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当前位置: 首页 > 文章 > 鸡γ-干扰素实时荧光定量RT-PCR检测方法的建立 中国兽医科学 2007,37 (7) 631-635
Position: Home > Articles > Establishment of a real-time fluorescent quantitative RT-PCR assay for the detection of chicken IFN-γ gene Chinese Veterinary Science 2007,37 (7) 631-635

鸡γ-干扰素实时荧光定量RT-PCR检测方法的建立

作  者：
王群;刘光亮;童铁钢;肖一红;吴东来
单  位：
中国农业科学院哈尔滨兽医研究所
关键词：
鸡γ-干扰素;实时荧光定量RT-PCR;检测方法
摘  要：
为建立一种检测鸡γ-干扰素的实时荧光定量RT-PCR方法,采用RT-PCR方法从ConA诱导活化的鸡脾淋巴细胞的总RNA中扩增得到鸡γ-干扰素(ChIFN-γ)和鸡的28 SrRNA(Ch28 S)基因,将其分别克隆至体外转录载体后经体外转录获得了ChIFN-γ和Ch28 S的RNA,采用各自的特异性引物及Taqman探针,以Ch28 S RNA作为内参进行一步法实时荧光定量RT-PCR,检测ChIFN-γ。结果表明:ChIFN-γ和内参Ch28 S的Ct值与标准品稀释梯度在1×102~1×107拷贝/μL范围分别呈良好的线性关系,r2均大于0.99。此方法用于检测ChIFN-γ,具有简便、高效、敏感、特异的特点。
译  名：
Establishment of a real-time fluorescent quantitative RT-PCR assay for the detection of chicken IFN-γ gene
作  者：
WANG Qun1,2,LIU Guang-liang1,2,TONG Tie-gang1,2,XIAO Yi-hong1,2,WU Dong-lai1,2(1.Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150001,China;2.Graduate School,Chinese Academy of Agricultural Sciences,Beijing 100081,China)
关键词：
ChIFN-γ;real-time fluorescent quantitative RT-PCR;detection method
摘  要：
To develop a real-time fluorescent quantitative RT-PCR assay for the detection of chicken IFN-γ,chicken interferon gamma(ChIFN-γ) and chicken 28 S rRNA(Ch28 S) genes were amplified from the total RNA of ConA-activated spleen lymphocytes by RT-PCR with specific primers respectively and then cloned into a pET-28a(+) vector.The RNAs of ChIFN-γ and Ch28 S genes were obtained from in vitro transcription systems using the recombinant plasmids as templates.The ChIFN-γ in the diluted by 10-fold standard samples was detected by the one-step real-time fluorescent quantitative RT-PCR with their specific primers and Taqman probe while the Ch28 S rRNA served as an internal control.The results showed that the Ct of ChIFN-γ or Ch28 S had a good linear relationship(r2>0.99) with the standard samples from 1×102 to 1×107copies/μL.It was concluded that a convenient,high performance and sensitive real-time fluorescent quantitative RT-PCR was established for the detection of ChIFN-γ gene.

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