目录
摘要Ⅱ
关键词Ⅱ
AbstractⅢ
引言
引言1
1材料与方法2
1.1试验材料 2
1.2.1培养供试菌株2
1.2.1准备供试植物3
1.2试验方法 3
1.2.1提取菌丝基因组DNA并检测浓度3
1.2.2设计恶疫霉LFRPA的引物和探针3
1.2.3建立和优化恶疫霉LFRPA体系4
1.2.4检测恶疫霉LFRPA的特异性和灵敏度4
1.2.5检测恶疫霉接种样品4
2结果与分析5
2.1特异性引物的设计和筛选5
2.2 LFRPA的特异性评估8
2.3恶疫霉LFRPA的灵敏度检测9
2.4扩增温度和反应时间的评估10
2.5恶疫霉侵染样品的LFRPA检测11
3讨论与结论 13
致谢14
参考文献15
基于重组酶聚合酶扩增技术的恶疫霉快速检测方法的建立
摘 要
恶疫霉（Phytophthora cactorum）寄主范围广泛，是引起我国梨、苹果、草莓、三七和人参等植物疫病的一种主要病原菌，一旦植物被侵染后，发病较快，并造成严重的经济损失。本研究的目的是建立一种准确的、快速的、便捷的和可视化的恶疫霉检测方法，并将其应用于恶疫霉的现场检测。该方法以Ypt1基因作为靶标设计特异性引物和探针，基于重组酶聚合酶扩增（Recombinase Polymerase Amplification，RPA）技术，并且结合测流层析（Lateral Flow，LF）技术和简易核酸提取技术，在3040℃的温度条件下反应20 min，就可以准确检测出恶疫霉。特异性试验表明，供测试的48个病原菌中，10 个恶疫霉菌株都呈现阳性检测结果，而其余病原菌均为阴性检测结果。同时，LFRPA的检测灵敏度可达到100 fg，而常规PCR检测的灵敏度仅为10 ng。此外，将恶疫霉接种到新鲜、健康的草莓叶片和果实上，然后应用建立的LFRPA检测方法对感病样品进行检测，均可以检测到恶疫霉。综上所述，本研究所建立的恶 *51今日免费论文网|www.51jrft.com +Q: *351916072* 
疫霉LFRPA检测方法具有很高的特异性、敏感性和准确度，操作方便，设备简单，适用于病害的田间现场诊断以及资源贫乏条件下病原菌的现场检测。
ESTABISHMENT OF A RAPID DETECTION METHOD FOR PHYTOPHTHORA CATORUM BASED ON RECOMBINASE POLYMERASE AMPLIFICATION ASSSY
ABSTRACT
Phytophthora cactorum has a wide range of hosts, and is the main pathogen of pear, apple, strawberry, Panax notoginseng, ginseng and other plants in China. Once the crops are infected, the disease tends to cause serious economic losses. The purpose of this study is to establish an accurate, efficient, convenient and visual detection method of P. cactorum, and apply it to the field diagnosis of disease. This detection method is based on the recombinase polymerase amplification （RPA） technology and lateral flow（LF）technology, and uses Ypt1 gene as the target to design specific primers and probes. By combining with simplified nucleic acid extraction technology, the LFRPA method can successfully detect P. cactorum at 3040℃ for 20 min. The specificity test showed that all the P. cactorum isolates produced positive results while all the other pathogens resulted in negative results. At the same time, the sensitivity of LFRPA can reach 100 fg, while the sensitivity of conventional PCR is 10 ng. In addition, this LFRPA method enabled detection of P. cactorum in diseased strawberry samples without specialized equipment. Consequently, the LFRPA method enables rapid and equipmentfree detection of P. cactorum, and has potential for further development as a diagnostic kit for application in resourcelimited settings.

原文链接：http://www.jxszl.com/nongxue/zwbh/607361.html