基于重组酶介导等温扩增-侧流层析技术丁香疫霉检测方法的建立毕业论文_林学毕业论文

基于重组酶介导等温扩增-侧流层析技术丁香疫霉检测方法的建立毕业论文

2021-04-20更新

摘 要

丁香疫霉 (Phytophthora syringae)主要危害柑橘果树,是我国对外公布的一种检疫性病害。本研究应用重组酶介导等温扩增(Recombinase Polymerase Amlification,RPA)技术,建立了以Ypt1基因为靶标的简单、快速和灵敏的丁香疫霉的检测方法。Ypt1是一个Ras相关的基因,其在酵母中编码一种与RAS相关的GTP结合蛋白。Ypt1包含有多个内含子,基因序列的保守序列和进化区域相互间隔,适合作为分子检测的靶标。在这项研究中,Ypt1基因用作靶序列,具体的RPA引物和探针依据重组酶介导等温扩增(RPA)而设计。结合测流层析试纸条技术建立反应体系,进行特异性与灵敏度实验。结果表明整个扩增检测过程仅需20分钟,即可通过试纸条的条带直接目测实验结果。当试纸条出现两条棕色条带,一条位于质控区内(Control line),一条位于检测区(Test line),则结果为阳性,表明样本中含有丁香疫霉(P. syringae);当质量控制区只出现一个棕色条带,试验区没有出现条带时,结果为阴性,表明样品中不含有丁香疫霉(p. syringae)。在特异性检测中,丁香疫霉菌株试纸条出现两条棕色条带,而其它疫霉、腐霉和真菌供试菌株在质控区只有一条带。在灵敏度检测中,PsyRPA-LFD技术最低检测限为 10 pg µL-1,普通PCR技术的最低检测限为100 pg µL-1,PsRPA-LFD技术检测灵敏度是 PCR方法检测灵敏度的10倍。同时人工接种柑橘叶片,PsRPA-LFD技术能快速检测出发病叶片。本研究建立的PsRPA-LFD技术可用于口岸和田间对丁香疫霉菌的快速检测。

关键词:丁香疫霉;RPA等温扩增技术;特异性;灵敏度

A novel recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay for detection of Phytophthora sojae

ABSTRACT

Phytophthora syringae is a quarantine disease that is mainly harmful to citrus fruit trees in China. In this study, a simple, rapid and sensitive detection method for Phytophthora syringae targeting Ypt1 gene was established using recombinant enzyme-mediated isothermal amplification (RPA) technology. Ypt1 is a Ras-related gene that encodes a Ras-related GTP binding protein in yeast. Ypt1 contains many introns, and the conserved sequence and evolution region of gene sequence are separated from each other, which is suitable as a target for molecular detection. In this study, Ypt1 gene was used as the target sequence, and specific RPA primers and probes were designed according to recombinant enzyme mediated isothermal amplification (RPA). The reaction system was established and the specificity and sensitivity experiments were carried out in combination with flow measurement chromatography test strip technology. The results show that the whole amplification detection process only takes 20 minutes, and the experimental results can be directly visually observed through the strips of the test paper strip. When two brown bands appear on the test strip, one in the Control line and the other in the Test line, the result is positive, indicating that the sample contains p syringae; When there is only one brown strip in the quality control area and no strip in the detection area, the result is negative, indicating that the sample does not contain Phytophthora syringae. In the specific detection, there are two brown bands on the test strip of Phytophthora syringae, while there is only one band in the quality control area for other tested strains of Phytophthora, Pythium and fungi. In sensitivity detection, the minimum detection limit of PsyRPA-LFD technology is 10 pg L-1, and that of common PCR technology is 100 pg L-1. The detection sensitivity of PsRPA-LFD technology is 10 times that of PCR method. At the same time, citrus leaves were inoculated artificially, and PsRPA-LFD technology could rapidly detect diseased leaves. PsRPA-LFD technology established in this study can be used for rapid detection of Phytophthora syringae in ports and fields.

Key words: Phytophthora syringae;RPA isothermal amplification technology;Specificity;Sensitivity

目 录

1 RPA重组酶聚合酶扩增检测技术的研究进展 – 1 –

1.1 重组酶聚合酶等温扩增(RPA)简介 – 1 –

1.1.1 RPA技术特点及原理 – 1 –

1.1.2 RPA技术的引物设计 – 2 –

1.1.3 RPA技术的检测方法 – 2 –

1.1.4 RPA技术的优缺点 – 3 –

1.2 RPA技术在病原微生物检测中的应用 – 3 –

1.2.1 病毒的检测 – 3 –

1.2.2 细菌的检测 – 4 –

1.2.3 寄生虫的检测 – 5 –

1.2.4 真菌的检测 – 5 –

2 丁香疫霉重组酶介导等温扩增-侧流层析检测技术的研究 – 7 –

2.1 实验材料和方法 – 7 –

2.1.1 供试菌株 – 7 –

2.1.2培养基的制备 – 8 –

2.1.3 DNA的提取 – 8 –

2.1.4引物与探针设计 – 9 –

2.1.5RPA引物设计与合成 – 10 –

2.1.6RPA技术反应体系的建立 – 10 –

2.1.7 RPA引物扩增产物的判断方法 – 10 –

2.1.8 RPA-LFD特异性验证 – 11 –

2.1.9 RPA-LFD灵敏度评价实验 – 11 –

2.2结果 – 11 –

2.2.1引物及探针的确定 – 11 –

2.2.2使用PCR技术对引物效果初步验证 – 11 –

2.2.3 不同反应时间下RPA-LFD扩增结果观察 – 12 –

2.2.4引物的特异性验证 – 13 –

2.2.5引物灵敏度的验证 – 14 –

2.2.6 人工接种柑橘进行实际样RPA-LFD检测 – 15 –

3结论 – 17 –

致 谢 – 18 –

参考文献 – 19 –

1 RPA重组酶聚合酶扩增检测技术的研究进展

1.1 重组酶聚合酶等温扩增(RPA)简介

1.1.1 RPA技术特点及原理

分子快速检测技术中致病疫霉菌检测的研究技术是分子生物学理论与技术的发展方向,PCR技术聚合酶链式反应(polymerase chain reaction,PCR)是目前最普遍使用的检测技术,包括荧光PCR、普通PCR和多重PCR等多种方法[1]。PCR技术的原理是以已知序列的核苷酸为引物,在聚合酶作用下对特定的DNA片段进行复制。无论是哪种检测技术关键点是对靶标的特异性和灵敏度的检测,靶标是80bp左右的长段NDA序列。很多靶标都适用于疫霉菌的检测,用于检测技术的常用靶标是核糖体转录间隔区域基因(ITS),目前该靶标已成功应用于冬生疫霉、橡树疫霉、大豆疫霉菌等多种疫霉菌的检测中,但它很难设计检测的引物。研究靶标是为了更好地检测DNA,并发掘更多重要信息,更快获得需要扩增的DNA片段。随着基因组学的发展,越来越多的疫霉菌的全基因组被测序,全基因组序列的获得将对研究疫霉菌,为实现病原菌快速、高效、灵敏、准确的检测技术提供有力的保障。

PCR技术有它的局限性,这几年来,恒温核酸扩增技术的出现解决了这个问题[2],它对简化了对高成本仪器的要求,显著缩短反应时间,因此备受关注。采用相比多种PCR技术更加简单方便,便宜节省费用的RPA技术来检测丁香疫霉可快速准确地降低丁香疫霉对柑橘果树的威胁。先前检测丁香疫霉的方法操作复杂成本太高、要大量的时间精力而且要求操作者具备对丁香疫霉大量的研究经验和实验操作技巧,相关知识。随着核酸扩增技术不断的发展,RPA检测已成功应用于丁香疫霉的检测。

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