摘要
本研究旨在建立一种共刺激分子CD80、CD86 SYBR GreenⅠ荧光定量PCR检测方法。根据GenBank中牛CD80、CD86的基因序列设计特异性引物,采用RT-PCR方法扩增牛CD80、CD86目的片段,将其克隆到pMD18-T载体上,提取阳性质粒进行测序鉴定,用得到的阳性重组质粒作为标准品,优化SYBR GreenⅠ荧光定量PCR反应条件,建立其标准曲线,对建立的方法进行特异性、重复性、敏感性试验。结果显示,所建立的方法特异性、稳定性良好,较敏感,可用于牛共刺激分子CD80、CD86的快速检测。
The purpose of this study was to establish a SYBR GreenⅠfluorescence quantitative PCR method for the detection of bovine costimulatory molecule CD80,CD86.The gene sequences of CD80,CD86 were found in GenBank,and specific primers were designed according to their respective gene sequences.The target fragment was amplified by PCR and cloned into pMD18-T vector.The positive plasmid was extracted and sequenced.The standard product was prepared by using the positive recombinant plasmid and the standard curve of SYBR GreenⅠfluorescence quantitative PCR was established.The established method was specific and repeated.The reliability of the fluorescence quantitative PCR method was analyzed and judged by specificity,repeatability and sensitivity test.The results showed that the established method was specific,stable and sensitive,it could be used for the rapid detection of bovine costimulatory molecule CD80,CD86.
作者
唐颖
邓宇
TANG Ying;DENG Yu(College of Animal Science,Xichang College,Xichang 615000)
出处
《中国奶牛》
2020年第6期1-5,共5页
China Dairy Cattle
