奶牛GPR109A基因SYBR GreenI实时荧光定量PCR检测方法的建立

【目的】建立奶牛G蛋白偶联受体109A基因（GPR109A）的实时荧光定量PCR检测方法,为开展奶牛产后脂类代谢紊乱及酮病发生机理研究奠定基础。【方法】根据Gen Bank中奶牛GPR109A基因和β-actin基因（内参基因）的m RNA保守序列设计合成两对引物,以奶牛肝脏组织c DNA为模板,PCR扩增目的基因后与载体连接构建重组质粒,以SYBR Green I实时荧光定量PCR进行扩增,绘制标准曲线,并对其特异性、敏感性、重复性等进行检验。【结果】GPR109A基因和β-actin基因的实时荧光定量PCR标准曲线方程分别为y=-3.309x＋10.92（R2=0.9985）和y=-3.2...

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Published in南方农业学报 Vol. 46; no. 3; pp. 512 - 517
Main Author 杜向宏 尹南男 左宗辉 杜玉兰 杨蓉 赵熠珩 陈亚明 何宝祥
Format Journal Article
LanguageChinese
Published 广西大学动物科学技术学院,南宁,530005 2015
Subjects
GPR109A基因
β-actin基因
奶牛
实时荧光定量PCR
敏感性
特异性
重复性
β-actin基因
dairy cow
实时荧光定量PCR
特异性
GPR109A基因
重复性
β-actin gene
real-time fluorescence PCR
repetition
奶牛
GPR109A gene
specificity
sensitivity
敏感性
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ISSN2095-1191
DOI10.3969/j:issn.2095-1191.2015.3.512

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Summary:【目的】建立奶牛G蛋白偶联受体109A基因（GPR109A）的实时荧光定量PCR检测方法,为开展奶牛产后脂类代谢紊乱及酮病发生机理研究奠定基础。【方法】根据Gen Bank中奶牛GPR109A基因和β-actin基因（内参基因）的m RNA保守序列设计合成两对引物,以奶牛肝脏组织c DNA为模板,PCR扩增目的基因后与载体连接构建重组质粒,以SYBR Green I实时荧光定量PCR进行扩增,绘制标准曲线,并对其特异性、敏感性、重复性等进行检验。【结果】GPR109A基因和β-actin基因的实时荧光定量PCR标准曲线方程分别为y=-3.309x＋10.92（R2=0.9985）和y=-3.289x＋9.794（R2=0.9997）,扩增效率分别为101.0%和100.0%。特异性试验结果显示,GPR109A基因和β-actin基因的溶解曲线峰单一,无引物二聚体和非特异性扩增产物生成;敏感性试验结果显示,线性扩增范围广,可检测到1.8×102个拷贝的GPR109A基因;重复性试验结果显示,各扩增反应的变异系数（CV）小于或等于1.7%。【结论】基于GPR109A基因建立的SYBR GreenⅠ实时荧光定量PCR具有操作简便、敏感性高、特异性强和重复性好等特点,可用于牛源性GPR109A基因表达量的快速检测和定量分析。
Bibliography:45-1381/S
dairy cow; GPR109A gene; β-actin gene; specificity; sensitivity; repetition; real-time fluorescence PCR
DU Xiang-hong,YIN Nan-nan,ZUOZong-hui,DU Yu-lan,YANGRong,ZHAOYi-heng,CHEN Ya-ming,HE Bao-xiang （College of Animal Science and Technology, Guangxi University, Nanning 530005, China）
[Objective]A real-time fluorescence PCR method for detection of G-protein-coupled receptor 109A（GPR109A） gene was established to provide foundation for further research on disorder of lipid metabolism and pathogenesis of ketosis in postpartum dairy cow. 【Method 】Two pairs of primers were designed according to the conservative sequence of bovine GPR109 A and β-actin（internal reference） gene published by Gen Bank. With c DNA from liver tissues of cow as the template, target genes were amplified by PCR and inserted into vector to establish the recombination plasmid. The recombination plasmids were used for amplifying by real-time PCR. Standard curve was made and the specificity, sensitivity and repeatability were tested. 【Res
ISSN:2095-1191
DOI:10.3969/j:issn.2095-1191.2015.3.512