应用反转录PCR和实时荧光定量PCR技术判定现场物证生物属性  

Determination of the Biological Attribute of Evidence at the Scene Using Reverse Transcription PCR and Real-time Fluorescent Quantitative PCR

作　　者：许炎[1] 张晨[1] 徐庆文[1] 黄江平[1] 刘亚楠[1] 邹凯南[1] 平原[1] 周怀谷[1] 

机构地区：[1]上海市公安局物证鉴定中心 法医物证学现场应用技术公安部重点实验室,上海200083

出　　处：《法医学杂志》2013年第4期259-262,共4页Journal of Forensic Medicine

基　　金：公安部科技强警基础工作专项项目(2012GABJC005)

摘　　要：目的探寻分子生物学方法判定刑事案件现场物证的生物属性的可行性。方法取30份现场生物检材,其中血液(斑)、唾液(斑)、精液(斑)各10份,分别进行常规检验法和分子生物学方法,通过DNA-RNA共提取技术,将其中的mRNA运用反转录PCR(reverse transcription PCR,RT-PCR)技术反转录成cDNA,根据3种生物检材不同的目标基因设计3对特异性引物,进行实时荧光定量PCR扩增,通过熔解曲线测定不同的熔解温度(Tm)和不同长度的扩增片段来区分生物检材。结果血液(斑)的Tm值为(84.5±0.2)℃,片段长度177 bp;唾液(斑)的Tm值为(76.9±0.3)℃,片段长度134 bp;精液(斑)的Tm值为(88.5±0.2)℃,片段长度294 bp。结论与常规检验法比较,联合应用RT-PCR和实时荧光定量PCR技术判定检材的生物属性特异性强、灵敏度高、结果稳定可靠,可应用于日常法医物证检验。Objective To explore the feasibility of biological method to identify the biological attribute of samples at crime scene.Methods Thirty samples of ten blood stains,ten saliva stains and ten semen stains were selected,and all the samples were processed by the routine method and biomolecular method,respectively.Both RNA and DNA were isolated using DNA-RNA co-extraction technology and the mRNA was converted into cDNA using reverse transcription PCR（RT-PCR）.Three pairs of specific primers were designed for blood stain,saliva stain and semen stain based on the different target genes in three specific tissues and the primers were amplified using real-time fluorescent quantitative PCR.The differences in these biological samples were evaluated by melting temperature（Tm） values and the size of the amplification fragment.Results The Tm values of blood stain,saliva stain and semen stain were（84.5±0.2） ℃,（76.9±0.3） ℃ and（88.5±0.2） ℃,respectively.The length of PCR fragments of them was 177 bp,134 bp and 294 bp,respectively.Conclusion Compared with the routine method,RT-PCR with real-time fluorescent quantitative PCR is highly specific,sensitive and reliable to identify the biological attribute of evidence,and can be potentially applied to determine evidence attribute in forensic practice.

关 键 词：法医遗传学 反转录PCR 实时荧光定量PCR 血液 唾液 精液 

分 类 号：R739.63[医药卫生—肿瘤]