人类RNA定量的方法学建立及其法医学体液斑鉴定的应用研究
发布时间：2018-10-31 08:39
【摘要】：目的:探索建立人类特异的RNA定量体系。克服在后续RNA鉴定体液斑过程中人类总RNA初始加样量的不可控制导致的假阴性,保证实验的可重复性。方法:通过文献检索和NCBI核酸数据库序列比对,针对组织、细胞广泛高水平表达的管家基因RNA转录子(COX1),设计合成人类特异的引物和TaqMan探针,在荧光定量PCR平台上利用标准品标准曲线绝对定量原理,建立人类特异的RNA定量体系。本研究着重构建、验证了TaqMan探针法建立的DNA标准品荧光定量体系,利用Primer3.0和primer express3.0设计出人类特异的引物、探针,并检验此引物在人类和其他10种灵长类之间的特异性。采用“一人独立十次试验”和“两人分别独立实验”检验DNA标准品的稳定性。在此基础上,构建了SYBR Green I染料法的DNA标准品荧光定量体系和尝试构建RNA标准品荧光定量体系以作参考。为进一步分析DNA标准品(TaqMan)荧光定量系统效能,样本模拟了真实案件检材状况,采取陈旧的血液、唾液和加速老化处理的精液各15例,筛选国际通用的体液鉴定遗传标记基因进行qRT-PCR。通过控制RNA初始模板上样量分析COX1定量系统有效性:?50pg RNA(Ribogreen Quant-it)用于cDNA合成;?COX1定量;?加入固定拷贝数的RNA用于cDNA合成:血液样本加入105拷贝RNA,唾液样本加入30000拷贝RNA,精液样本加入1000拷贝RNA。对比分析COX1定量前后数据变化,验证此定量系统的灵敏性、稳定性和重复性。结果:COX1定量体系在人类特异性、灵敏性、稳定性上都充分展示了极好的系统性能。特异性:即便在10种灵长类动物中也保持了高度特异性。一方面可以有效地排除案件样本中细菌、真菌等非人生物RNA的干扰,一方面作为种属鉴定工具,比鲁米诺等预实验试剂更灵敏、准确,具有广泛适用性。灵敏性:当统一加入50pg RNA时,三种样本的组织特异性基因Ct值均跨越范围大,荧光定量曲线无规律分散分布。血液SPTB基因Ct值跨越34.53-42.4,相差7.87个循环;唾液HTN3基因Ct值跨越31.62-37.22,相差5.6个循环;精液PRM2基因Ct值相差13.370个循环。利用COX1标准品体系定量可知,样本所含拷贝数千差万别:15个血液样本拷贝数相差11863.243,唾液样本拷贝数相差31918.901,精液样本拷贝数相差387.741。将三组样本初始加样量统一调整至固定拷贝数,组织特异基因均发生明显变化。血液样本Ct值幅度缩减为3.9062个循环。33%的阴性样本转变为阳性;唾液样本Ct值聚集在27-28,31.5-33循环之间;精液样本Ct值缩减至7.804。以上Ct值变化可以直观的从荧光定量曲线分散程度观察到。以上数据充分证明了COX1定量系统具有极高的灵敏性。稳定性:在保存长达半年标准品的10次独立试验中,10次绝对定量标准曲线仅有微小变化,斜率、截距、R2以及效率的变异系数分别为:1.31、0.79、0.03和1.82。检验手法差异的2人独立试验中,荧光定量曲线也高度重合,利用两组样本数据制作绝对定量标准曲线时,数据点位几乎都在拟合的线性方程上,R2=0.9953。COX1标准品的稳定性确保了实验的可重复性以及数据的有效性。结论:本研究初步建立了人类特异RNA定量体系,通过RNA鉴定体液体系进行验证,初步证明了RNA定量体系具有高度特异性、灵敏性和稳定性。
[Abstract]:Objective: To explore a human-specific RNA quantitative system. overcomes the false negative caused by the uncontrollable control of the initial sample addition amount of human total RNA in the subsequent RNA identification body fluid spot process, and ensures the repeatability of the experiment. Methods: Through literature search and NCBI nucleic acid database sequence alignment, we designed and synthesized human-specific primers and TaqMan probes, using the absolute quantitative principle of standard curve on fluorescence quantitative PCR platform. Establish a human-specific RNA quantitative system. In this study, the fluorescence quantitative system of DNA standard established by TaqMan probe method was established, and human-specific primers and probes were designed by Primer3.0 and primer express 3.0. The specificity of this primer between human and other 10 primates was examined. The stability of the DNA standard was examined using 鈥淥ne person's stand-alone test鈥，
本文编号：2301544
[Abstract]:Objective: To explore a human-specific RNA quantitative system. overcomes the false negative caused by the uncontrollable control of the initial sample addition amount of human total RNA in the subsequent RNA identification body fluid spot process, and ensures the repeatability of the experiment. Methods: Through literature search and NCBI nucleic acid database sequence alignment, we designed and synthesized human-specific primers and TaqMan probes, using the absolute quantitative principle of standard curve on fluorescence quantitative PCR platform. Establish a human-specific RNA quantitative system. In this study, the fluorescence quantitative system of DNA standard established by TaqMan probe method was established, and human-specific primers and probes were designed by Primer3.0 and primer express 3.0. The specificity of this primer between human and other 10 primates was examined. The stability of the DNA standard was examined using 鈥淥ne person's stand-alone test鈥，
本文编号：2301544
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