摘要
对GenBank上已发布的虎、狮、豹等猫科物种的线粒体基因序列进行多重比对,基于虎特异性单核苷酸多态性(Single nucleotide polymorphism,SNP)位点设计并合成一对用于虎种源性成分检测的特异性引物和双标记探针,同时以肌肉生长抑制素基因(myostatin,MSTN)作为内参靶标合成引物和不同荧光素标记的探针。经体系优化,建立了可以用于检测虎源性成分的双重荧光PCR方法。该方法具有良好的特异性和灵敏度,能够有效检测动物制品中的虎源性成分并可对采样和核酸提取进行质量控制;通过对采集的15份虎毛、虎粪便、豹毛及猫毛等样品进行检测鉴定,结果显示建立的双重荧光PCR方法可特异性检测鉴定出虎源性成分,与其他猫科动物样品无交叉反应。本研究建立的虎源性成分双重荧光PCR鉴定方法可用于口岸截获的疑似虎制品的精准快速检测、野生动物追踪溯源。本研究为保护濒危虎物种、打击走私虎制品提供一种特异性更强的快检手段。
Based on multiple alignment of mitochondrial gene of feline species such as tiger,lion and leopard published on GenBank,a pair of specific primers and a double labeled probe were designed and synthesized according to a tiger-specific SNP site for the detection of tiger-derived materials.At the same time,another pair of primers and the probe labeled with different fluorescein,which were targeted to the myostatin gene,were synthesized as the internal amplification control(IAC).By optimizing the reaction system,a duplex real-time PCR was established to detect tiger-derived materials.With good specificity and sensitivity,it could detect tiger-derived materials in animal products effectively and could control the quality of sampling and nucleic acid extraction.A total of 15 samples including tiger hair,tiger feces,leopard hair and cat hair were detected by the developed real-time PCR.The results showed that it could detect and identify tiger-derived materials specifically,and had no cross reaction with samples from other felines.The duplex real-time PCR can be used for accurate and rapid detection of suspected tiger products intercepted at ports and tracking of wild animals established in this study.This study provided a more specific rapid detection method for protecting endangered tiger species and combating smuggling of tiger products.
作者
赵相鹏
蒲静
种焱
高志强
杜思乐
白子龙
赖平安
汪琳
ZHAO Xiang-Peng;PU Jing;CHONG Yan;GAO Zhi-Qiang;DU Si-Le;BAI Zi-Long;LAI Ping-An;WANG Lin(Science and Technology Research Center of China Customs,Beijing 100026)
出处
《中国口岸科学技术》
2022年第12期85-91,共7页
China Port Science and Technology
基金
海关总署科技项目(2020HK140)
关键词
动物制品
虎源性成分
荧光PCR鉴定
animal products
tiger derived materials
real-time PCR identification
