报告题目：Point-of-Care Testing for Pathogenic Bacteria Using Functional Nucleic Acids
传统 DNA 提取技术需要好几个小时，最近，北卡罗莱纳州立大学（North Carolina State University）的研究人员开发了一种新技术，能在一分钟内从植物组织中提取收集 DNA，相关论文于 6 月 9 日发表在《美国化学会·纳米》上。DNA 提取是鉴定植物病害的第一步，新方法使用了微针贴片（microneedle patch），有助于开发小型化、可以用于现场即时检测的植物病害诊断工具。
Abstract: We have interested in developing point-of-care testing that use two classes of synthetic DNA molecules: DNA aptamers (DNA-based molecular receptors) and DNAzymes (catalytic DNAs). We have developed DNAzymes that are capable of tracking E. coli (a common water/food-borne pathogen) and aptamer that can recognize C. difficle (a common cause for healthcare-associated infections in North America and Europe). Furthermore, we have been interested in engineering molecular devices in which a key player is a DNA aptamer or ligand-responsive DNAzymes to achieve ultra-sensitive bioanalytical applications. For example, we have developed several biosensing DNA devices that are built with various aptamer/DNAzyme-containing complexes and can be activated by a biomarker or a bacterial pathogen.
刘猛，大连理工大学教授，博士生导师，入选中组部第十三批千人计划。博士毕业于大连理工大学环境工程专业，2013年至2016年在McMaster大学生物化学与生物医学系和生物界面研究所从事博士后研究，研究领域包括功能核酸、生物传感和纸基微流控，在Accounts of Chemical Research, Nature Communications, Angewandte Chemie International Edition, ACS Nano等期刊发表SCI论文30余篇，申请美国专利7项，多篇论文被CBC News, Science News, TheStar, Science Daily, Newswise等科技媒体和网站予以亮点报道。
"快速检测的障碍之一是从植物样品中提取高质量的 DNA 需要耗费较长时间，我们的技术则为这一问题提供了一个快速、简单的解决方案。"魏青山说。
"一些植物病害有类似的症状。如导致历史上着名的爱尔兰饥荒的马铃薯晚疫病，和其姊妹物种烟草疫霉（Phytophthora nicotianae）引起的疫病症状就非常相似，"北卡罗莱纳州立大学植物病理学系教授、论文的共同通讯作者琼·里斯塔伊诺（Jean Ristaino）说，"DNA 分子检测是目前鉴定植物疾病的标准方法。我们的新技术非常重要，因为在从样品中提取 DNA 之前，你无法对马铃薯晚疫病或任何其他植物病害菌株进行扩增或基因测定。”
实验室通常使用十六烷基三甲基溴化铵（cetyltrimethylammonium Ammonium Bromide, CTAB）法从植物样品中提取 DNA，该方法必须在实验室中完成，需要大量设备，并且至少要耗费 3～4 小时。CTAB提取是一个多步骤过程，包括从植物组织研磨到有机溶剂萃取和离心机纯化的整个流程。
图片来源| DOI: 10.1021/acsnano.9b00193
相比之下，新的 DNA 提取技术只涉及微针贴片和水性缓冲溶液。微针贴片仅邮票大小，由一种低廉的聚合物制成，贴片一侧的表面由数百个 0.8 毫米长的微针组成。
农民或研究人员可以将微针贴片应用于他们怀疑产生病害的植物。应用时，只要将贴片按压到植物叶面可疑区域几秒钟，然后将其剥离；接下来用缓冲溶液冲洗贴片，将微针头上吸附的 DNA 遗传物质收集入无菌容器中。整个过程大约只需要一分钟。
加州大学洛杉矶分校（University of California, Los Angeles）生物工程学教授、该论文的共同通讯作者顾臻说："很高兴看到微针贴片技术在农业和植物科学领域的新应用。”
"在实验测试中，我们发现与传统的 CTAB 提取相比，微针技术提取的样品杂质含量略高，"魏青山说，"然而，微针技术的纯度水平与实验室其他常用的 DNA 提取方法相当。最重要的是，我们发现微针技术和 CTAB 提取之间的轻微纯度差异并没有干扰后续 DNA 放大检测的准确度。”
"事实上，微针提取的采样体积偏小似乎不是问题，"北卡罗莱纳州立大学化学和生物分子工程系的博士生拉杰什·保罗（Rajesh Paul）说，"在最近的一项盲测中，微针技术成功地从所有野外采集的受感染番茄叶片中提取了病原体 DNA。”保罗是也是该论文的第一作者。
New Microneedle Technique Speeds Plant Disease Detection
Researchers have developed a new technique that uses microneedle patches to collect DNA from plant tissues in one minute, rather than the hours needed for conventional techniques. DNA extraction is the first step in identifying plant diseases, and the new method holds promise for the development of on-site plant disease detection tools.
“When farmers detect a possible plant disease in the field, such as potato late blight, they want to know what it is right away; rapid detection can be important for addressing plant diseases that spread quickly,” says Qingshan Wei, an assistant professor of chemical and biomolecular engineering at North Carolina State University and co-corresponding author of a paper on the work.
“One of the obstacles to rapid detection is the amount of time it takes to extract DNA from a plant sample, and our technique provides a fast, simple solution to that problem,” Wei says.
“Some plant diseases have similar leaf symptoms, such as late blight caused by the famed Irish famine pathogen Phytophthora infestans, and Phytophthora blight caused by a sister species P. nicotianae,” says Jean Ristaino, William Neal Reynolds Distinguished Professor of Plant Pathology at NC State and co-corresponding author of the paper. “The gold standard for disease identification is a molecular assay. Our new technique is important because you can’t run an amplification or genotyping assay on strains of P. infestans, or any other plant disease, until you’ve extracted DNA from the sample.”
Typically, DNA is extracted from a plant sample using a method called CTAB extraction, which has to be done in a lab, requires a lot of equipment, and takes at least 3 to 4 hours. CTAB extraction is a multi-step process involving everything from tissue grinding to organic solvents and centrifuges.
By contrast, the new DNA extraction technique involves only a microneedle patch and an aqueous buffer solution. The patch is about the size of a postage stamp and is made of an inexpensive polymer. The surface on one side of the patch is made up of hundreds of needles that are only 0.8 millimeters long.
A farmer or researcher can apply the microneedle patch to a plant they suspect is diseased, hold the patch in place for a few seconds, then peel it off. The patch is then rinsed with the buffer solution, washing genetic material off of the microneedles and into a sterile container. The entire process takes about a minute.
“It is exciting to see the new application of microneedle patch technology in agriculture and plant science,” says Zhen Gu, a professor of bioengineering at the University of California, Los Angeles and co-corresponding author of the paper, who developed several microneedle-based drug delivery systems for human health.
“In experimental testing, we found that the microneedle technique does result in slightly higher levels of impurities in the sample, as compared to CTAB,” Wei says. “However, the microneedle technique’s purity levels were comparable to other, validated laboratory methods of DNA extraction. Most importantly, we found that the slight difference in purity levels between the microneedle and CTAB samples did not interfere with the ability to accurately test the samples by a PCR or LAMP assay.”
“The fact that microneedles extract a smaller sampling volume seems not to be an issue,” says Rajesh Paul, a Ph.D. student at NC State and first author of the paper. “The microneedle technique successfully extracted pathogen DNA from all field-collected infected tomato leaves in a recent blind test.”
“DNA extraction has been a significant hurdle to the development of on-site testing tools,” Wei says. “We are now moving forward with the goal of creating an integrated, low-cost, field-portable device that can perform every step of the process from taking the sample to identifying the pathogen and reporting the results of an assay.”
The paper, “Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases,” is published in the journal ACS Nano. The paper was co-authored by Amanda Saville, lab manager in Ristaino’s lab; Jeana Hansel, a graduate student at NC State; Carmin Ball and Alyssa Williams, undergraduates at NC State; Yanqi Ye, a former graduate student in the Joint Biomedical Engineering Department at NC State and the University of North Carolina, Chapel Hill; Xinyuan Chang, an undergraduate at Tianjin University who worked as a visiting researcher at NC State; and Guojun Chen, postdoctoral researcher at UCLA.
The work was done with support from the NC State Chancellor’s Faculty Excellence Program on Emerging Plant Disease and Global Food Security, the Kenan Institute for Engineering, Technology & Science at NC State, and the USDA Integrated Pest Information Platform for Extension and Education, under grant number 2015-0097.