Hanson Erin K, Ballantyne Jack
National Center for Forensic Science, Orlando, FL 32816-2367, USA.
National Center for Forensic Science, Orlando, FL 32816-2367, USA ; Department of Chemistry, University of Central Florida, Orlando, FL 32816-2366, USA.
F1000Res. 2013 Dec 20;2:281. doi: 10.12688/f1000research.2-281.v2. eCollection 2013.
Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen). The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence.
确定证据物品上生物材料的性质对于了解犯罪相关情况可能至关重要。然而，传统的基于蛋白质的方法无法鉴定所有体液和组织，因此需要开发基于分子的策略来最终鉴定所有法医相关的生物体液和组织。信使核糖核酸（mRNA）分析就是这样一种基于分子的方法。目前的mRNA体液鉴定分析涉及毛细管电泳（CE）或定量逆转录聚合酶链反应（qRT-PCR）平台，每个平台都有其自身的局限性。这两个平台都需要使用昂贵的荧光标记引物或探针。基于CE的分析需要单独的扩增和检测步骤，从而增加了分析时间。对于qRT-PCR分析，由于每个标记需要不同的荧光染料，因此单个反应中只能包含3-4个标记。为了简化mRNA分析方法，并减少分析时间和成本，我们开发了用于鉴定常见法医相关生物体液和组织的单重和多重体液高分辨率熔解（HRM）分析方法。纳入的生物标志物包括白细胞介素19（阴道分泌物）、白细胞介素1F7（皮肤）、δ-氨基-γ-酮戊酸合成酶2（血液）、基质金属蛋白酶10（经血）、组胺酶3（唾液）和转谷氨酰胺酶4（精液）。HRM分析只需要未标记的PCR引物和一种单一的饱和嵌入荧光染料（Eva Green）。每个体液特异性标志物都可以通过独特的熔解峰轻松识别。通常，HRM分析用于检测单个基因靶点的变体或亚型。然而，我们独特地开发了双重和三重HRM分析方法，以允许在每个反应中同时检测多个靶点。在此，我们描述了所开发的HRM分析方法的开发过程和初步性能评估。结果表明，HRM分析方法在快速且相对廉价地筛查生物证据方面具有潜在用途。
