Alves Cíntia, Pereira Rui, Prieto Lourdes, Aler Mercedes, Amaral Cesar R L, Arévalo Cristina, Berardi Gabriela, Di Rocco Florencia, Caputo Mariela, Carmona Cristian Hernandez, Catelli Laura, Costa Heloísa Afonso, Coufalova Pavla, Furfuro Sandra, García Óscar, Gaviria Anibal, Goios Ana, Gómez Juan José Builes, Hernández Alexis, Hernández Eva Del Carmen Betancor, Miranda Luís, Parra David, Pedrosa Susana, Porto Maria João Anjos, Rebelo Maria de Lurdes, Spirito Matteo, Torres María Del Carmen Villalobos, Amorim António, Pereira Filipe
Instituto de Investigação e Inovação em Saúde, I3S, Universidade do Porto, Portugal; Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal.
Instituto de Investigação e Inovação em Saúde, I3S, Universidade do Porto, Portugal; Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal.
Forensic Sci Int Genet. 2017 May;28:219-224. doi: 10.1016/j.fsigen.2017.03.003. Epub 2017 Mar 7.
DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.
DNA是一种可用于需要鉴定物种的法医调查的强大工具。然而，有必要开发并验证能够在法医研究中所要求的高标准下，对降解的和/或低质量DNA样本产生结果的方法。在此，我们描述了一项自愿合作实验，以测试最近开发的插入/缺失物种鉴定法（SPInDel）。SPInDel试剂盒通过生成数字图谱来鉴定物种，该图谱结合了在单一反应中扩增的六个线粒体核糖体RNA（rRNA）基因区域的长度，随后进行毛细管电泳。该实验于2014年由国际法医遗传学协会（GHEP-ISFG）西班牙语和葡萄牙语工作组的一个工作委员会组织开展，该工作组于2013年成立。来自10个国家的24个参与实验室被要求使用SPInDel引物混合物和10个目标物种的对照样本，鉴定来自之前GHEP-ISFG能力验证测试的11个DNA样本中的物种。还向参与者提供了一个计算机软件，以协助结果分析。24个实验室中的22个正确鉴定出了所有样本，包括DNA含量低的样本（毛发）以及唾液和血液的混合物。在报告的241个SPInDel图谱中的238个（98.8%）中获得了正确的物种鉴定结果。三例假分类案例由两个实验室造成。考虑到只有一个实验室未能在一个样本中检测出混合物，SPInDel在混合物中的物种鉴定方面是有效的。这一结果表明，SPInDel是一种无需DNA测序即可进行混合物分析的有效方法，其优势在于能在单一反应中鉴定出不止一个物种。错误等位基因（5.0%）和缺失等位基因（2.1%）的低频率并未干扰正确的物种鉴定，这证明了使用基于多个位点分析的方法的优势。总体而言，使用不同基因分型平台的实验室都能轻松实施SPInDel方法，结果解读简单直接，且SPInDel软件使用过程中没有任何问题。这项合作实验的结果表明，SPInDel方法可成功应用于法医案件调查。
