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一种用于昆虫凭证标本及相关微生物群落的基于螯合树脂的无损、快速、廉价、无毒的DNA提取方案。

A non-destructive, fast, inexpensive, non-toxic chelating resin-based DNA extraction protocol for insect voucher specimens and associated microbiomes.

作者信息

Brown Morgan E, Ottati Sara, Trivellone Valeria

机构信息

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.

出版信息

J Insect Sci. 2025 May 9;25(3). doi: 10.1093/jisesa/ieaf062.


DOI:10.1093/jisesa/ieaf062
PMID:40459989
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12132034/
Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ee2/12132034/1a4d1abd900c/ieaf062_fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ee2/12132034/1a4d1abd900c/ieaf062_fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6ee2/12132034/1a4d1abd900c/ieaf062_fig1.jpg

相似文献

[1]
A non-destructive, fast, inexpensive, non-toxic chelating resin-based DNA extraction protocol for insect voucher specimens and associated microbiomes.

J Insect Sci. 2025-5-9

[2]
Interrelationships between "Candidatus Phytoplasma asteris" and its leafhopper vectors (Homoptera: Cicadellidae).

J Econ Entomol. 2007-10

[3]
Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Infect Immun. 2018-4-23

[4]
Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells.

Appl Environ Microbiol. 2018-4-2

[5]
Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

BMC Microbiol. 2015-9-30

[6]
Effect of two strains of Flavescence dorée phytoplasma on the survival and fecundity of the experimental leafhopper vector Euscelidius variegatus Kirschbaum.

J Invertebr Pathol. 2005-6

[7]
Host plant determines the phytoplasma transmission competence of Empoasca decipiens (Hemiptera: Cicadellidae).

J Econ Entomol. 2011-4

[8]
Bacteriophage-Host Association in the Phytoplasma Insect Vector .

Pathogens. 2021-5-17

[9]
Evidence suggesting interactions between immunodominant membrane protein Imp of Flavescence dorée phytoplasma and protein extracts from distantly related insect species.

J Appl Microbiol. 2019-10-8

[10]
Immunofluorescence Assay to Study Early Events of Vector Salivary Gland Colonization by Phytoplasmas.

Methods Mol Biol. 2019

本文引用的文献

[1]
Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction.

PeerJ. 2024

[2]
Simple, Robust Invertebrate DNA Barcoding: Chelex-Based DNA Extraction and Optimized COI Amplification.

Methods Mol Biol. 2024

[3]
Pathogen prospecting of museums: Reconstructing malaria epidemiology.

Proc Natl Acad Sci U S A. 2024-4-9

[4]
Testing the effectiveness of different wash protocols to remove body surface contaminants in invertebrate food web studies.

PeerJ. 2023

[5]
Comparison of Traditional and Next-Generation Approaches for Uncovering Phytoplasma Diversity, with Discovery of New Groups, Subgroups and Potential Vectors.

Biology (Basel). 2022-6-28

[6]
Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

PLoS One. 2022

[7]
A novel and non-invasive method for DNA extraction from dry bee specimens.

Sci Rep. 2022-7-8

[8]
Disentangling bias for non-destructive insect metabarcoding.

PeerJ. 2022-2-23

[9]
Contribution of sample processing to gut microbiome analysis in the model Lepidoptera, silkworm .

Comput Struct Biotechnol J. 2021-8-17

[10]
Quality Control of DNA Extracted from All-Cell Pellets After Cryopreservation for More Than 10 Years.

Biopreserv Biobank. 2022-6

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