基于功能化磁珠快速提取植物病毒RNA方法的建立与优化

植物病毒病是制约作物生产的主要病害之一。及时明确其病毒病原和发展规律是有效控制其大规模传播的前提。而现有植物病毒病检测技术存在周期长、步骤繁琐、检测环境严苛等缺点。本研究以烟草花叶病毒 (Tobacco mosaic virus，TMV)为模型，基于碱基互补配对原则设计针对TMV的功能化磁珠（CMBs-ACPTMV）进行RNA提取，并对功能化磁珠的制备条件、提取反应条件以及灵敏性和稳定性等性能进行优化分析。结果表明，当添加4 μmol捕获探针（ACPTMV）、0.08 mg羧基磁珠（CMBs）时，所制备的CMBs-ACPTMV吸附RNA的能力最好；当提取时间为3 min时，CMBs-ACPTMV提取RNA的效果最好，而改变CMBs-ACPTMV的提取温度时其提取能力无明显变化；性能评价分析发现，CMBs-ACPTMV的灵敏度可达2.5 ng/μL，且检测稳定性较好。与常规RNA提取技术相比，CMBs-ACPTMV在检测时间和样品消耗量上具有突出优势。本研究所建立的功能化磁珠提取法快速、安全和简便，只需简易设备便可实现植物病毒RNA的快速提取，具有广阔的应用前景。

Establishment and Optimization of a Method for Rapid Extraction of Plant Virus RNA Based on Functional Magnetic Beads

Plant virus diseases are one of the major diseases restricting crop production. Timely identification of their pathogen and development rules is the prerequisite for effective control of their large-scale spread. However, long cycle, tedious steps and strict detection environment were the disadvantages existing in the detection technology of plant viru- disease. In this study, Tobacco Mosaic virus (TMV) was used as a model to be extract RNA based on CMBs-ACPTMV, which was design based on the principle of complementary base pairing. Meanwhile, the experimental conditions were optimized and analyzed, including the preparation conditions of functionalized magnetic beads, the reaction conditions during extraction, and the sensitivity, stability and other properties of the method. The results showed the ability to capture RNA of CMBs-ACPTMV were best when prepared with 4 μmol capture probe (ACPTMV) and 0.08 mg carboxyl magnetic beads (CMBs); After 3 min of extraction, CMBs-ACPTMV has the best RNA extraction effect, but when the extraction temperature of CMBs-ACPTMV was changed, its extraction capacity showed no significant change; In the comprehensive performance evaluation, the sensitivity of CMBs-ACPTMV can reach 2.5 ng/μL, and the detection stability is good. Compared with conventional RNA extraction technology, CMBs-ACPTMV has outstanding advantages in detection time and sample consumption. The functional magnetic beads extraction method established in this study is fast, safe and simple. It can achieve rapid extraction of plant virus RNA with simple equipment, which has a broad application prospect.