牛脊柱畸形综合征HRM-非标记探针检测方法的建立与应用

Development and application of high resolution melting analysis with unlabeled probe for detecting complex vertebral malformation

为建立用于牛脊柱畸形综合征（CVM）检测的高分辨率熔解曲线（HRM）非标记探针法,根据Gen Bank中SLC35A3基因序列设计PCR引物及非标记性探针,构建G/G型、G/T型、T/T型3种标准质粒,利用质粒作为标准品建立HRM特征图谱。采用优化的HRM-非标记探针法对305份荷斯坦奶牛血样及冷冻精液样品中SLC35A3位点的基因型进行检测,同时采用测序法进行验证。结果,305份样本中9份冻精样本为CVM携带者,携带率为2.95%,检测结果与测序法检测结果一致,准确率达100%。上述研究结果表明,本研究建立的针对牛CVM中SLC35A3基因G-T位点突变检测的HRM-非标记探针方法是一种简单快速、灵敏特异、高效准确的牛CVM有害基因分型检测技术。

This experiment was conducted to establish a method of high resolution melting analysis（HRM）with unlabeled probe for detection of CVM alleles.According to CVM SLC35 A3 gene sequences published in Gen Bank,PCR primers and unlabeled probes were designed and optimized.Three standard plasmids including G/G type,G/T type and T/T type were constructed and the plasmids were used as the standard of the HRM characteristic plots.The optimized HRM with unlabeled probe method was used to detect genotypes of the SLC35 A3 loci among 305 blood and frozen semen samples of Holstein calves,then verified by DNA sequencing method.In result,9 semen samples were CVM carriers,and carrying rate was 2.95%.The test results from HRM-unlabeled probes were consistent with the sequencing.In conclusion,this study provided a HRM-unlabeled probe method for detection of CVM SLC35 A3 gene mutation,which is a simple,fast,sensitive,specific,efficient and accurate detecting technique.