唾液特异性mRNA荧光复合检测体系的构建  

Construction of a fluorescent detection system for salivay sepecific mRNA

作　　者：赵一霞 郝文静 李文 杨栋辉 胡胜[1] 邓锴[2] 季安全[1] 孙启凡[1] Zhao Yixia;Hao Wenjing;Li Wen;Yang Donghui;Hu Sheng;Deng Kai;Ji Anquan;Sun Qifan(Institute of Forensic Science,Ministry of Public Security&National Engineering Laboratory for Forensic Science,MPS Key Laboratory of Forensic Genetics,Beijing 100038,China;Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]公安部物证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 [2]湖北医药学院,湖北十堰442000

出　　处：《中国法医学杂志》2021年第1期41-46,共6页Chinese Journal of Forensic Medicine

基　　金：国家重点研发计划(2017YFC0803503);基本科研业务费(2018JB006,2018JB036)。

摘　　要：目的基于唾液中特异表达的mRNA标记分子,建立五重荧光复合检测系统,实现对唾液的有效鉴定。方法采集健康志愿者唾液样本,提取样本总RNA,利用终点逆转录-聚合酶链反应(Endpoint Reverse Transcription-Polymerase Chain Reaction,Endpoint RT-PCR)技术扩增3个唾液特异mRNA标记分子MUC7、HTN3、STATH和2个管家基因GAPDH、ACTB,通过毛细管电泳分离检测特异性产物,构建起五重mRNA荧光复合检测体系。同时使用100份样本和6份室温晾干1M、6M的唾液斑样本对体系的特异性、重复性和灵敏度进行验证。结果在40份唾液样本中均检测到唾液mRNA标记分子MUC7、HTN3、STATH以及管家基因GAPDH、ACTB共5个特异性的峰,而在外周血、月经血、精液和阴道分泌物样本中仅检测到管家基因GAPDH、ACTB,检测灵敏度可达2ng总RNA,且在室温晾干1M、6M的唾液斑样本中仍能检测到唾液特异性标记分子。结论本研究建立五重m RNA荧光复合扩增检测系统可以有效的将唾液与其他4种法医学常见的体液区分。Objective To efficiently and objectively assess the salivary stains,a pentaplex mRNA fluorescent system was developed that based on specific RNA biomarkers.Methods Saliva samples were collected from healthy volunteers,total RNA was extracted from the samples.An endpoint RT-PCR assay was developed that simultaneously amplifies five m RNA markers.This multiplex assay analyses three saliva markers(MUC7,HTN3,STATH)and two housekeeping genes(GAPDH and ACTB).Then 100 samples and 6 saliva stains dried at room temperature for 1 M and 6 M were used to verify the specificity,repeatability and sensitivity of the system.Results The saliva markers and housekeeping genes were detected in all saliva samples,while only housekeeping genes were detected in peripheral blood,menstrual blood,semen and vaginal secretion samples.The assay has good sensitivity as RNA profile was obtained when using>2 ng total RNA.Moreover,salivary specific markers could still be detected in saliva stain samples.Conclusion The pentaplex mRNA fluorescent system can effectively distinguish saliva from other four common forensic body fluids.

关 键 词：法医物证学 体液斑迹鉴定 mRNA分析 多重PCR复合检测体系 唾液 

分 类 号：DF795.1[医药卫生—法医学]