摘　　要：肺表面活性蛋白A（surfactant protein A,SP-A）具有维持肺内稳态、防御病原感染以及协助免疫应答等重要作用。本试验建立了定量检测绵羊SP-A基因及内参基因B2M（beta 2 microglobulin）m RNA表达水平的SYBR Green I实时荧光定量RT-PCR方法,并采用该方法对健康滩羊、小尾寒羊和滩寒杂交羊三个绵羊品种肺脏中SP-A m RNA的表达情况进行了检测。结果显示,SP-A基因和B2M基因的扩增效率分别为94.1%和95.9%;线性范围分别在1×10-1～1×10-6和1×10-1～1×10-7;相关系数分别为0.998和0.999;熔解曲线分别在84.5℃和83.0℃出现一个特异峰;滩羊SP-A m RNA的表达水平略高于小尾寒羊和滩寒杂交羊。本试验建立的绵羊SP-A基因实时荧光定量RT-PCR方法特异性好、检测线性范围广,为绵羊SP-A m RNA表达水平的检测提供了重要方法。Surfactant protein A（SP-A） plays important roles in maintenance of lung homeostasis,host defense against infection and immune response.In present study,we developed a SYBR Green-based real-time RT-PCR for quantitative detection of m RNA expression of sheep SP-A gene and reference gene B2M（beta 2 microglobulin）.Subsequently,the method was used to determine the SP-A m RNA levels in lungs of three different sheep species,Tan sheep,Han sheep and Tan×Han hybrid.In result,the amplification efficiency of the real-time RT-PCR was 94.1 % for SP-A gene and 95.9 % for B2 M gene,respectively.The assay showed a wide linear range（from 1×10-1to 1×10-6for SP-A and from 1×10-1to 1×10-7for B2M） with good correlation coefficients（0.998 for SP-A and 0.999 for B2M）.Melting curve analysis ofthe real-time RT-PCR showed single peaks for the two genes with Tmvalues of 84.5 ℃ for SP-A and 83.0 ℃for B2 M,respectively.The expression of SP-A m RNA in lung of Tan sheep was slightly higher than that of Han sheep and Tan×Han hybrid.In conclusion,the developed real-time RT-PCR was specific with a wide linear range,and could be used as a useful tool for quantitative detection of SP-A m RNA in sheep.

关 键 词：荧光定量RT-PCR 绵羊 肺表面活性蛋白A MRNA 

分 类 号：S852.23[农业科学—基础兽医学]