Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system
Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system
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DOI：
10.1007/s12024-016-9763-3
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发表时间：
2016-06-01
影响因子：
1.8
通讯作者：
Hsieh, Hsing-Mei
中科院分区：
文献类型：
--
作者：
Lin, Yu-Chih;Tsai, Li-Chin;Hsieh, Hsing-Mei
了解复杂体液混合物的组成可能对法医调查有很大帮助。然而，许多传统的测试本质上是推定的，可能会导致模糊的结果。本研究的目的是通过分析不同生物体液的DNA甲基化图谱，建立一种可靠的方法来鉴定各种生物体液。本研究从Infinium Human Megylation450 BeadChip的开放数据库中分离了唾液、静脉血、阴道液和精液的8个生物体液特异性甲基化标志物。将这些生物体液特异性标记物、确认亚硫酸氢盐转化的对照标记物和性别标记物结合到一个10链甲基化特异性的聚合酶链式反应单碱基延伸(MSP-SBE)体系中。对从静脉血、精液、阴道液、唾液和经血中分离的65个DNA样本进行分析，结果显示所有8个标记物都能检测到它们设计的体液。明确的体液识别既来自单一的体液来源，也来自复杂的混合物。为每个标记设计了一个阈值，以最大限度地减少错误纳入的机会。使用来自真实指控性侵犯案件的五个非证明性样本证明了该分析和应用于法医实践的有效性。结果表明，本研究建立的10-plex MSP-SBE系统用于体液鉴定具有较高的灵敏度和特异性，可应用于法医实践。
Knowledge of the composition of complex body fluid mixtures may aid forensic investigations greatly. However, many of the traditional tests are presumptive in nature and can lead to ambiguous results. The aim of this study is to establish a reliable method to identify various biofluids via analysis of their DNA methylation profiles.A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids, and semen were isolated from the open database of Infinium HumanMethylation450 BeadChip. These biofluid-specific markers, a control marker to confirm bisulfite conversion, and a gender marker, were combined into a 10-plex methylation-specific PCR single-base-extension (MSP-SBE) system.Analysis of 65 DNA samples isolated from venous blood, semen, vaginal fluid, saliva, and menstrual blood that had been treated with bisulfite, resulted in all eight markers detecting the body fluid to which they were designed. Unambiguous body fluid identification occurred from both single sources of body fluids and complex mixtures. A threshold was devised for each marker to minimize the chance of a false inclusion. The efficacy of the assay and application to forensic practice was demonstrated using five non-probative samples from real alleged sexual assault cases. The system unambiguously determined the biofluid types for the non-probative forensic samples that previously resulted in inconclusive or conflicting results using traditional tests.The results demonstrated the 10-plex MSP-SBE system established in this study is both sensitive and specific when applied to body fluid identification and can be readily adopted into forensic practice.