一种复合miRNAs检测体系的构建及在体液组织属性来源鉴定中的应用研究  

A Single-tube 10-miRNAs Assay for Identifying Five Human Body Fluids by Capillary Electrophoresis

作　　者：孙启凡[1] 马温华[1] 胡胜[1] 李冉冉 赵一霞 王乐[1] 叶健[1] 李彩霞[1] 季安全[1] SUN Qifan;MA Wenhua;HU Sheng;LI Ranran;ZHAO Yixia;WANG Le;YE Jian;LI Caixia;JI Anquan(Institute of Forensic Science,Key Laboratory of Forensic Genetics,Ministry of Public Security&National Engineering Laboratory for Forensic Science,Beijing 100038,China)

机构地区：[1]公安部物证鉴定中心法医遗传公安部重点实验室现场物证溯源技术国家工程实验室,北京100038

出　　处：《中国司法鉴定》2020年第3期28-34,共7页Chinese Journal of Forensic Sciences

基　　金：国家重点研发计划(2017YFC0803503);公安部科技强警基础工作专项(2016GABJC017);基本科研业务费课题(2016JB038)。

摘　　要：目的选取5种常见体液的10条特异性miRNAs基因,通过逆转录-终点法PCR技术构建复合扩增体系,并对体系的灵敏度进行测试,之后使用该体系检测静脉血、精液、月经血、唾液和阴道分泌液中miRNAs的表达情况。方法首先设计10条miRNAs标记的逆转录引物和cDNA扩增引物,构建10个标准miRNAs标记的荧光复合体系,并进行灵敏度测试。然后使用miRNeasy Mini Kit(QIAGEN)提取体液样本的RNA,运用荧光复合体系进行检测,并对检测结果进行分析。结果本研究得到10个标准miRNAs标记的分型结果,标准miRNAs标记的荧光复合体系的检测灵敏度为总miRNAs模板量≥1ng。静脉血样本10条miRNA检测结果:miR-451、miR-144-3p、miR-144-5p,峰值在3000~7000 rfu之间;月经血样本分型结果:miR-214、miR-451、miR-144-3p、miR-205-5p、miR-203-3p,峰值在800~7000 rfu之间;阴道分泌物样本分型结果:miR-144-3p、miR-203-3p,miR-205-5p,峰值在600~3000 rfu之间。唾液样本分型结果:miR-451、miR-144-3p、miR-205-5p、miR-203-3p,峰值在600~7000 rfu之间;精液样本分型结果:miR-888、miR-144-3p、miR-891a,峰值在1000~7000 rfu之间。结论本研究建立的该体系能够对5种法医学常见体液(静脉血、唾液、精液、月经血和阴道分泌物)进行区分鉴定,为犯罪现场体液斑痕鉴定提供了新的检测方法。Objective Ten specific miRNAs genes of 5 common body fluids were selected,and a complex amplification system was constructed by reverse transcription-endpoint PCR technology,and the sensitivity of the system was tested.Then the system was used to detect venous blood,semen,menstrual blood,saliva and the expression of miRNAs in vaginal secretions.Methods First,we design 10 miRNAs-labeled reverse transcription primers and cDNA amplification primers,construct 10 standard miRNAs-labeled fluorescent complex systems,and perform sensitivity tests.Then we use the miRNeasy Mini Kit(QIAGEN)to extract RNA from body fluid samples,use a fluorescent composite system for detection,and analyze the detection results.Results In this study,the typing results of 10 standard miRNAs were obtained.The detection sensitivity of the standard miRNAs-labeled fluorescent complex system was that the total amount of miRNAs template was≥1ng.Test results of 10 miRNAs in venous blood samples:miR-451,miR-144-3p,miR-144-5p,with a peak value between 3000 and 7000 rfu;menstrual blood sample typing results:miR-214,miR-451,miR-144-3p,miR-205-5p,miR-203-3p,the peak value is between 800 and 7000 rfu;the typing results of vaginal secretions:miR-144-3p,miR-203-3p,miR-205-5p,and the peak value is between 600 and 3000 rfu.Saliva sample typing results:miR-451,miR-144-3p,miR-205-5p,miR-203-3p with peak values between 600 and 7000 rfu;semen sample typing results:miR-888,miR-144-3p and miR-891a with peak values between 1000 and 7000 rfu.Conclusion The research established in this study can distinguish and identify five common body fluids of forensic medicine(venous blood,saliva,semen,menstrual blood and vaginal secretions),which provides a new detection method for the identification of body fluid spots on crime scenes.

关 键 词：法医遗传学 MIRNAS 体液来源鉴定 逆转录-终点法PCR 毛细管电泳 

分 类 号：DF795.4[医药卫生—法医学]