高分辨率溶解曲线分析结合COLD-PCR快速检测绵羊低丰度FecB基因

发布时间：2018-01-24 23:04

  本文关键词： 绵羊 FecB基因 COLD-PCR 高分辨率熔解曲线分析　出处：《中国畜牧杂志》2017年06期 　论文类型：期刊论文

【摘要】：本文利用高分辨率熔解曲线分析结合COLD-PCR建立绵羊骨形态发生蛋白受体IB(BMPR-IB)基因低丰度A746G点突变的检测方法,为Fec B基因大面积高通量筛查提供方便快捷和低成本的检测技术。实验分别提取绵羊BMPR-IB基因A746G点突变阳性纯合子和阴性纯合子的基因组,分别PCR扩增223 bp的目的片段,测定浓度和纯度后,分别稀释至0.05 ng/μL,将阳性纯合子用阴性纯合子进行系列稀释作为模板标准品。根据模板标准品序列设计合成3对引物,以标准品为模板进行COLD-PCR,其产物进行高分辨率熔解曲线分析,生成的归一化温度转移差异图(Normlized and Temp-Shifted Differines Plot)即为标准曲线图,作为待测样品Fec B检测的依据,对其可靠重复性做了进一步验证。结果表明:2对引物COLD-PCR HRM标准曲线图曲线分离清晰可靠,检测下限均为0.5%,与传统q PCR HRM相比,灵敏度提高到4倍。本实验建立的绵羊低丰度Fec B基因检测方法可以用于绵羊Fec B基因大面积高通量快速筛查工作。
[Abstract]:A method for detection of low abundance A746G point mutation of bone morphogenetic protein receptor (BMP) receptor IBP BMPR-IBB in sheep was established by high resolution fusion curve analysis and COLD-PCR. Fec. Large area high-throughput screening of B gene provides a convenient and low-cost detection technique. The genome of positive homozygote and negative homozygote of sheep BMPR-IB gene A746G were extracted respectively. The target fragment of 223bp was amplified by PCR and diluted to 0.05ng / 渭 L after determination of concentration and purity. The positive homozygote was diluted with negative homozygote as template standard, and three pairs of primers were designed and synthesized according to the template standard product sequence. The products were analyzed by high-resolution melting curve. Normlized and Temp-Shifted Differines Plotole is the standard curve. As the basis of Fec B detection, the reliability and repeatability of Fec B were further verified. The results showed that the standard curve of COLD-PCR HRM curve of two pairs of primers was clear and reliable. The detection limit was 0.5, compared with the traditional Q PCR HRM. The sensitivity was increased to 4 times. The low abundance Fec B gene detection method developed in this experiment can be used for the rapid screening of sheep Fec B gene in large area and high throughput.
【作者单位】： 新疆石河子大学动物科技学院;
【分类号】：S826
【正文快照】： 绵羊产羔率属于阈性状,由多个基因控制。其中,绵羊骨形态发生蛋白受体IB(BoneMorphogenetic Protein Receptor IB,BMPR-IB)基因发生第六外显子A746G单碱基突变后被称作Fec B基因,是影响绵羊产羔率的最重要的主效基因,增羔效应最明显,应用最广泛。Davis等[1]报道,一个拷贝的Fec，

本文编号：1461276