Oliveira Pablo Viana, de Almeida Francine Alves Nogueira, Lugon Magda Delorence, Britto Karolinni Bianchi, Oliveira-Costa Janyra, Santos Alexandre Rosa, Paneto Greiciane Gaburro
Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil.
Polícia Civil do Estado do Rio de Janeiro, Instituto Médico Legal Afrânio Peixoto, Rio de Janeiro, Rio de Janeiro, Brazil.
PeerJ. 2020 Nov 30;8:e9680. doi: 10.7717/peerj.9680. eCollection 2020.
Forensic entomology is the study of insects and other arthropods used in the solution of crimes. Most of entomological evidences strongly depend on accurate species identification. Therefore, new methods are being developed due to difficulties in morphological identification, including molecular methods such as High-Resolution Melting. In this study, we reported a new HRM primer set to identify forensically important Calliphoridae (blowflies) from Brazil. For such purpose, Calliphoridae species of forensic importance in Brazil were listed and confirmed by specialists. Mitochondrial COI sequences of those species were downloaded from databases and aligned, and polymorphic variations were selected for distinction between species. Based on it, HRM primers were designed. Forty-three fly samples representing six species were tested in the HRM assay. All samples had the COI gene sequenced to validate the result. Identifying and differentiating the six species proposed using a combination of two amplicons was possible. The protocol was effective even for old insect specimens, collected and preserved dried for more than ten years, unlike the DNA sequencing technique that failed for those samples. The HRM technique proved to be an alternative tool to DNA sequencing, with advantage of amplifying degraded samples and being fast and cheaper than the sequencing technique.
法医昆虫学是研究用于犯罪侦破的昆虫和其他节肢动物的学科。大多数昆虫学证据在很大程度上依赖于准确的物种鉴定。因此，由于形态学鉴定存在困难，新的方法正在不断开发，包括高分辨率熔解等分子方法。在本研究中，我们报告了一套新的高分辨率熔解引物组，用于鉴定巴西具有法医重要性的丽蝇科（绿头苍蝇）。为此，列出了巴西具有法医重要性的丽蝇科物种，并经专家确认。从数据库中下载并比对这些物种的线粒体细胞色素氧化酶亚基I（COI）序列，选择多态性变异以区分不同物种。在此基础上设计了高分辨率熔解引物。在高分辨率熔解分析中对代表六个物种的43只苍蝇样本进行了测试。对所有样本的COI基因进行测序以验证结果。使用两个扩增子的组合来鉴定和区分这六个物种是可行的。该方案即使对于收集并干燥保存了十多年的陈旧昆虫标本也有效，而DNA测序技术对这些样本则无法奏效。高分辨率熔解技术被证明是DNA测序的一种替代工具，具有能够扩增降解样本、速度快且比测序技术成本低的优势。
