丁香疫霉快速检测技术研究及樟疫霉效应子基因Avh2和Avh9功能初步研究毕业论文_林学毕业论文

丁香疫霉快速检测技术研究及樟疫霉效应子基因Avh2和Avh9功能初步研究毕业论文

2021-04-20更新

摘 要

丁香疫霉(Phytophthora syringae,PSY)是一种有害的植物检疫性生物,其分布范围较广,但在我国禁止进境。其主要侵染的寄主包含苹果、桃、梨、柑橘、丁香、橡树等14个科29个属植物,可以使寄主的根茎叶发生病变。病菌侵染柑橘类水果,可引起褐腐、枝干流胶、根颈部脚腐和根腐,严重影响果实产量和品质,带来重大经济损失,严重威胁世界柑橘生产。柑橘的各大产区遍布世界的各个范围,这种疫霉也随之分布范围变广,但是仍未未在我国发现这种菌。本次研究使用一种快速检测方法,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、特异和灵敏的丁香疫霉的检测技术体系。LAMP技术采用的是特异性识别靶序列上的6个位点、4条引物及一种具有链置换活性的DNA聚合酶,温度掌控在60~65℃之间保持稳定,并在75min左右进行PCR扩增,扩增效率可达到高达109~1010个数量级,其优点在于特异性较强,灵敏度也比较高,更主要是操作简单以及检测结果快速。这种技术已经广泛的被用于原核生物如细菌,病毒,一些真核生物如寄生虫的检测中,在植物病原物和人畜的病害诊断和快速检测等的领域都具有很宽广的应用前景。该技术的出现,为丁香疫霉的快速诊断提供了新的可视化检测技术。

关键词:丁香疫霉、检测靶标、LAMP、环介导等温扩增技术

Rapid detection of Phytophthora syringae

ABSTRACT

Phytophthora syringae is a prohibited plant quarantine pest in China. The main host includes 14 families, 29 families, including orange apple, peach, pear, clove, acorn and so on. It can cause many diseases of the root, leaf and fruit of the host. The pathogen can infect citrus fruit brown rot, branch dry glue, root neck foot rot and root rot, affect fruit yield and quality, bring serious economic loss, and seriously threaten the production of Citrus in the world. Its distribution ranges from all citrus producing areas in the world, but has not yet been found in China.This study used a rapid detection method for Phytophthora syringae, mainly using a new nucleic acid amplification technology, namely loop mediated isothermal amplification.A simple, rapid, specific and sensitive detection system for Phytophthora syringae was established. The ring mediated isothermal amplification technique uses 4 primers and a DNA polymerase with chain replacement activity to identify 6 loci specifically on the target sequence and amplify the nucleic acid around 60min at the isothermal condition (60~65℃). The amplification efficiency can reach 109~1010 orders of magnitude, which has high specificity, high sensitivity, isothermal and simple operation. The test results are fast and so on. The technology has been widely used in the detection of virus, bacteria, parasites, bacteria and other diseases. It has a broad application prospect in the field of rapid detection and disease diagnosis of human and animal and plant pathogens. The emergence of this technology provides a new visual detection technology for the rapid diagnosis of Phytophthora syringae .

Key words: Phytophthora syringae, target detection, Loop-mediated isothermal amplification

目 录

1 引言…………………………………………………………………………………………………………..……6

1.1 丁香疫霉的介绍………………………………………………………………………………………………..6

1.1.1 丁香疫霉的分布及危害………………………………………………………………………………1

1.1.2 丁香疫霉的定殖与扩散………………………………………………………………………………1

1.1.3 菌落性状及病菌形态特征…………………………………………………………………………..2

1.1.4 丁香疫霉的检测进展研究…………………………………………………………………………..2

1.2 环介导等温扩增技术(LAMP)简介………………………………………………………………….4

1.2.1 LAMP技术特点及原理………………………………………………………………………………4

1.2.2 LAMP技术的引物设计………………………………………………………………………………4

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

1.4 LAMP技术的研究展望……………………………………………………………………………………. 5

2 实验材料和方法…………………………………………………………………………………………………………..6

2.1 实验材料……………………………………………………………………………………………………………6

2.1.1 培养基的制备………………………………………………………………………………………………6

2.1.2 菌丝粉的收集和DNA的提取……………………………………………………………………….6

2.1.3 材料与试剂………………………………………………………………………………………………….7

2.2实验方法………………………………………………………………………………………………………………7

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