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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 explore a novel molecular biology method for rapid identification of common mouse animals. Methods Cytochrome b (Cytb) gene of mitochondrial DNA (mtDNA) was used as a target gene. According to the sequence published by Gen Bank, sequencing primers were designed and the target genes of five common mouse species were amplified and sequenced. Based on the sequencing results and sequences published by Gen Bank, primers were designed and analyzed for high-resolution melting curve analysis. Using freshly extracted genomic DNA as a template, according to a 1:10 ratio gradient dilution, seven concentration gradients were tested for sensitivity. Results Cytb sequences were amplified from 5 kinds of mouse animals by sequencing primers. The melting curves of 5 kinds of mouse animals were amplified by primers, and 5 kinds of mouse animals were identified according to the curve. The detection sensitivity was 10-2ng / μl. Conclusion Using high-resolution melting curve analysis technology, five common mouse-shaped animals can be quickly and accurately identified.