应用MGB荧光探针技术确定猪KIT基因拷贝重复数

摘要

The main objective of the present study was to establish a simple, rapid method for detecting copy number variations (CNVs) in porcine KIT gene. TaqMan-MGB probes and primers were designed according to sequence of exon 2 of KIT gene, and a real-time fluorescence quantitative PCR procedure was established for the CNVs detection. The results showed that amplification curves of KIT obtained by TaqMan-MGB probe were a series of parallel curves corresponding to 2-fold serial dilutions of DNA samples, Ct values between groups were significantly different (P＜0. 001) , and the coefficient-of-variations were low (from 0. 12％ to 0. 26％). The amplification effiaencies of the KIT and ESR were approximately equal. The CNVs in KIT of 50 pigs were estimated by cluster analysis, assigned to 2, 3, 4, 5 or 6 copies, respectively. The real-time quantitative PCR using TaqMan-MGB probe is a simple, rapid method with high resolution and stability to measure CNVs in KIT, and it could be carried out in common laboratories.%旨在建立一种简便、快速检测猪KIT基因拷贝重复数(CNVs)的方法并应用于未知样品的检测.在KIT外显子2中设计TaqMan-MGB探针和引物,建立荧光实时定量PCR检测KIT基因CNVs的方法.结果表明,TaqMan-MGB探针扩增KIT基因目的片段,不同DNA梯度浓度Ct值差异极显著(P＜0.001),变异系数低(0.12%～0.26%);KIT与内标基因ESR的扩增效率相差只有2.4%;采用聚类分析推测了待测的50枚样品KIT的CNVs分别归属于2、3、4、5、6个拷贝.本研究建立的TaqMan-MGB探针实时荧光定量PCR检测KIT基因CNVs的方法,具有简便、快速、特异性和稳定性好的特点,适合于普通实验室应用.