17个STR基因座快速多重扩增体系的建立及应用

The construction of a rapid multiplex PCR amplification system of 17 STR loci and its application in forensic science

目的 建立17个STR基因座多重PCR快速扩增体系.方法 采用人血样本提取DNA并定量,用于多重快速扩增体系分型准确性检测;采用标准品9948,设定稀释度,检测体系灵敏度;采用定量男女DNA样本按11种比例混合,检测体系对混合样本的分型能力;在标准品中加入干扰物质血红素和腐植酸,检测体系的抗干扰能力;对5种非人样本进行检测,评价体系的特异性;对实际案例进行检验,评价体系实际应用价值.结果 采用本文体系,在65min 内,用0.5 ～ 2ngDNA模板量能获得较好的扩增效果,分型结果准确稳定,扩增均衡;种属特异性好;血红素≤50 μmol/L,腐植酸≤25ng/μL时可不受干扰准确分型;男女混合样本中单一样本量不低于1/10即可进准确进行判断;对实际案例常见生物检材的检验结果良好.结论 本文17个STR基因座快速多重扩增体系可显著缩短扩增时间,技术性能符合实际检案要求,可在实践中选用.

Objective To develop a rapid multiplex PCR amplification system of 17 STR loci. Methods Accuracy detection of the rapid multiplex PCR amplification system was carried out in a sample of human blood after extracted and quantified, sensitivity was studied using 9948 DNA standard with a serial dilution. Mixture studies were conducted using quantified male/female DNA with 11 mixture ratios. Inhibitor tolerance was carried out by adding hematin and humic acid into the system. Species specificity were studied using non-human DNA samples, casework samples were tested to validate the system＇ s applicability. Results The rapid multiplex PCR amplification system possesses the qualities of accurate typing, well balance, and high species specificity when using 0. 5 ～ 2ngDNA template in 65 minutes. Full profiles were obtained until the concentration of hematin increased to 50μmoL/L or humic acid increased to 25ng/μL. All alleles were called for the 1 ： 10 mixture of male/female samples. A variety of routine casework samples were successfully tested. Conclusion The established 17 locus rapid multiplex PCR amplification system can significantly shorten the amplification time and meet the requirements of practical applications and could be used for forensic science.