摘要
为采用SYBR GreenⅠ染料建立检测水貂IFN-α、IFN-β和IFN-γmRNA荧光定量RT-PCR检测方法,根据GenBank中登录的MiIFN-α和MiIFN-β、MiIFN-γ和3-磷酸甘油脱氢酶(GAPDH)基因序列,分别设计合成特异性引物,通过RT-PCR扩增MiIFN-α、MiIFN-β、MiIFN-γ和MiGAPDH的基因片段,构建到pMD18-T载体中制备质粒标准品,建立了相应基因mRNA的荧光定量RT-PCR检测方法并建立标准曲线。结果表明,MiGAPDH、MiIFN-α和MiIFN-β和MiIFN-γ基因的Ct值与标准品稀释度在10拷贝质粒/μL^107拷贝质粒/μL内均呈良好的线性关系,相关系数(R2)均为1.00,熔解曲线均呈单一熔解峰,检测下限为10拷贝质粒/μL,重复性试验表明组内、组间变异系数均小于4.5%。临床样品检测结果表明水貂感染肠炎病毒后,MiIFN-α、MiIFN-β和MiIFNγ相对表达水平均在6d达到最高峰值,MiIFN-α相对表达量要比MiIFN-β和MiIFN-γ相对表达量高两个数量级,并且在感染期(4d^6d)MiIFN-α相对表达量明显提高。本研究为水貂IFN mRNA的定量分析提供了有效工具。
In this study,the methods of SYBR GreenⅠreal-time quantitative RT-PCR assay for detection of mink IFN-α,IFN-βand IFN-γ（MiIFN-α,MiIFN-βand MiIFN-γ）mRNA were established using four pairs of specific primers designed according to the MiIFN-α,MiIFN-βand MiIFN-γgene sequences,with the mink glyceraldehyde-3-phosphate dehydrogenase（MiGAPDH）mRNA as an internal control.The target genes were amplified by RTPCR and inserted to pMD18-T vector as the standard recombinant plasmids for the establishing the real-time PCR standard curves.The results showed that the Ct values of MiIFN-α,MiIFN-β,MiIFN-γand MiGAPDH mRNA had good linear relationships（R2=1.00）with the standards from 10copies/μL to 107 copies/μL,and the melting curve showed a single peak.And the sensitivity assay results showed that the genetic test minimum value ware 10copies/μL.The coefficient variations of inter-assay and intra-assay were both less than 4.5%.The result of clinical test showed that the relative expressions of MiIFN-α,MiIFN-βand MiIFN-γreached the highest level.The expressions of MiIFN-αwas 100 times higher than that of MiIFN-βand MiIFN-γand increased significantly during the infection period.The detection methods provide an effective tool for quantitative analysis of mink IFN-α,IFN-βand IFN-γmRNA.
出处
《动物医学进展》
北大核心
2015年第11期1-6,共6页
Progress In Veterinary Medicine
基金
吉林省科技发展计划项目(20130206026NY
20140520172JH)
吉林市杰出青年项目(2013625018)
吉林市科技发展计划(2013222014)
