Paul Rajesh, Ostermann Emily, Gu Zhen, Ristaino Jean B, Wei Qingshan
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina.
Bioengineering Department, University of California, Los Angeles, Los Angeles, California.
Curr Protoc Plant Biol. 2020 Mar;5(1):e20104. doi: 10.1002/cppb.20104.
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.
从受感染的植物标本中分离高质量的DNA是植物病原体分子检测的关键步骤。然而，从被坚硬的多糖细胞壁包围的植物细胞中分离DNA涉及复杂的步骤，并且需要台式实验室设备。因此，植物DNA提取目前仅限于设备完善的实验室，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。为克服这一障碍，已开发出一种从植物叶片组织中提取DNA的简单方法。由聚乙烯醇（PVA）制成的微针（MN）贴片可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贴片穿透植物叶片组织，打破坚硬的植物细胞壁以分离细胞内DNA。提取的DNA无需额外纯化即可进行聚合酶链反应（PCR）扩增。这种微创方法已成功从受感染的番茄叶片中提取出致病疫霉DNA。此外，MN贴片可直接用于在田间从其他植物病原体中分离DNA。因此，它极有可能成为一种用于植物病原体检测的快速现场样品制备技术。©2020约翰威立国际出版公司。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
