24个基因座复合扩增系统的应用

Application of the 24-locus multiplex system

目的建立24个基因座的复合扩增系统,并对其性能指标进行评价。方法选择24个基因座(包含D8S1179、D5S818、D2S1338、D18S51、D6S1043、D2S441、D3S1358、vWA、D19S433、D16S539、CSF1PO、Penta D、D22S1045、D13S317、D1S1656、D7S820、TPOX、Penta E、D10S1248、TH01、D12S391、D21S11、FGA等23个STR基因座及1个Amelogenin基因座);合成引物,上游端标记荧光染料;收集DNA数据库建库血痕及口腔拭子样本及相关11种动物样本,采用本文方法进行复合扩增;并对方法的准确性、灵敏度、稳定性、种属特异性、检材适用性、混合样本的检验等系统性能指标进行验证。结果本文复合扩增系统对最长保存9年的各种血痕检材完整分型成功率为100%,且均衡性良好,无非特异性扩增,口腔拭子的成功率为97.8%,灵敏度达125pg,对含有抑制剂样本,在血红素浓度≤600μmol/L,腐殖酸浓度≤50ng/μL时检出效果稳定,种属特异性好。结论本文24个基因座复合扩增系统在DNA数据库建设中具有较好的应用价值。

Objective To develop a new 24-locus multiplex system and evaluate its technical performance. Methods Amelogenin and 23 STR loci（ including D8S1179,D5S818,D2S1338,D18S51, D6S1043,D2S441,D3S1358,vWA,D19S433,D16S539,CSF1PO,Penta D,D22S1045,D13S317,D1S1656, D7S820,TPOX,Penta E,D10S1248,TH01,D12S391,D21S11 and FGA） were chosen to be co-amplified and detected. Primers were designed and synthesized respectively. All forward primers were labeled with a fluorescent dye. A total of 736 unrelated individual blood samples and buccal swabs and 11 related animal samples were collected and tested. The accuracy,sensitivity,stability,species specificity,adaptability and ability to detect mixture samples of the system were assessed. Results The success rate to obtain full profiles was 100% for various years of bloodstains with well balance and no unspecific amplification,and the success rate of buccal swabs was 97. 8%. The system showed high sensitivity（ 125pg）,high stability（ hematin ≤ 600μmol / L and humic acid ≤ 50ng / μL） and high species specificity. Conclusion The results demonstrate that the 24-locus multiplex system is a valuable and reliable tool for forensic DNA databasing.