鲤春病毒G蛋白基因实时荧光定量RT-PCR标准曲线的构建  

Construction of real-time RT-PCR standard curve for G protein gene of Spring viremia of carp virus

作　　者：夏明 王冬雪 李月红[1] 康元环[1] 贾俊鹏 王伟利[2] 钱爱东[1] 孟庆峰[1,2] 单晓枫[1] XIA Ming;WANG Dongxue;LI Yuehong;KANG Yuanhuan;JIA Junpeng;WANG Weili;QIAN Aidong;MENG Qingfeng;SHAN Xiaofeng(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Jilin Entry-Exit Inspection and Quarantine Bureau Inspection and Quarantine Technology Center,Changchun 130062,China)

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]吉林出入境检验检疫局检验检疫技术中心,长春130062

出　　处：《黑龙江畜牧兽医》2018年第19期108-110,114,240,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(30972191)

摘　　要：为了更加精确地对鲤春病毒（Spring viremia of carp virus,SVCV）进行定量检测,试验以Taq Man探针法为基础,提取鲤春病毒总RNA,反转录为cDNA后进行PCR扩增,得到目的条带并回收,将目的片段与pMD18-T载体连接导入大肠杆菌DH5α感受态细胞中,提取质粒并进行实时荧光定量RT-PCR测定,利用反应得到的Ct值与各浓度梯度质粒模板拷贝数的对数值绘制标准曲线。结果表明：以鲤春病毒cDNA为模板进行PCR扩增,获得清晰的目的条带,与预期相同;对鲤春病毒G蛋白重组质粒进行实时荧光定量RT-PCR扩增,所得扩增曲线有较好的重复性,各浓度梯度扩增曲线有明显的规律性,低浓度质粒模板扩增明显;获得的标准曲线有明显的线性关系,曲线回归系数为0.992。说明实时荧光定量RT-PCR方法敏感性较高、稳定性好、可靠精确,可以作为鲤春病毒检测的定量方法。The aim of the present study was to quantitatively detect the Spring viremia of carp virus（SVCV）. Based on the TaqMan probe method, the total RNA of SVCV was extracted, reverse transcription into cDNA and amplified by PCR. The target band was obtained and recovered, and the target fragment was inserted into the pMD18-T vector. The ligation products were transformed into competent cell E. coli DH5α, and the plasmids were extracted and detected by real-time RT-PCR. Then, the logarithmic values of Ct and the copy numbers of plasmid templates with different concentration gradients were used to draw the standard curve. The results showed that clear target bands were obtained by PCR using the cDNA of SVCV as template, which was the same as expected. The G protein recombinant plasmid of SVCV was amplified by real-time RT-PCR, and the amplification curve was reproducible. The amplification curves of each concentration gradient had obvious regularity, and the amplification of plasmid template at low concentration was obvious. The standard curve obtained had obvious linear relationship, and the regression coefficient of the curve was 0.992. The results indicated that the real-time fluorescence quantitative RT-PCR method had high sensitivity, good stability, reliability and accuracy, and could be used as a quantitative method for the detection of SVCV.

关 键 词：鲤春病毒 G蛋白基因 实时荧光定量RT-PCR 标准曲线 

分 类 号：S852.618[农业科学—基础兽医学]