AbstractLoop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops.

AbstractLoop介导的等温扩增（LAMP）代表了一种有价值的DNA/RNA检测技术，以其异常的灵敏度，特异性，速度，准确性和可承受性而闻名。这项研究的重点是优化基于LAMP的方法，以检测向日葵霜霉病的偶然病原体Plasmopara halstedii的早期迹象，向日葵霜霉病是向日葵作物的严重威胁。

Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined.

具体而言，从halstedii基因组DNA设计了一组六个LAMP引物（两个外部，两个内部和两个环），靶向核糖体大亚基（LSU）。这些引物通过计算机分析和使用目标和非目标物种DNA的实验验证进行了验证。确定了包括反应条件（温度，时间）和组分浓度（镁，Bst DNA聚合酶，引物和dNTP）的优化。

Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique.

通过琼脂糖凝胶电泳进行这些优化的验证。此外，评估了各种比色化学物质（中性红，羟基萘酚蓝，SYBR Safe，噻唑绿）以促进方法分析，并优化了实时分析，提出了使用LAMP技术检测向日葵霜霉病的多种方法。

The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability..

通过检测低至0.5 pg/μl的halstedii DNA浓度，证实了该方法的分析灵敏度。这项开创性的研究通过LAMP方法建立了halstedii检测方法，在其领域中独树一帜。LAMP协议的精确度，稳健性和实用性使其成为关注向日葵霉菌的研究的理想选择，由于其操作简便和可靠性，强调了其推荐用途。。

IntroductionHelianthus annuus L., commonly known as sunflower, ranks one of the most important oilseed crops grown worldwide. Since 2017, global annual sunflower cultivation has consistently increased1. In 2023, oilseed production worldwide constituted 61% soybean, 12.4% rapeseed, 7.9% groundnut, and 7.8% sunflower2.

简介向日葵，俗称向日葵，是世界上最重要的油料作物之一。自2017年以来，全球向日葵年度种植量一直在增加1。2023年，全球油籽产量占大豆的61%，油菜的12.4%，花生的7.9%和向日葵的7.8%。

Except for Antarctica, Helianthus annuus is extensively cultivated across all continents as a source of edible oil and food3.Sunflower cultivation is economically significant worldwide but faces several pathogenic factors that cause substantial yield losses. Among these, sunflower downy mildew stands prominent4.

除南极洲外，向日葵作为食用油和食物的来源广泛种植在各大洲。向日葵种植在世界范围内具有重要的经济意义，但面临着导致大量产量损失的几种致病因素。其中，向日葵霜霉病占主导地位4。

Downy mildew in sunflowers, caused by Plasmopara halstedii (Farlow) Berlese & de Toni (PHAL), originates from a biotrophic oomycete5. This pathogen is an obligate biotroph, exhibiting prominent symptoms such as stunting in infected sunflowers, rosette formation, spotting on leaves, and white sporangia layers protruding from the lower leaf surface5.

向日葵中的霜霉病由Plasmopara halstedii（Farlow）Berlese＆de Toni（PHAL）引起，起源于生物营养卵菌5。这种病原体是一种专性生物营养体，表现出明显的症状，如受感染的向日葵发育迟缓，玫瑰花结形成，叶片斑点，以及从叶片下表面突出的白色孢子囊层5。

The disease can also spread asymptomatically, posing risks during seed trade, leading to significant losses. Consequently, the spread of P. halstedii-resistant strains might occur, as reported by Martínez et al.23. Between 2006 and 2014, there were 35 pathotypes documented, 41 in 2014, and 50 as of 20185,6,7.

这种疾病也可能无症状传播，在种子贸易过程中构成风险，导致重大损失。因此，正如Martínez等人报道的那样，可能会发生halstedii耐药菌株的传播。在2006年至2014年间，记录了35种病理类型，2014年为41种，截至20185,6,7年为50种。

The variability of P. halstedii's pathogenicity, its development of new pathotypes resistant to fungicides used in field management, and the widespread prevalence of fungicide-resistant pathotypes have been reported8.Detection tests for both symptomatic and asymptomatic PHAL presence are necessary during the transportation of sunflower seeds in agricultural fields and trade.

据报道，halstedii致病性的变异性，其对田间管理中使用的杀菌剂具有抗性的新致病型的发展以及抗杀菌剂致病型的广泛流行8。在运输葵花籽期间，有症状和无症状的PHAL存在的检测测试是必要的。在农田和贸易中。

Several methods have been developed for detecting this pathogen in sunflowers, including the Enzyme-Linked Immunosorbent Assay .

已经开发了几种检测向日葵中这种病原体的方法，包括酶联免疫吸附测定法。

Table 1 Primer set designed specifically for PHAL ribosomal DNA gene large subunit.Full size tableLAMP reaction optimizationLAMP reaction optimization studies were conducted using Bst 2.0 Warm Start DNA Polymerase (8,000 U/ml) (NEW ENGLAND BIOLABS, Cat no: M0538) in a thermal cycler (APPLIED BIOSYSTEM, SimpliAmp) and a real-time thermal cycler device (BIO-RAD, CFX96 Touch Real-Time PCR Detection System).Different reaction conditions were evaluated to optimize the LAMP reaction.

表1专门为PHAL核糖体DNA基因大亚基设计的引物组。全尺寸台灯反应优化使用Bst 2.0温启动DNA聚合酶（8000 U/ml）（NEW ENGLAND BIOLABS，目录号：M0538）在热循环仪（APPLIED BIOSYSTEM，SimpliAmp）和实时热循环仪设备（BIO-RAD，CFX96 Touch real-time PCR Detection System）中进行LAMP反应优化研究。评估不同的反应条件以优化LAMP反应。

These conditions included temperature (63 °C, 65 °C, 67 °C, 70 °C), reaction duration (15, 35, 45, 60 min), Bst 2.0 DNA polymerase concentrations (6, 8, 10, 12 U), LAMP primer concentrations [F3/B3 (0.1, 0.2, 0.4 μM), FIP/BIP (0.8, 1.6, 3.2 μM), LF/LB (0.2, 0.4, 0.8 μM)], and reagent concentrations [Mg2+ (6, 8, 10, 12 mM), dNTPs (1.0, 1.2, 1.4, 1.6 mM)].

这些条件包括温度（63°C，65°C，67°C，70°C），反应持续时间（15,35,45,60分钟），Bst 2.0 DNA聚合酶浓度（6,8,10,12 U），LAMP引物浓度[F3/B3（0.1,0.2,0.4μM），FIP/BIP（0.8,1.6,3.2μM），LF/LB（0.2,0.4,0.8μM）]和试剂浓度[Mg2+（6,8,10,12 mM），dNTPs（1.0,1.2,1.4,1.6毫米）]。

In all experiments, the template DNA concentration was maintained at 50 ng, and reactions were conducted in a 1X reaction buffer [20 mM Tris–HCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tween 20, pH 8.8 at 25 °C] with a final reaction volume of 25 µl adjusted with PCR-grade water.The optimization process was carried out in a systematic manner.

在所有实验中，模板DNA浓度保持在50 ng，反应在1X反应缓冲液[20 mM Tris-HCl，10 mM（NH4）2SO4、2 mM MgSO4、0.1%吐温20，25°C pH 8.8]中进行，最终反应体积为25µl，用PCR级水调节。优化过程以系统的方式进行。

Initially, temperature optimization was performed by varying the temperature while keeping all other components constant to determine the optimal temperature. Following this, magnesium ion concentration was optimized by varying Mg2+ levels, and then Bst 2.0 DNA polymerase concentration was adjusted.

最初，通过改变温度来进行温度优化，同时保持所有其他组分恒定以确定最佳温度。之后，通过改变Mg2+水平来优化镁离子浓度，然后调整Bst 2.0 DNA聚合酶浓度。

After establishing the optimal conditions for temperature, Mg2+, and polymerase concentration, primer concentrations were optimized, and finally, dNTP concentration was varied. After amplification, all reactions were incubated at 80 °C for 10 min to inactivate the enzyme. Throughout this study, all reactions were perf.

在确定温度，Mg2+和聚合酶浓度的最佳条件后，优化引物浓度，最后改变dNTP浓度。扩增后，将所有反应在80°C下孵育10分钟以使酶失活。在整个研究过程中，所有反应都很好。

Table 2 The variables and optimum values from the optimization studies for the detection of P. halstedii pathogen using the LAMP method are highlighted in bold.Full size tableThe products of the optimized LAMP reaction exhibited ladder-like bands as expected in positive reactions during agarose gel electrophoresis, while no bands were observed in NTC shown in Fig. 1.Fig.

表2使用LAMP方法检测halstedii病原体的优化研究的变量和最佳值以粗体突出显示。全尺寸表优化的LAMP反应的产物在琼脂糖凝胶电泳期间在阳性反应中显示出预期的梯形条带，而在NTC中未观察到条带，如图1所示。

1Agarose gel electrophoresis image of optimized LAMP reaction products for the detection of PHAL. M: Marker 100 bp (HIBRIGEN, MG-LDR-100), 1–2: PHAL (The presence of a positive reaction was visualized as a pattern resembling a ladder), 3: NTC. Samples were run on a 2% agarose gel at 100 V for 45 min.Full size imageVisualization results of LAMP reactionsIn this study, various optimizations were conducted to enable the analysis of LAMP products.

1用于检测PHAL的优化LAMP反应产物的琼脂糖凝胶电泳图像。M： 标记100 bp（HIBRIGEN，MG-LDR-100），1-2:PHAL（阳性反应的存在被视为类似阶梯的模式），3:NTC。样品在2%琼脂糖凝胶上于100 V电泳45分钟。LAMP反应的全尺寸图像可视化结果在这项研究中，进行了各种优化以分析LAMP产品。

The optimal concentrations determined for Neutral Red, Hydroxynaphthol Blue, SYBR Safe DNA Gel Stain and Thiazole Green were 120 μM, 150 μM, 100X, 1000X, respectively.When visualizing LAMP reactions using Neutral Red with optimized indicators, positive samples transitioned from a light orange color before the reaction to pink after the reaction, while no color change was identified in the NTCs before or after the reaction.

中性红，羟基萘酚蓝，SYBR安全DNA凝胶染色剂和噻唑绿的最佳浓度分别为120μM，150μM，100X，1000X。当使用中性红和优化的指示剂可视化LAMP反应时，阳性样品从反应前的浅橙色转变为反应后的粉红色，而在反应前后NTC中未发现颜色变化。

Hydroxynaphthol Blue optimized visualization studies, where PHAL genomic DNA was used in positive samples, observed a color change from purple-magenta to blue, while the color remained purple-magenta in the NTC. Both Neutral Red and Hydroxynaphthol Blue chemical indicators were added before the reaction commenced, and the analyses were evaluated by the naked eye shown in Fig. 2.Fig.

羟基萘酚蓝优化的可视化研究（其中PHAL基因组DNA用于阳性样品）观察到颜色从紫色品红色变为蓝色，而NTC中的颜色保持紫色品红色。在反应开始之前加入中性红和羟基萘酚蓝化学指示剂，并通过肉眼评估分析，如图2所示。

2Obtained LAMP reaction results with the optimized dye concentrations. In the presence of PHAL genome in the reaction, Neutral Red produced a color gradient from ora.

2用优化的染料浓度获得LAMP反应结果。在反应中存在PHAL基因组的情况下，中性红从ora产生颜色梯度。

Data availability

数据可用性

Data is provided within the manuscript or supplementary information files.

数据在手稿或补充信息文件中提供。

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Download referencesAcknowledgementsThe research was funded by the Research Fund of Istanbul University under Project Number FYL 2022-39343. Oğuzhan YENİ, who conducted his thesis as a graduate student during this study, received a 2210A success scholarship from The Scientific and Technological Research Council of Türkiye and we express our gratitude for the support provided by The Scientific and Technological Research Council of Türkiye.

下载参考文献致谢该研究由伊斯坦布尔大学研究基金资助，项目编号为FYL 2022-39343。在这项研究中，作为研究生完成了他的论文，Oğuzhan YENİ获得了蒂尔基耶科学技术研究委员会颁发的2210A成功奖学金，我们感谢蒂尔基耶科学技术研究委员会提供的支持。

We thank Önder BAYDEMİR (Directorate of Trakya Agricultural Research Institute, Türkiye) and Assoc. Prof. Arzu ÇELİK OĞUZ (Ankara University, Faculty of Agriculture, Plant Protection Department, Türkiye) for their support in providing experimental materials. The authors have no conflict of interest to declare.Author informationAuthors and AffiliationsInstitute of Science, Program of Molecular Biotechnology and Genetics, Istanbul University, Istanbul, TurkeyOğuzhan YeniInstitute of Science, Program of Biotechnology and Genetics, Trakya University, Edirne, TurkeyMutlu ŞenFaculty of Engineering, Department of Genetics and Bioengineering, Trakya University, Ahmet Karadeniz Yerleskesi, Edirne, TurkeySemra HasançebiFaculty of Science, Department of Molecular Biology and Genetics, Istanbul University, Vezneciler, 34134, Istanbul, TurkeyNeslihan Turgut KaraAuthorsOğuzhan YeniView author publicationsYou can also search for this author in.

我们感谢Önder BAYDEMİR（图尔基耶特拉基亚农业研究所董事会）和助理教授ArzuÇELİK OĞUZ（图尔基耶安卡拉大学农业学院植物保护系）在提供实验材料方面的支持。作者没有利益冲突要申报。作者信息作者和附属机构伊斯坦布尔大学分子生物技术与遗传学项目科学研究所，伊斯坦布尔，土耳其尤赞-耶尼科学研究所，特拉基亚大学生物技术与遗传学项目，埃迪尔内，土耳其穆特卢恩工程学院，特拉基亚大学遗传与生物工程系，Ahmet Karadeniz Yerleskesi，埃迪尔内，土耳其塞姆拉-哈桑比比科学学院，伊斯坦布尔大学分子生物学与遗传学系，维兹奈克勒，34134，伊斯坦布尔，土耳其尤兹利汉-图尔古特卡拉奇作者，奥扎恩-耶尼维尤在中搜索此作者。

PubMed Google ScholarMutlu ŞenView author publicationsYou can also search for this author in

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PubMed Google ScholarSemra HasançebiView author publicationsYou can also search for this author in

PubMed Google ScholarSemra HasançebiView作者出版物您也可以在

PubMed Google ScholarNeslihan Turgut KaraView author publicationsYou can also search for this author in

PubMed Google ScholarNeslihan Turgut KaraView作者出版物您也可以在

PubMed Google ScholarContributionsO.Y.: Methodology, investigation, analysis, writing—original draft preparation; M.Ş.: Metodology, revised the manuscript; S.H.: Metodology, revised the manuscript; N.T.K.: conceptualization and revised the manuscript, and supervision. All authors contributed to the concept and design of the study.Corresponding authorCorrespondence to.

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Reprints and permissionsAbout this articleCite this articleYeni, O., Şen, M., Hasançebi, S. et al. Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection.

转载和许可本文引用本文Yeni，O.，Şen，M.，Hasançebi，S。等人。用于向日葵霉变病检测的环介导等温扩增测定的优化。

Sci Rep 14, 23224 (2024). https://doi.org/10.1038/s41598-024-72228-yDownload citationReceived: 23 May 2024Accepted: 04 September 2024Published: 05 October 2024DOI: https://doi.org/10.1038/s41598-024-72228-yShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

科学报告1423224（2024）。https://doi.org/10.1038/s41598-024-72228-yDownload引文接收日期：2024年5月23日接受日期：2024年9月4日发布日期：2024年10月5日OI：https://doi.org/10.1038/s41598-024-72228-yShare本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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KeywordsLoop-mediated isothermal amplification (LAMP)Plant pathogensSunflower downy mildew

关键词LOOP介导等温扩增（LAMP）植物病原真菌霜霉病

Plasmopara halstedii

霍尔斯单轴霉

Plant disease detection

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