Brown Morgan E, Ottati Sara, Trivellone Valeria
Illinois Natural History Survey, Prairie Research Institute, University of Illinois Urbana-Champaign, Urbana-Champaign, IL, USA.
Department of Entomology, University of Illinois Urbana-Champaign, Urbana-Champaign, IL, USA.
J Insect Sci. 2025 May 9;25(3). doi: 10.1093/jisesa/ieaf062.
Identifying a DNA extraction method that yields high quantity and quality DNA is a crucial component of molecular ecological studies; and the best suited method can vary greatly depending on research priorities. Here, we propose a nondestructive extraction method for insect museum vouchers aimed at analyzing gut-associated microbiomes. The leafhopper Euscelidius variegatus (Kirschbaum) (Hemiptera: Cicadellidae) associated with the bacterial plant pathogen Flavescence dorée phytoplasma, a member of the genus 'Candidatus Phytoplasma' (Mollicutes: Acholeplasmataceae), was used as an experimental model. We developed and refined a resin-based DNA extraction protocol by testing the effects of prelysis bleaching and postlysis proteinase K inactivation on DNA quality and yield. We found that bleaching did not compromise the integrity of insect and associated bacterial DNA and that excluding the inactivation of proteinase K did not interfere with quantitative polymerase chain reaction analysis. Based on our findings, we recommend a DNA extraction protocol for insect voucher specimens and associated microbiomes that includes a prelysis bleaching step to chemically degrade external contaminants without proteinase K inactivation, thereby reducing processing time. Our refined protocol resulted in a high DNA yield, which we successfully analyzed using quantitative polymerase chain reaction analysis and other downstream molecular applications, including targeted high-throughput sequencing.
确定一种能提取高质量和高产量DNA的方法是分子生态学研究的关键组成部分;最适合的方法会因研究重点的不同而有很大差异。在此,我们提出一种针对昆虫博物馆标本的非破坏性提取方法,旨在分析与肠道相关的微生物群落。以与细菌性植物病原体黄萎病植原体(‘Candidatus Phytoplasma’属,软壁菌门:无胆甾原体科)相关的叶蝉Euscelidius variegatus(Kirschbaum)(半翅目:叶蝉科)作为实验模型。我们通过测试裂解前漂白和裂解后蛋白酶K灭活对DNA质量和产量的影响,开发并完善了一种基于树脂的DNA提取方案。我们发现漂白不会损害昆虫及相关细菌DNA的完整性,并且不进行蛋白酶K灭活不会干扰定量聚合酶链反应分析。基于我们的研究结果,我们推荐一种针对昆虫标本及其相关微生物群落的DNA提取方案,该方案包括一个裂解前漂白步骤,以化学方式降解外部污染物,且不进行蛋白酶K灭活,从而减少处理时间。我们完善后的方案能产生高产量的DNA,我们已使用定量聚合酶链反应分析及其他下游分子应用(包括靶向高通量测序)成功对其进行了分析。
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