绵羊BMP15基因B2突变可视化检测技术的建立  

Establishment of a Visual Detection Technique for B2 Mutation of BMP15 Gene in Sheep

作　　者：胡瑞瑞 刘莉 郭涛 李雅心 王小奎[1] 胡圣伟[1] HU Ruirui;LIU Li;GUO Tao;LI Yaxin;WANG Xiaokui;HU Shengwei(College of Life Sciences, Shihezi University,Shihezi 832000,China)

机构地区：[1]石河子大学生命科学学院,石河子832000

出　　处：《中国畜牧兽医》2021年第3期810-817,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31660644);新疆生产建设兵团国际科技合作计划项目(2018BC011)。

摘　　要：骨形态发生蛋白15(bone morphogenetic protein 15,BMP15)基因突变对绵羊排卵率、产仔数以及繁殖力有很大影响,本研究旨在建立快速可视化检测BMP15基因B2突变的技术。将改良的扩增阻滞突变系统(amplification refractory mutation system,ARMS)与核酸染料SYBR GreenⅠ结合,针对BMP15基因B2突变设计ARMS特异性引物,使引物下游3′端的最后一个碱基为A,并在下游引物3′端的第3位碱基处设计额外的错配,以提高引物的特异性;经ARMS PCR扩增后,向产物中加入SYBR GreenⅠ,通过肉眼观察PCR管内的颜色变化来检测BMP15基因的B2突变。经测序发现所采集的样本均为野生型,因此,利用重叠延伸PCR构建BMP15基因B2突变型模板,测序结果显示BMP15基因B2突变型模板构建成功。ARMS PCR结果显示,ARMS特异性引物能够只扩增突变型模板,而不会扩增野生型模板,且扩增效果良好。ARMS PCR扩增后加入SYBR GreenⅠ发现已知野生型模板与阴性对照一致,呈SYBR GreenⅠ原始颜色橙黄色,而已知突变型模板呈亮绿色,且色差明显,肉眼可辨。用建立的可视化检测BMP15基因B2突变的技术检测50个小尾寒羊基因组DNA(随机向其中20个样本中加入构建的BMP15基因B2突变型模板),将可视化检测的突变型样本编号与混合样本时记录的编号对比,结果表明该技术能够准确检测绵羊BMP15基因的B2突变,且准确率高达100%。将构建的BMP15基因B2突变型的模板进行梯度稀释,对本试验可视化检测体系的灵敏度进行检测,发现ARMS可视化检测技术的灵敏度达到0.006 ng/μL。因此,本研究建立的技术能够可视化地检测绵羊BMP15基因B2突变,且准确率高,有望为高繁殖力绵羊的选育提供技术支持。Bone morphogenetic protein 15(bone morphogenetic protein 15,BMP15)gene mutation had a great impact on sheep ovulation rate,litter size and fecundity.Therefore,this study was aimed to establish a rapid visual detection technology for B2 mutation of BMP15 gene.Combine the improved amplification refractory mutation system(ARMS)with the nucleic acid dye SYBR GreenⅠ,and designed ARMS-specific primers for the B2 mutation of BMP15 gene,made the last base at the 3′end of the primer as A,meanwhile designed additional mismatches at the third base of the 3′end of the downstream primer to improve primer specificity.After ARMS PCR,added SYBR GreenⅠto the product,and detected the B2 mutation of BMP15 gene by visually observing the color change of the PCR tube.It was found through sequencing that the collected samples were all wild-type.Therefore,the B2 mutation of BMP15 gene template was constructed by overlap extension PCR.The sequencing results showed the B2 mutation of BMP15 gene template was successfully constructed.ARMS PCR results showed that ARMS-specific primers could only amplify mutant templates,could not amplify wild-type templates,and the amplification effect was good.After ARMS PCR,SYBR GreenⅠwas added and it was found that the known wild-type template was consistent with the negative control and showed the original color of SYBR GreenⅠorange-yellow,while the known mutant template was bright green with obvious color changes.Using the established visual detection technology to detect 50 Small-tailed Han sheep genomic DNA(20 samples were added with the constructed B2 mutant template of BMP15 gene),and compare the number of the mutant sample detected visually with the number recorded when the sample was mixed.The results showed that this technology could accurately detect the B2 mutation of BMP15 gene,and the accuracy was as high as 100%.The constructed BMP15 gene B2 mutant template was serially diluted to detect the sensitivity of the visual detection system in this experiment.It was found that the sensi

关 键 词：绵羊 BMP15基因 B2突变 可视化检测 ARMS SYBR GreenⅠ 

分 类 号：S826[农业科学—畜牧学]