Development of a multiplex RT-PCR assay and statistical evaluation of its use in forensic identification of vaginal fluid
Development of a multiplex RT-PCR assay and statistical evaluation of its use in forensic identification of vaginal fluid
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DOI：
10.1016/j.legalmed.2020.101715
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发表时间：
2020-07-01
期刊：
影响因子：
1.5
通讯作者：
Sakurada, Koichi
中科院分区：
文献类型：
--
作者：
Akutsu, Tomoko;Yokota, Isao;Sakurada, Koichi
从性侵犯案件样本中鉴定出阴道液，为阴道性交提供了重要的证明证据。本研究的目的是建立一个更具体的程序，以确定阴道液的法医目的。阴道液标志物候选人进行了定量评价，这些标志物（ESR1，SERPINBI3，KLK13，CYP2B7P1，MUC4）已被同时扩增的多重逆转录-聚合酶链反应（RT-PCR）程序。使用芯片电泳自动分离和定量每个扩增子。随后，在本研究中，使用各种法医相关体液详细评估了开发的多重程序的可检测性和交叉反应性。然后，通过Youden指数为每个标记物设定阴道液阳性检测的截止值。使用似然比（LR）对多重RT-PCR检测区分阴道和其他体液的能力进行了统计学评价，该似然比（LR）是使用贝叶斯估计方法估计的，以考虑检测频率的不高。当5个标记物均为阳性时，获得了较高的LR（LR = 4.33 × 10（9）; 95%可信区间，3.95 × 10（7）-2.87 × 10（12））。在各种条件下使用阴道液样品验证所开发的程序。高LR被发现为老年阴道液污渍，虽然每个扩增子峰低。它还能够识别与其他体液混合的阴道污渍。总之，多重RT-PCR为基础的程序，然后使用LR的统计评价可能是一个强大的工具，用于客观鉴定阴道液。
The identification of vaginal fluid from casework samples of sexual assaults provides important probative evidence of vaginal intercourse. The aim of this study was to establish a more specific procedure for identifying vaginal fluids for forensic purposes. Vaginal fluid marker candidates have been evaluated quantitatively and five of these markers (ESR1, SERPINBI3, KLK13, CYP2B7P1, MUC4) have been amplified simultaneously by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) procedure. Each amplicon has been separated and quantified automatically using chip electrophoresis. Subsequently, in the present study, detectability and cross-reactivity of the developed multiplex procedure were assessed in detail using various forensically relevant body fluids. Then, a cutoff value for the positive detection of vaginal fluids was set for each marker by Youden index. The ability of the multiplex RT-PCR assay to distinguish between vaginal and other body fluids was evaluated statistically using a likelihood ratio (LR) that was estimated using a Bayesian estimation approach to consider the infrequency of detection. A high LR was obtained when all five markers showed positive results (LR = 4.33 x 10(9); 95% credible interval, 3.95 x 10(7) -2.87 x 10(12)). The developed procedure was validated using vaginal fluid samples under various conditions. High LRs were found for aged vaginal fluid stains, although each amplicon peak was low. It was also able to identify vaginal stains mixed with other body fluids. In conclusion, the multiplex RT-PCR-based procedure followed by the statistical evaluation using LR could be a powerful tool for the objective identification of vaginal fluids.