快速PCR方法在法医DNA检验中的研究进展

摘要

目前法医DNA检验应用的短串联重复序列(short tandem repeat,STR)复合扩增系统均依赖于荧光标记的多重PCR方法.常规DNA检验流程包括提取、定量、扩增、分离和检测,而多重PCR扩增常常是整个过程中的限速步骤,完成28～30个循环大约需要3h.以往常利用商业化的热循环仪和热稳定DNA聚合酶,但需要很长的PCR循环时间才能够确保100～400 bp的目标片段得到有效且均衡的复合扩增.随着各种缩短扩增时间方法的出现,STR复合扩增不再需要3h,而是可以减少到几十分钟甚至十几分钟.现主要总结了快速PCR、直接PCR、芯片PCR以及其他快速扩增等方法的研究现状与进展,尤其是在法医DNA检验领域,同时对比了几种常见快速PCR扩增方法,可见缩短扩增时间对DNA检验的意义重大.未来,以芯片为主导、快速检验为目的的全自动、便携式、集成化的DNA分析系统,将使得法医DNA检验从实验室走进案件现场,甚至日常生活,实现真正的快速即时检验.%To date,multiplex PCR with fluorescently labeled primers has been used as an essential method for the amplification of short tandem repeats (STRs) in forensic DNA testing:nevertheless,the multiplex PCR is commonly identified as the bottleneck in the process of traditional STR workflow,which includes extraction,quantitation,amplification,separation and detection.It often takes up to three hours to complete 28～30 cycles of PCR.In order to ensure that all of the targets (typically 100～400 bp) can be effectively amplified in a balanced manner,long PCR cycling times are required by the use of thermal cyclers and heat stable polymerases.However,with the advent of modified polymerases and faster thermal cyclers,the time required for the PCR is no longer on the order of three hours,but as little as a few minutes.The application of rapid PCR,direct PCR,rapid PCR on chip and additional rapid PCR protocols in the field of DNA rapid testing,especially the progress of forensic DNA rapid detection are summarized here.Besides,it also summarizes the comparison of rapid PCR protocols for STR typing.So far,shortening the amplification time is the mainstream direction of forensic DNA technology research.In the future,the microfluidic chip-based fully automatic,portable and integrated DNA analysis system will extend the forensic DNA testing from the laboratory to crime scene,even to the daily life,and achieve a rapid real-time detection.