摘要
为建立SYBR Green I实时荧光PCR检测鉴定狮(Panthera Leo)制品的方法,本文在豹属动物线粒体细胞色素b基因上设计狮特异性引物,建立并优化了SYBR Green I实时荧光PCR检测鉴定体系,采用17种血液、组织和骨粉类样品的DNA,通过扩增曲线和融解曲线分析检测的特异性和灵敏度,并用狮肉粉和牛肉骨粉系列百分比混合物进行抗干扰能力分析。结果显示,狮血液和组织DNA样品均出现特异性的实时扩增曲线,平均熔解温度在84±0.2℃。虎、豹、熊等14种哺乳动物DNA样本的检测结果均为阴性。灵敏度检测显示,本实验所建立的SYBRGreen I实时荧光PCR方法能够检测出10 pg/μL的狮组分DNA。抗干扰能力显示在骨粉中狮的成分最低检出含量为0.1%。这些结果说明,本文建立的SYBR Green I实时定量PCR方法具有方便、经济、灵敏度高和重复好等特性,可用于狮源性制品的检测鉴定。
SYBR Green 1 Real-Time PCR system for assay products（muscle,fur,blood and bone）of the lion（Panthera leo）was constructed on the basis of a pair of primers,specific for the lion,designed according to the sequence of cytochrome 6 gene of mitochondria DNA of Panthera animals.PCR amplification showed following results：samples of lion blood and tissue DNA were amplified,but those of 14 other mammals（3 subspecies of tiger,2 subspecies of leopards,bears,catde, dogs etc）failed to amplify;sensibility was 0.1%lion material in a mixture of bone powder of mammals and at the concentration of 10 pg/ul of lion DNA.
基金
公益性行业科研专项"食品中濒危物种资源分子指纹研究"(2007GYJ037)
