多重位点特异性PCR快速检测牛肉中常见掺假动物源性成分

目的:基于动物线粒体cytb基因的多态性位点,建立一种特异性多重PCR体系检测牛肉、猪肉和鸡肉的方法。方法:提取肉类的基因组DNA,利用不同物种mtDNA cytb基因序列的SNP位点的差异,设计特异性引物。进行多重PCR扩增,利用扩增产物片段大小不同,检测牛肉中常见的掺假动物源性成分。通过灵敏性试验,确定最低检测量。结果:实验设计的引物特异性良好,在同一反应体系中,在同一退火温度52℃条件下,牛肉DNA扩增后产生149 bp的特异性条带,猪肉DNA扩增片段为261 bp,鸡肉DNA扩增片段为554 bp,未发生非特异性扩增。且检测的最低浓度达到100 pg/μL,具有高度的灵敏性和适用性。结论:根据动物线粒体cytb基因的差异性位点,开发的多重PCR体系,可一次性地同时检测牛肉、猪肉和鸡肉,可快速、灵敏、高通量地分析食品中掺假动物成分的来源。

Rapid Detection of Common Adulterated Components in Beef by Multiple Allele-specific Polymerase Chain Reaction

Objective:To establish a specific multiplex PCR system for the detection of beef,pork and chicken based on the polymorphic site of the animal mitochondrial cytb gene. Methods:The genomic DNA of meat was extracted and specific primers were designed using SNP sites of mtDNA cytb gene sequences of different species. Multiple PCR amplification was carried out to detect adulterated animal-derived components commonly found in beef by using different sizes of amplified product fragments. The minimum detection amount was determined by a sensitivity test. Results:The primers of the experimental design had good specificity. In the same reaction system and the same annealing temperature of 52 ℃,the beef DNA amplified to produce 149 bp specific bands,the pork DNA amplified fragment was 261 bp,the chicken DNA amplified fragment was 554 bp,and no non-specific amplification occurred. And the lowest concentration detected reaches the 100 pg/μL,and had a high sensitivity and applicability. Conclusion:According to the differential sites of animal mitochondrial cytb gene,a multiplex PCR system was developed to simultaneously detect beef,pork and chicken at one time. It can quickly,sensitively and efficiently analyze the source of adulterated components in food.