基于TaqMan探针的虎制品实时荧光定量PCR的特异性鉴定

根据虎线粒体细胞色素b核苷酸保守序列设计引物和探针,建立了一种快速鉴定虎制品的TaqMan实时荧光定量PCR方法。以梯度稀释的含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示,5.0×10⁵~ 5.0×10¹拷贝/μL范围内定量PCR均有"S"型扩增曲线,检测灵敏度为50拷贝/μL。对豹、狮子、猫等7种非虎哺乳动物DNA样本的检测结果均为阴性。本研究建立的实时荧光定量PCR方法具有灵敏度高、特异性和重复性好、方便经济的特性,在虎制品的检测与鉴定中具有良好的应用前景。

Real-time Quantitative RT-PCR Assay for Specific Identification of Panthera tigris Materials

Primers and probes were designed based on the conservative sequence of cytochondriome cytochrome b,a real-time quan quantitative RT-PCR assay was developed for indentification of Panthera tigris.The recombinant plasmid containing the target sequence was constructed to detect the sensitivity and prepare the standard curve.The real-time RT-PCR assay had a detection limit of 50 copies/μL,and with a dynamic range of detection between 5×105 to 5×101 copies /μL.The primers and probe were specific for Panthera tigris’ DNA and did not react with other non-Panthera tigris’ DNA.This real-time RT-PCR assay with high sensitivity,specificity and accuracy is considered to be a powerful tool for the rapid indentification and quantification of Panthera tigris.