Quantitative evaluation of candidate genes and development of a multiplex RT-PCR assay for the forensic identification of vaginal fluid
Quantitative evaluation of candidate genes and development of a multiplex RT-PCR assay for the forensic identification of vaginal fluid
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DOI：
10.1016/j.fsigss.2017.09.078
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发表时间：
2017-12-01
期刊：
影响因子：
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通讯作者：
Sakurada, Koichi
中科院分区：
文献类型：
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作者：
Akutsu, Tomoko;Watanabe, Ken;Sakurada, Koichi
从含有精液的混合样本中鉴定出阴道液为性侵犯中的阴道性交提供了重要的证明证据。在基于mRNA的体液鉴定研究中，阴道特征基因，如MUC 4和HBD 1，已被用于鉴定阴道液;然而，由于特异性和可检测性不完全，这些不足以区分阴道液和其他体液。本研究的目的是开发一个更具体的程序来识别阴道液。首先，使用实时逆转录-聚合酶链反应（RT-PCR）对先前报道的和新选择的候选基因进行定量评价。然后，我们开发了一种多重RT-PCR方法，同时检测可能的候选人。通过芯片电泳检测和定量每个扩增子。此外，我们使用各种体液和老化阴道液污渍检查了开发的多重RT-PCR检测的特异性和稳健性。作为这种实时RT-PCR检测的结果，我们选择了五个候选基因MUC 4，CYP 2B 7 P1，KLK 13，ESR 1和SERPINB 13-根据其特异性和敏感性。成功地实现了这些基因的同时扩增，并通过芯片电泳实现了每个片段的自动分离和定量。阴道特征基因多重检测的特异性和可检测性与实时RT-PCR几乎相当，可应用于室温保存1.5年的阴道液样本。尽管鉴别标准有待确定，但本研究建立的多重RT-PCR方法可作为鉴别阴道分泌物的有效工具。
The identification of vaginal fluid from a mixed sample containing semen provides important probative evidence of vaginal intercourse in sexual assaults. In studies on the mRNA-based identification of body fluids, vaginal characteristic genes, such as MUC4 and HBD1, have been used to identify vaginal fluid; however, these are insufficient to discriminate vaginal fluid from other body fluids because of incomplete specificity and detectability. The aim of this study was to develop a more specific procedure for identifying vaginal fluid. First, previously reported and newly selected candidate genes were evaluated quantitatively using real-time reverse transcription-polymerase chain reaction (RT-PCR). Then, we developed a multiplex RT-PCR assay to detect probable candidates simultaneously. Each amplicon was detected and quantified by chip electrophoresis. Furthermore, we examined the specificity and robustness of the developed multiplex RT-PCR assay using various body fluids and aged vaginal fluid stains. As a result of this real-time RT-PCR assay, we selected five candidate genes-MUC4, CYP2B7P1, KLK13, ESR1, and SERPINB13-on the basis of their specificity and sensitivity. Then, the simultaneous amplification of these genes was performed successfully, and each fragment could be separated and quantified automatically by chip electrophoresis. The specificity and detectability of the multiplex detection of vaginal characteristic genes were almost comparable to those of real-time RT-PCR and it could be applied to vaginal fluid samples stored at room temperature for 1.5 years. Although discrimination criteria should be set, the multiplex RT-PCR assay developed in this study could be an effective tool for the identification of vaginal fluid.