高分辨率熔解曲线分析技术在鼠形动物鉴定中的应用

The application of high resolution melting curve analysis technology in the identification of rat-shaped animals

目的探索一种能够快速鉴定常见鼠形动物的新型分子生物学方法。方法以线粒体基因组(mt DNA)细胞色素b(Cytochrome b,Cytb)基因为靶基因,根据Gen Bank公布的序列,设计测序引物,扩增5种常见鼠形动物的目的基因并测序。依据测序结果和Gen Bank公布的序列,设计分析引物,进行高分辨率熔解曲线分析。以新鲜提取的基因组DNA为模板,按照1∶10的比例梯度稀释,检测7个浓度梯度,进行敏感性检测。结果测序引物扩增出5种鼠形动物Cytb序列,分析引物扩增出5种鼠形动物的熔解曲线,并可根据曲线鉴别5种鼠形动物的种类,检测灵敏度为10-2ng/μl。结论利用高分辨率熔解曲线分析技术,可以快速准确鉴定5种常见鼠形动物。

Objective To develop a molecular biology method to identify rat-shaped animals quickly. Methods With mitochondrial genome（mt DNA） Cytochrome b（Cytochrome b,Cytb） as a target gene,sequencing primers were designed according to the sequences published in Gen Bank. Amplification and sequencing were applied to 5 kind of rat-shaped animals. The enotyping primers were designed according to the sequenced and Gen Bank sequences,and high resolution melting curve analysis was run. DNA template was 10-flod diluted serially and amplified to get its sensitivity. Results The sequencing primers were able to amplify Cytb sequences of 5 kind of rat-shaped animals,the genotyping primers were able to amplify 5 kinds of melting curves and identify all the rat-shaped animals,the detection limit was 10-2ng/μl. Conclusion The 5 rat-shaped animals could be identified with the high resolution melting curve analysis technology accurately.