本文關(guān)鍵詞： 綿羊 FecB基因 COLD-PCR 高分辨率熔解曲線分析　出處：《中國畜牧雜志》2017年06期 　論文類型：期刊論文

【摘要】：本文利用高分辨率熔解曲線分析結(jié)合COLD-PCR建立綿羊骨形態(tài)發(fā)生蛋白受體IB(BMPR-IB)基因低豐度A746G點突變的檢測方法,為Fec B基因大面積高通量篩查提供方便快捷和低成本的檢測技術(shù)。實驗分別提取綿羊BMPR-IB基因A746G點突變陽性純合子和陰性純合子的基因組,分別PCR擴增223 bp的目的片段,測定濃度和純度后,分別稀釋至0.05 ng/μL,將陽性純合子用陰性純合子進行系列稀釋作為模板標準品。根據(jù)模板標準品序列設(shè)計合成3對引物,以標準品為模板進行COLD-PCR,其產(chǎn)物進行高分辨率熔解曲線分析,生成的歸一化溫度轉(zhuǎn)移差異圖(Normlized and Temp-Shifted Differines Plot)即為標準曲線圖,作為待測樣品Fec B檢測的依據(jù),對其可靠重復(fù)性做了進一步驗證。結(jié)果表明:2對引物COLD-PCR HRM標準曲線圖曲線分離清晰可靠,檢測下限均為0.5%,與傳統(tǒng)q PCR HRM相比,靈敏度提高到4倍。本實驗建立的綿羊低豐度Fec B基因檢測方法可以用于綿羊Fec B基因大面積高通量快速篩查工作。
[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.
【作者單位】： 新疆石河子大學動物科技學院;
【分類號】：S826
【正文快照】： 綿羊產(chǎn)羔率屬于閾性狀,由多個基因控制。其中,綿羊骨形態(tài)發(fā)生蛋白受體IB(BoneMorphogenetic Protein Receptor IB,BMPR-IB)基因發(fā)生第六外顯子A746G單堿基突變后被稱作Fec B基因,是影響綿羊產(chǎn)羔率的最重要的主效基因,增羔效應(yīng)最明顯,應(yīng)用最廣泛。Davis等[1]報道,一個拷貝的Fec，

本文編號：1461276