Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration
Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration
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通过多重逆转录-PCR 评估和同时测定直肠粘膜标记物，作为肛门渗透性侵犯的生物学证据
DOI：
10.1016/j.fsigen.2022.102712
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Hirotaro Iwase
中科院分区：
文献类型：
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作者：
Tomoko Akutsu;Hisako Saito;Ken Watanabe;Kochi Toyomane;Takayuki Yamagishi;Hirotaro Iwase
肛门插入性侵犯与儿童性虐待或男性受害者密切相关。然而，由于用于识别直肠粘膜的程序尚未充分发展，因此难以通过使用从可疑物体表面收集的生物样本来证明这种行为。因此，对于直肠粘膜的特异性鉴定，筛选了报告在直肠中特征性表达的mRNA标记物，并开发了多重RT-PCR程序用于同时测定这些候选标记物。使用直肠粘膜样本和法医相关体液评价直肠粘膜候选标志物的可检测性和特异性。稀释或混合样本也进行了测试，以评估这一程序的法医案例工作的适用性。结果表明，采用多重RT-PCR结合毛细管电泳和片段分析技术，成功地对所选候选基因（PHGR 1、MUC 13、CLCA 1、MEP 1A、CDX 1和ZG 16）和参考基因进行了同时扩增和测定。应用临界值，其他体液均未与直肠粘膜候选标志物发生交叉反应。由于一些候选标志物的低灵敏度和可检测性可以通过它们的同时检测来补偿，因此认为所有六种候选标志物都适用于作为直肠粘膜标志物。此外，不应直接对可疑粪便样本进行检测，因为这些标记物在粪便样本中可能是阳性的。开发的多重RT-PCR程序可能不适合于微小或稀释的样品;然而，它可能对性侵犯相关体液的污染有抵抗力。总之，同时测定选定的直肠粘膜标记物与从可疑物体表面收集的生物样品可能有利于对肛门渗透性侵犯的刑事调查。
Sexual assault with anal penetration is closely related to child sexual abuse or male victims. However, it is difficult to prove such an act by using biological samples collected from the surface of a suspected object because procedures for identifying rectal mucosa have not been developed sufficiently. Therefore, for the specific identification of rectal mucosa, mRNA markers reported to be characteristically expressed in the rectum were screened and a multiplex RT-PCR procedure was developed for the simultaneous determination of those candidate markers. The detectability and specificity of rectal mucosa candidate markers were evaluated using rectal mucosa samples and forensically relevant body fluids. Diluted or mixed samples were also tested to evaluate the applicability of this procedure for forensic casework. As a result, simultaneous amplification and determination of the selected candidates (PHGR1,MUC13, CLCA1,MEP1A,CDX1, and ZG16) and reference gene were successfully performed using a multiplex RT-PCR assay combined with capillary electrophoresis and fragment analysis. Applying the cutoff values, none of the other body fluids cross-reacted with rectal mucosa candidate markers. Because the low sensitivity and detectability of some candidate markers could be compensated for by their simultaneous detection, all six candidate markers were considered to be applicable as rectal mucosa markers. Besides, the developed assay should not be performed on suspicious fecal samples directly because these markers could be positive in the fecal samples themselves. The developed multiplex RT-PCR procedure might not be suitable for minute or diluted samples; however, it might be resistant to contamination with sexual assault-related body fluids. In conclusion, the simultaneous determination of selected rectal mucosa markers with a biological sample collected from the surface of a suspected object could be beneficial for criminal investigation of sexual assault with anal penetration.