miniSTR荧光检测体系的建立及法医学应用

Establishment of miniSTR Fluorescent Detection System and Its Forensic Application

目的建立检测片段均小于150bp的miniSTR荧光检测体系,提高对微量降解检材DNA的检测效能。方法应用Primer Premier 5软件设计、FastPCR 6.0筛选引物,组合成用四色荧光标记引物的miniSTR复合扩增体系。优化PCR检测条件和引物浓度,在3100-Avant仪上用POP4胶进行电泳检测。分型结果用DNA标准品9947A和007进行验证,并通过检测新鲜血样、疑难微量检材评估该体系的法医学应用效能。结果建立的miniSTR荧光检测体系(D12ATA63、D2S1776、D1GATA113、D4S2408、D17S974、D20S482、D3S3053、Amelogenin、D6S474、D9S1122)中各基因座的检测片段均小于150 bp,各等位基因扩增均衡性良好,无非特异性扩增产物,等位基因频率分布符合Hardy-Weinberg平衡,累积个体识别率为0.999999983,三联体累积非父排除率为0.9968。能成功检测腐败肌肉组织、低拷贝数DNA检材以及在40%甲醛溶液中固定12d的人体组织。结论 miniSTR荧光检测体系可独立应用于降解DNA样本的个体识别鉴定或补充应用于亲权鉴定,提高对微量、降解检材DNA的检测能力。

Objective To establish miniSTR fluorescent detection system with all detected fragments be- low 150bp and to enhance the efficiency of detecting the degraded DNA samples. Methods All candi- date primers were designed by Primer Premier 5 and screened by FastPCR 6.0. The miniSTR multiplex system was established by these selected loci labeling by four fluorescent dye. The parameters of PCR and primer concentrations were subsequently optimized. The electrophoresis was fulfilled under POP4 on 3100-Avant and the typing data was validated by standard DNA 9947A and 007. Fresh blood samples and difficult degraded DNA samples were tested to evaluate the usefulness of the system. Results All amplicons in the established miniSTR fluorescent detection system （D12ATA63, D2S1776, DIGATA113, D4S2408, D17S974, D20S482, D3S3053, Amelogenin, D6S474, D9Sl122） were less than 150bp. The profile showed a balanced peak height without extra stutter by optimal protocol. Allele frequencies showed no deviations from Hardy-Weinberg equilibrium. The system showed accumulated probability of discrimina- tion 0.999 999 983 and accumulated triplet excluding probability of paternity 0.996 8. It could detect cor- rupt muscle tissue, low copy number DNA samples and human tissues fixed by 40% formaldehyde solu- tion for 12 days. Conclusion The miniSTR fluorescent detection system could be solely used for person- al identification of degraded DNA samples or complementally used for paternity tests. And the system could enhance the ability of detecting the trace and degraded DNA.