Y染色体短串联重复序列微流控芯片复合扩增检测体系研究

Study of The Y-STR Multiplex Microfluidic Chip Rapid Amplification System

目的构建Y染色体短串联重复序列(Y-STR)微流控芯片扩增检测试剂,并进行性能验证,实现Y-STR基因座的快速全集成检测。方法使用Y-STR微流控芯片检测体系,对其灵敏度、成功率和分型准确率、峰平衡性、精准性和准确性、检材适应性、混合物检测能力和抗抑制性进行验证评估。结果DNA标准品9948的模板量≥8 ng,血卡片数≥3片以及口腔拭子刮擦次数≥7次时可获得Y-STR完整分型;165份样本的全集成检测成功率为91.52%,分型准确率为99.74%;不同荧光通道之间的峰高比值为89.81%;10次运行的等位基因分型标准品的片段大小标准差均在0.5 bp以内,20份样本全集成检测的等位基因片段和相应的等位基因标准品之间的片段准确性均在0.5 bp以内;能够对口腔拭子、血卡、唾液卡、烟蒂、血棉签、布片精斑等检材进行准确分型;混合样本中较小贡献者与较大贡献者在1∶3的比例时可获得完整基因分型;在不同浓度的腐殖酸(50~400 mg/L)、靛蓝(20~100 nmol/L)、血红蛋白(100~500μmol/L)等抑制物的干扰下,该体系可获得完整基因分型。结论该体系可应用于国产Quick TargSeq法医DNA现场快速检测系统使用,可在法医学实践中选用。

Objective At present,the matching reagents of commercially available rapid DNA instruments based on microfluidics chip technology are autosome short tandem repeat(STR)individual identification reagents.The non-recombining part of the human Y chromosome is widely used in forensic DNA analysis,particularly in cases where standard autosomal DNA profile is uninformative.Y-STR loci are useful markers to identify males and male lineages in forensic practice.In order to achieve rapid and fully integrated detection of Y-STR loci,this study constructed the RTyper Y27 microfluidic chip rapid detection system and validated the performance of this system.Methods The system was verified and evaluated by sensitivity,success rate,typing accuracy,peak height balance,sizing precision and accuracy,mock case sample tests,mixture detection ability,and inhibition tolerance.Results Complete Y-STR profiles can be obtained when the template amount of DNA standard 9948 was≥8 ng,the number of blood cards was≥3 pieces,and the number of oral swab scrapings was≥7 times.The success rate of fully integrated detection was 91.52%,and the concordance rates was 99.74%for 165 testing samples.The success rate of 115 blood spots in these samples was 90.43%,with a typing accuracy of 99.65%,the success rate of 50 buccal swabs was 94%,with a typing accuracy of 99.92%.There was no significant difference in typing accuracy between blood spots and buccal swab samples.The peak height ratio between different fluorescence channels was 89.81%.The standard deviation of allelic ladder for 10 runs was within 0.5 bp.The size differences between allele and corresponding allele in allelic ladder was within 0.5 bp.The maximum precision CV values within and between batches were 0.48%and 0.68%,respectively,which were lower than 15%.These data indicate that the system has good accuracy and precision.The system was capable of accurately typing oral swabs,blood cards,saliva cards,cigarette butts,blood swabs and seminal stains.Complete Y-STR profiles can be obtained and distinguish at the 1∶3 ratio of minor and major contributors in artificial male DNA mixtures.Complete Y-STR genotyping can be obtained under the interference of inhibitors,such as different concentrations of humic acid(50-400 mg/L),indigotin(20-100 nmol/L)and hemoglobin(100-500μmol/L).Conclusion In this study,the RTyper Y27 microfluidic chip rapid amplification system is combined with the Quick TargSeq 1.0 integrated system,and the Y-STR profile can be obtained in approximately 2 h.Through a series of verification experiments,the results show that the system has good repeatability,accuracy and stability,can meet the on-site Y-STR detection requirements,and can be used in forensic practice.