DNA Extraction from Plant Leaves Using a Microneedle Patch

Q1 Agricultural and Biological Sciences Current protocols in plant biology Pub Date : 2020-02-19 DOI:10.1002/cppb.20104

Rajesh Paul, Emily Ostermann, Zhen Gu, Jean B. Ristaino, Qingshan Wei

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引用次数: 16

Abstract

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc.

Basic Protocol: Microneedle patch-based DNA extraction

Support Protocol 1: Microneedle patch fabrication

Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA

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用微针贴片提取植物叶片DNA

从植物感染标本中分离高质量DNA是植物病原分子检测的重要步骤。然而，从被刚性多糖细胞壁包围的植物细胞中分离DNA涉及复杂的步骤，需要台式实验室设备。因此，植物DNA提取目前仅限于设备齐全的实验室，样品制备已成为植物病原体现场分子检测的主要障碍之一。为了克服这一障碍，一种从植物叶片组织中提取DNA的简单方法被开发出来。聚乙烯醇(PVA)制成的微针(MN)贴片可以在一分钟内从不同植物物种中分离出植物或致病DNA。在DNA提取过程中，聚合物MN贴片渗透到植物叶片组织中，打破坚硬的植物细胞壁，分离细胞内DNA。提取的DNA是聚合酶链反应(PCR)扩增，无需额外的纯化。该方法成功地从侵染番茄叶片中提取了疫霉DNA。此外，MN片段还可用于田间直接从其他植物病原体中分离DNA。因此，它有很大的潜力成为一种快速，现场样品制备技术的植物病原体检测。©2020 by John Wiley &基本方案:基于微针贴片的DNA提取支持方案1:微针贴片制作支持方案2:微针贴片提取DNA的实时PCR扩增

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.