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用于……的可视化环介导等温扩增技术(Visual LAMP)和环介导等温扩增技术- TaqMan 分析方法的开发与比较

Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for .

作者信息

Cui Shuning, Ma Haoze, Wang Xinyue, Yang Han, Wu Yuanzheng, Wei Yanli, Li Jishun, Hu Jindong

机构信息

Ecology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250013, China.

出版信息

Microorganisms. 2024 Jun 28;12(7):1325. doi: 10.3390/microorganisms12071325.

Abstract

Strawberry anthracnose caused by spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the β-tubulin sequence of . Both methods can detect genomic DNA at very low concentrations (10 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/μL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for . The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of in China and control strawberry anthracnose in the field.

摘要

由[具体病原菌名称]引起的草莓炭疽病已在全球范围内给草莓生产造成了重大损失。草莓炭疽病主要发生在幼苗期和定植初期,在华北地区是主要病原菌,土壤中产生的菌丝体、无性态核和分生孢子是主要感染源。检测土壤中的病原菌对于预测炭疽病的流行至关重要。在本研究中,针对[病原菌名称]的β-微管蛋白序列开发了可视化环介导等温扩增(LAMP)检测法和环介导等温扩增法结合TaqMan探针(LAMP-TaqMan)检测法。两种方法均可检测土壤中极低浓度(10拷贝/克)的[病原菌名称]基因组DNA,而可视化LAMP和LAMP-TaqMan检测法的检测限均为50拷贝/微升,超过了传统PCR和qPCR技术的灵敏度,且两种方法对[病原菌名称]均表现出高特异性。对这两种方法进行了比较:LAMP-TaqMan由于加入了荧光分子信标而表现出增强的特异性,而可视化LAMP仅需在恒温下进行简单孵育,结果通过颜色变化判断;因此,对仪器的要求相对简单且用户友好。总之,这两种检测方法将有助于监测中国[病原菌名称]的种群数量并控制田间草莓炭疽病。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/35dc/11279180/0f3983bd7c1f/microorganisms-12-01325-g001.jpg

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