38重InDel快速族群推断体系的优化及验证

Optimization and Developmental Validation of 38-plex InDel Panel for Ancestry Inference

目的对构建的38重InDel快速族群推断体系进行优化,根据DNA分析方法科学工作组(Scien⁃tific Working Group on DNA Analysis Method,SWGDAM)指南进行性能验证,并对其应用于东亚、欧洲、非洲及其混合人群族群推断的准确性进行验证。方法以DNA标准品9947A为模板,通过调整引物平衡性、Mg2+终浓度、优化PCR热循环参数和扩增体积等建立最优扩增条件,比较样本的等位基因丢失、非特异性扩增以及推断样本来源是否与已知信息匹配,评价该体系的相关性能。结果本体系的最佳模板用量为0.125~2 ng DNA,InDel分型结果准确,扩增均衡性好,种属特异性好;对混有血红蛋白(≤80μmol/L)、靛蓝(≤40 mmol/L)、钙离子(≤1.0 mmol/L)以及腐殖酸(≤90 ng/μL)等抑制剂的样本具有一定耐受性;可以直接扩增检测血卡、唾液卡,且族群推断结果准确;能够区分两样本的混合DNA样本;对实际案例常见生物检材的检验结果良好。结论38重InDel快速族群推断体系分型结果准确可靠,体系性能符合SWGDAM指南要求,可以准确推断未知个体的非洲、欧洲、东亚及欧亚混合人群来源,可用于法医鉴定实践。

Objective The previously established 38-plex InDel system was optimized and its performance was validated according to the Scientific Working Group on DNA Analysis Method(SWGDAM)application guidelines.The ancestry inference accuracy of individuals from East Asian,European,African and mixed populations was verified.Methods DNA standard sample 9947A was used as the template to establish the optimal amplification conditions by adjusting primer balance,Mg2+final concentration and optimizing PCR thermal cycle parameters and amplification volume.The allelic dropout,nonspecific amplification and whether the origin of the inferred samples matched the known information were compared to evaluate the performance of this system.Results The optimal dosage of this system was 0.125-2 ng DNA template.The results of InDel typing were accurate,the amplification equilibrium was good,and the species specificity was good.This system showed certain tolerance to DNA samples including the inhibitor such as hemoglobin(≤80μmol/L),indigo(≤40 mmol/L),calcium ion(≤1.0 mmol/L),and humic acid(≤90 ng/μL).The system enabled the direct amplification of DNA from saliva and blood on filter paper,and the results of ethnic inference were accurate.The system successfully detected the mixed DNA sample from two individuals.The test results of the system for common biological materials in practical cases were accurate.Conclusion The results of the 38-plex InDel system are accurate and reliable,and the performance of the system meets the requirement of the SWGDAM guidelines.This system can accurately differentiate the ancestry origins of individuals from African,European,East Asian,and Eurasian populations and can be implemented in forensic practice.