三类斑迹多重荧光扩增体系的构建及应用研究  

Research on The Construction and Application of Multiple Fluorescence Amplification System for Three Kinds of Stains

作　　者：白一帆 赵禾苗 陈静 刘洪迪 杨瑞琴[1] 王冲 BAI Yi-Fan;ZHAO He-Miao;CHEN Jing;LIU Hong-Di;YANG Rui-Qin;WANG Chong(School of Criminal Investigation,People’s Public Security University of China,Beijing 100038,China;Key Laboratory of Forensic Biological Evidence,Institute of Forensic Science,Ministry of Public Security,Beijing 100038,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,法医遗传学公安部重点实验室,北京100038

出　　处：《生物化学与生物物理进展》2025年第4期982-994,共13页Progress In Biochemistry and Biophysics

基　　金：中央级公益性科研院所基本科研业务费(2023JB005)资助项目。

摘　　要：目的利用体液斑迹的组织特异性RNA标记,构建一套基于毛细管电泳平台,可同时检测唾液-精液-阴道分泌物特异性标记的多重复合扩增体系,旨在用于对犯罪现场发现的可疑体液斑迹进行组织来源鉴定。方法收集唾液、精液、阴道分泌物样本,通过引物筛选、体系调配以及PCR条件优化,构建出一套多重荧光复合扩增体系,并对该体系的特异度、灵敏度以及对混合样本的检出能力进行探究。结果本复合体系对上述3类体液均表现出较高的特异性,每类体液标记物与其他两类间无交叉反应,对3类体液的最低检测阈值分别达到了0.0029、0.0015和0.42 mg/L。同时,本体系对混合样本的检验也有较高的区分度,能够应用于实际样本。结论该复合扩增检测体系可实现唾液、精液与阴道分泌物的正确筛检,灵敏度满足实际工作需求,操作简便,为办案实践提供了一种体液斑迹的分析筛选方法。Objective A multiplex amplification system was constructed based on the capillary electrophoresis platform for simultaneous detection of saliva,semen,and vaginal secretions using tissue-specific RNA markers.The aim of this study is to identify the tissue origin of suspicious body fluid stains found at crime scenes and determine whether the body fluid stains at the crime scene are one or several types among saliva,semen,and vaginal secretions.Methods Thirty saliva samples,forty semen samples,and forty vaginal secretion samples(half from 2015 and half from 2024)were collected from healthy adult volunteers.Through primer designing,system formulation,and PCR condition optimization,a multiplex fluorescent amplification system was constructed.The specificity,sensitivity,and detection ability for mixed samples of this system were investigated,and it was tested using real crime scene materials.In the primer design stage,to reduce the requirements for RNA template quality,the amplification products were set within 80-300 bp.In the system formulation stage,dominant and subordinate primers were mainly considered.By reducing the concentration of dominant primers and increasing that of subordinate primers,a capillary electrophoresis spectrum with an appropriate peak height ratio was finally obtained.Additionally,gradient experiments were designed to adjust the concentrations of PCR reagents and PCR amplification conditions,and multiple versions of DNA amplification enzymes were optimized to achieve the best experimental results.Results Through statistical analysis,there was no significant difference in the capillary electrophoresis of the 3 types of body fluid samples from the two years(2015 and 2024),demonstrating that the sample preservation method in this study can preserve samples for a relatively long time.The composite amplification system constructed in this study exhibited high specificity for all 3 types of body fluid,with no cross-reactions between the markers of each type of body fluid.The minimum detection thresho

关 键 词：法医物证学 体液斑迹 MRNA 

分 类 号：D919.4[医药卫生—法医学]