荧光STR直接复合扩增试剂缓冲体系的研制

目的研制适用于数据库样本荧光STR直接复合扩增体系。方法针对常规血卡、FTA和903血卡样本,配制扩增缓冲液基准母液,采用不同配方的扩增缓冲体系进行直接扩增及检测。考察不同种类增强剂、4种商业化DNA聚合酶、不同复性温度和终延伸时间对检材的检测效果,并验证优化体系的适应性。结果采用本文所建体系对各类血卡样本进行检验,均可获得样本清晰、完整的STR分型。体系选择BSA\Tween20\DMSO\甘油等增强剂组合、Typer热启动聚合酶1.5U/10μL、57～59℃复性温度、30～50min终延伸时间,采用10μL体系即可对直径1.2mm FTA卡血样进行有效分型。结论本文所研制的缓冲体系能够满足常规血卡、FTA和903血卡样本直接扩增检验的需要。

Development and application of a fluorescent STR multiplex assay for the direct amplification of forensic database samples

Objective To develop a direct multiplex amplification reagent system and examine its validity using forensic DNA database samples.Methods Several direct PCR buffer based on standard buffer were prepared.The performance of buffer were tested by direct amplification of different samples,such as blood stains on filter paper,FTA card and 903 card.The effects of different PCR enhancers,DNA polymerase,annealing temperature and final extended time on amplification were examined.The suitability of optimized reagent system was also investigated.Results With the assay established by this study,reliable,complete and high quality STR profiles were obtained from forensic database samples.Under 57~59℃ annealing temperature and 30~50min final extended times,complete STR profiles could be obtained from 1.2mm FTA bloodstains using 10μL of reaction volume of optimized reagent system containing BSA,Tween 20,DMSO,Glycerol and Typer DNA polymerase.Conclusion The developed reagent provides a accurately,quickly and efficiently approach for high-throughput identification of forensic DNA database samples.