摘 要

樟疫霉( Phytophthora cinnamomi)属于卵菌门( Oomycota) 、卵菌纲( Oo- mycetes) 、霜霉目( Peronosporales) 、腐霉科( Pythia- ceae) 、疫霉属( Phytophthora) 。樟疫霉能在全球范围内对造林树种、果树、农作物引起的重要病害。樟疫霉可感染樟属植物引起根部腐烂以及樟属植物溃疡等病害，会对樟属植物造成毁灭性的冲击。樟疫霉引起的病害症状与其他病菌引起的症状相似，而且其分离和培养较困难，控制樟疫霉的感染与传播最有效的途径就是对樟疫霉进行快速检验检疫，防止病原菌的传播和扩散。本次研究使用的是对樟疫霉的一种快速检测方法，主要运用一种新型的核酸扩增技术即环介导等温扩增技术（LAMP：Loop-mediated isothermal amplification），建立简单、快速、特异和灵敏的樟疫霉的检测技术体系。结果表明在64℃的等温环境下仅需60min，并且在实验前的体系中加入染料羟基苯酚蓝（HNB）作为指示剂，就可以直接通过肉眼来观察颜色的变化判定是否发生了LAMP反应。在特异性的检测中，樟疫霉菌株在HNB显色反应中观察到阳性反应，而其他的疫霉如大豆疫霉、致病疫霉、恶疫霉和其他的真菌供试菌株没有这种阳性显色反应。在灵敏度检测中，Pcinn-10079-LAMP技术最低检测限为100 pg/μL。该技术的出现为樟疫霉的快速诊断提供了新的可视化检测技术。

关键词：樟疫霉、检测靶标、LAMP、羟基苯酚蓝（HNB）

APPLICATION OF LOOP-MEDIATED ISOTHERAL AMPLICATION (LAMP) IN RAPID DETECTION OF PHYTOPHTHORA CINNAMOMI

ABSTRACT

Phytophthora cinnamomi causes important disease in ornamental, agronomic, and forest ecosystems worldwide. It is difficult to judge which pathogens caused disease only by according symptom. Traditionally, baiting and morphological characteristic have been used to detect P.cinnamomi in soil, water, and plant material but may take a considerable amount of time, and specific taxonomical expertise is required to correctly identify the isolate to species level. Early detection, accurate identification, traceability, and elimination are important aspects in the control of P.cinnamomi. Technological advances in PCR-based methods and Real-time PCR technology allow fast, accurate detection and quantification of plant pathogen and are now being applied to practical problem. However, these methods might not be suitable for developing countries because of high-tech equipment required, elaborate and complicated assay procedures, expensive reagents, time requirements, and the frequency of false positives. Therefore, there is a growing demand for simple and economical molecular tests. This paper reports on the development of a Loop-mediated isothermal amplification(LAMP) assay targeting IGS sequences.. The IGS-LAMP assay efficiently amplified the target element in less than 60 min at 64°C and was evaluated for specificity and sensitivity. The specificity was evaluated against P. ramorum, Phytophthora spp. , Pythium spp., and true fungi isolates. The experimental results can be directly visually by the naked eye. A positive colour (sky blue) was only observed in the presence of P. cinnamomi .Hydroxynaphthol blue (HNB) prior to amplification, whereas none of other isolates showed a colour change. The detection limit of specific LAMP assay was 10 fg μL^-1 . Establishment of LAMP provides a new alternative method for the rapid detection of P. cinnamomi.

Key words: Phytophthora cinnamomi, target detection, LAMP, HNB

目 录

1.环介导等温扩增技术（LAMP）的研究进展……………………………………………………1

1.1 樟疫霉的分布与危害……………………………………………………………………1

1.2 环介导等温扩增技术（LAMP）简介……………………………………………………1

1.2.1 环介导等温扩增技术（LAMP）特点………………………………………………1

1.2.2 LAMP技术的引物设计…………………………………………………………… 2

1.2.3 环介导等温扩增技术（LAMP）原理………………………………………………3

1.3 LAMP产物扩增的判断方法………………………………………………………………5

1.4 本实验的目的和意义……………………………………………………………………6

2.樟疫霉菌的环介导等温扩增（LAMP）技术的研究………………………………………………7

2.1 实验材料和方法…………………………………………………………………………7

2.1.1 培养基的制备………………………………………………………………………7

2.1.2 实验所用菌株以及菌丝粉的收集…………………………………………………7

2.1.3 基因组的提取………………………………………………………………………8

2.1.4 实验所需试剂、仪器与耗材………………………………………………………8

2.1.5 靶标基因的发掘……………………………………………………………………9

2.1.6 LAMP引物设计与合成………………………… ………… ……………… ……10

2.1.7 可视化LAMP反应体系的建立…………………………………………… … … 10

2.1.8 LAMP引物扩增产物的判断方法……………………………………………… … 11

2.1.9 LAMP引物特异性评价试验… ……………………………………………… … 12

2.1.10 LAMP引物灵敏度评价试验…………………………………………………… 12

2.2 结果……………………………………………………………………………………12

2.2.1 LAMP引物所在靶基因的位置和引物序列 ………………………………… …12

2.2.2 所设计引物效果初步验证…………………………………………………… 14

2.2.3 LAMP 引物的特异性验证………………… …………………………………… 14

2.2.4 LAMP 引物灵敏度的验证………………… …………………………………… 15 结论……………………………… ………………………………………………………………16

致谢……………… ………………………………………………………………………………19参考文献……… …………………………………………………………………………………20

1.环介导等温扩增技术（LAMP）的研究进展

1.1 樟疫霉的分布与危害

樟疫霉可以感染樟属植物引起植物根部腐烂以及枝干的溃疡，会对樟属植物造成毁灭性打击^[1]。据报道，樟疫霉已经使澳洲西南地区森林结构和植物种群发生变化，已经严重威胁到澳洲地区自然生态系统以及生物多样性^[2-3]，并且已逐渐向全世界范围蔓延。樟疫霉引起的危害，其寄主广泛。在我国，樟疫霉还会侵害雪松，造成雪松疫霉腐烂病^[4]。目前对该病害还没有有效的治理方法，加强检疫，防止病原菌的传播扩散是控制该病的最有效措施。为此应加强疫情监测，建立快速的疫霉检测方法，为病害的风险及研究决策提供依据，从而有利于降低樟树疫病引起的损失。

1.2 环介导等温扩增技术（LAMP）简介