miR-451a和miR-21-5p双重实时荧光定量PCR检测体系的建立及应用  

Establishment and Application of a Duplex Real Time Fluorogenic Quantitative PCR Assay System for miR-451a and miR-21-5p

作　　者：胡舒啸 陈惠香 胡胜 赵一霞 季安全 李洋 廉洁[1] 孙启凡 HU Shu-Xiao;CHEN Hui-Xiang;HU Sheng;ZHAO Yi-Xia;JI An-Quan;LI Yang;LIAN Jie;SUN Qi-Fan(School of Investigation,People’s Public Security University of China,Beijing 100038,China;MPS’s Key Laboratory of Forensic Genetics,National Engineering Laboratory for Crime Scene Evidence Investigation and Examination,Institute of Forensic Science,Ministry of Public Security(MPS),Beijing 100038,China;School of Forensic Medicine,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]中国人民公安大学侦查学院,北京100038 [2]公安部鉴定中心,现场物证溯源技术国家工程实验室,法医遗传学公安部重点实验室,北京100038 [3]山西医科大学法医学院,太原030001

出　　处：《生物化学与生物物理进展》2024年第3期706-715,共10页Progress In Biochemistry and Biophysics

基　　金：国家重点研发计划(2022YFC3341002);公安部科技强警基础工作专项(2022JC11)资助项目。

摘　　要：目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。Objective Body fluid stains left at crime scenes are frequently trace amounts,while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample,as multiple miRNA markers are the basis for the identification.Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples,a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously,and the system should be further verified by actual sample for the body fluid identification.Methods The duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes,and the concentrations of the primers and probes were both optimized.The specificity,sensitivity and reproducibility of the system were validated,while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio.Results The optimized assay system exhibited excellent specificity and repeatability,with coefficients of variation consistently below 8%for both intra-and inter-batch variability.The amplification efficiency of miR-451a and miR-21-5p reached 71.77%and 74.81%,respectively,with high and relatively consistent results.By utilizing this duplex real time fluorogenic quantitative PCR assay system,a total of 58 body fluid samples were analyzed,exhibiting a discrimination rate of 100%between blood and non-blood samples,as well as between peripheral blood and menstrual blood samples.Moreover,the results,obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system,showed no statistically significant difference with randomly selected blood samples(n=20).Compared to previous single real time fluorogenic quantitative PCR assay system,the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement.A minimum i

关 键 词：法医遗传学 体液鉴别 双重实时荧光定量PCR MICRORNA 

分 类 号：DF795.2[医药卫生—法医学]