摘要
目的利用线粒体的特点构建一种特异性好、灵敏度高、稳定性好、操作简便,适用于法医学种属鉴定的复合扩增体系。方法查找Gene bank上常见动物mt DNA序列,用序列比对软件,对人和动物序列进行分析,选出COX1、12SrRNA、16SrRNA区域。使用primer5和primer3(online)设计引物,COX1的引物有人类特异性,12SrRNA和16SrRNA的引物作为内参对照可以匹配人和大部分常见动物。收集人和恒河猴,猪、牛、羊、兔、鲈鱼、鲍鱼、鸭、鸡、狗、鼠11种动物的肌肉或血液样本,用Chelex-100法提取DNA,复合扩增COX1、12SrRNA、16SrRNA基因片段,琼脂糖凝胶电泳分离扩增产物。结果人的样本出现三条带,分别为COX1、12SrRNA、16SrRNA的扩增产物,大小分别为202、231、395bp;11种动物出现两条带,分别为12SrRNA和16SrRNA的扩增产物,大小分别在230、395bp左右。复合扩增体系灵敏度为2.5pg,而特异性引物COX-1的灵敏度为10-8pg。结论该复合扩增体系可以明确区分人与动物,灵敏度高,可应用法医学中的种属鉴定。
Objective To establish a multiplex amplification system with high specificity, sensitivity and reliability for species identification. Methods The mitochondrial DNA （mtDNA） sequences of human and ordinary animals were acquired from online GenBank and analyzed with Clustal W software. The primers were designed using primer 3.0 and primer 5.0 according to the gene sequence. The primer for COX1 was of human specificity, and the primers for 12S rRNA and 16S rRNA were non-specific to human and most of the known animals and were used as internal controls. The muscle or blood samples of rhesus, pig, cattle, sheep, rabbits, weever, abalone, duck, chicken, dog and mouse were collected, and their DNA was extracted using Chelex-100 method. The PCR products were separated with agarose gel eleetrophoresis. Results Human samples showed three bands resulted from 12SrRNA, 16SrRNA and COX1, and the corresponding fragment size was 235bp, 395bp and 202bp respectively. The other eleven animals showed only two bands resulted from 12SrRNA and 16SrRNA, and the corresponding fragment size was 230bp and 395bp respectively. The sensitivity of the multiplex system and individual COX1 specific primer was 2.5 pg and 10^-8 pg of DNA respectively. Conclusion The multiplex amplification system can exactly and sensitively distinguish human from animals, and can be applied to forensic species identification.
出处
《中国法医学杂志》
CSCD
2015年第6期560-562,566,共4页
Chinese Journal of Forensic Medicine
