Federal Criminal Police Office, 65173 Wiesbaden, Germany.
Federal Criminal Police Office, 65173 Wiesbaden, Germany.
Forensic Sci Int Genet. 2014 Sep;12:185-91. doi: 10.1016/j.fsigen.2014.06.006. Epub 2014 Jun 17.
A reliable method to provide molecular biology products free of contaminating DNA is of forensic interest. Ethylene oxide (EO) treatment has been demonstrated as an effective method in published studies. This study aimed to address some additional experiments that are closer to forensic practice. In the first part of this study, different consumables such as cotton swabs, latex gloves and micro test tubes were spiked with saliva, blood and skin cells to mimic a real-life contamination scenario. EO treatment was performed for a period of 3, 5, 7, and 10h, respectively. For comparison, gamma and electron beam treatment was applied. In the second part of this study, a cell culture line (K562) was used to apply defined cell counts on cotton swabs followed by EO treatment for 3 and 5h. After extraction of samples, the DNA content was quantified using a real-time PCR based system. STR analysis was performed using a latest generation STR kit to meet current sensitivity limits. A good correlation of real-time PCR results and STR results was observed. This work confirmed the findings of earlier studies showing that chemical EO treatment is much more successful in reducing the amount of PCR-amplifiable DNA than ionising radiation. Furthermore, the efficacy of EO treatment is affected by the nature of the samples. DNA in saliva was more susceptible to damage by EO gas than DNA in blood. Our results show, that accessibility of the sample to EO gas has a strong influence on the method's efficiency. While treatment of samples on cotton swabs packed into gas-permeable bags was very successful, samples inside a closed micro test tube were resistant to the same treatment conditions. Our work with defined K562 cell numbers and multi-copy quantitative PCR could show that a 5h EO treatment results in a 10(5) fold reduction of PCR-amplifiable DNA. Corresponding STR-PCR results also show only sporadic allele calls in the Mini-loci range, providing a reliable interpretation of forensic analysis. Finally, we do recommend an EO treatment of forensic consumables and a multi-copy quantitative PCR approach to establish reliable treatment conditions.
提供无污染 DNA 的分子生物学产品的可靠方法是法医学感兴趣的问题。环氧乙烷 (EO) 处理已在已发表的研究中证明是一种有效的方法。本研究旨在解决更接近法医学实践的一些额外实验。在本研究的第一部分中，不同的消耗品，如棉签、乳胶手套和微量离心管，分别用唾液、血液和皮肤细胞污染，以模拟现实生活中的污染情况。分别进行了 3、5、7 和 10 小时的 EO 处理。为了比较，还应用了γ射线和电子束处理。在本研究的第二部分中，使用细胞培养系 (K562) 将定义的细胞计数应用于棉签上，然后进行 3 和 5 小时的 EO 处理。提取样品后，使用基于实时 PCR 的系统定量 DNA 含量。使用最新一代 STR 试剂盒进行 STR 分析，以满足当前的灵敏度限制。实时 PCR 结果和 STR 结果之间存在良好的相关性。这项工作证实了早期研究的发现，即化学 EO 处理在减少可 PCR 扩增 DNA 的量方面比电离辐射更成功。此外，EO 处理的效果受样品性质的影响。唾液中的 DNA 比血液中的 DNA 更容易受到 EO 气体的损伤。我们的结果表明，样品对 EO 气体的可及性对方法的效率有很大影响。虽然在透气袋中包装的棉签上进行的样品处理非常成功，但在密封的微量离心管中的样品对相同的处理条件具有抗性。我们使用定义的 K562 细胞数量和多拷贝定量 PCR 可以表明，5 小时的 EO 处理导致可 PCR 扩增 DNA 减少 105 倍。相应的 STR-PCR 结果也仅显示 Mini-loci 范围内的零星等位基因调用，为法医分析提供可靠的解释。最后，我们确实建议对法医消耗品进行 EO 处理并采用多拷贝定量 PCR 方法来建立可靠的处理条件。
