Fast detection by loop-mediated isothermal amplification (LAMP) of the three begomovirus species infecting tomato in Panama

Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) of genus Begomovirus (family Geminiviridae) are the only three begomovirus species detected infecting tomato (Solanum lycopersicum L.) in Panama. PYMPV, ToLCSiV and TYMoV induce symptoms of stunting, yellowing, curling, distortion of leaves and reduction of fruit size and cause important economic loses. A loop-mediated amplification under isothermal conditions (LAMP) assay was developed for the individual detection of these three begomovirus species by using a set of three primer pairs specific per each one of them. Amplification products were visualized by gel electrophoresis or direct Gel-Red staining of DNA into the reaction tube. PYMPV, ToLCSiV and TYMoV were detected in total DNA extracts obtained from different plant tissues such as leaves, stems, flowers, fruits and roots of infected tomato plants collected in different production regions of Panama. LAMP sensitivity was similar to that of conventional PCR but, the first procedure was faster and cheaper than the last one. Moreover, all three viruses were successfully detected by LAMP and not by conventional PCR from sap extracts obtained from leaf tissues of infected tomato plants which were embedded into 3MM Whatman paper and stored several days, facilitating the samples processing as well as the material movement among different laboratories. Therefore, LAMP is a specific, rapid and cheap procedure to detect all three begomoviruses infecting tomato in Panama and it is suitable for field surveys and sanitation programs.     

基于环介导等温扩增技术检测雪松疫霉根腐病菌

雪松疫霉根腐病菌(Phytophthora lateralis Tucker et Milbrath)是一种重要的毁灭性植物病原菌,也是我国进境植物检疫性有害生物。目前,我国未有该病害发生的报道,为了防止P.lateralis的传入和扩散,需对其进行快速、准确的检测。本文利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),以Ypt1基因为靶标序列,设计LAMP特异性引物,建立LAMP反应体系,并对灵敏度和特异性进行检测。结果表明:整个检测过程仅需80 min,即可通过肉眼观察直接判定检测结果。在特异性检测中,P.lateralis菌株都能观察到天蓝色的阳性反应,而其他疫霉菌和真菌供试菌株均呈阴性反应。在灵敏度检测中,最低检测限为100 pg/μL。该方法的建立为P.lateralis的检疫鉴定及其所致病害的快速诊断提供了新技术。     

Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP)

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.     

Detection of Ralstonia solanacearum by loop-mediated isothermal amplification

Ralstonia solanacearum is a pathogenic bacterium that causes wilt in over 200 plant species. Here we report a rapid and sensitive detection of R. solanacearum using an isothermal method for copying DNA known as loop-mediated amplification (LAMP). A set of four primers was designed to replicate the gene coding for the flagellar subunit, fliC, and conditions for detection were optimized to complete in 60 min at 65 degrees C. Magnesium pyrophosphate resulting from the amplification reaction could be detected optically as an increase in the solution turbidity, and the DNA products spread in a reproducible ladder-like banding pattern after electrophoresis in an agarose gel. Replication of the fliC gene was detected only from R. solanacearum. The detection limit of this LAMP assay was between 10(4) to 10(6) colony forming units/ml, and the technique may be useful for developing rapid and sensitive detection methods for the R. solanacearum pathogen in soil and water.     

世界主要储粮书虱环介导等温扩增试剂盒研发与应用

为利用分子生物学技术快速准确鉴定国内外主要储粮书虱,基于环介导等温扩增 （loop-mediated isothermal amplification, LAMP）技术拟研发一款世界主要储粮书虱LAMP试剂盒,利用该LAMP试剂盒对10种储粮书虱的DNA粗提液进行测试,并结合DNA条形码技术对采自中国山东省和内蒙古自治区粮库的未知种类储粮书虱成虫样品进行鉴定。结果显示,本试验研发的LAMP试剂盒可准确鉴定10种储粮书虱;使用LAMP试剂盒和DNA条形码技术鉴定未知种储粮书虱样品的结果一致,即山东省储粮书虱样品为嗜虫书虱 Liposcelis entomophila,内蒙古自治区储粮书虱样品为啮书虱 L. corrodens,表明本试验研发的世界主要储粮书虱LAMP试剂盒可以快速、精准完成主要储粮书虱的鉴定,且操作便捷,可为海关口岸和基层仓储保护部门书虱鉴定工作提供技术支持。     

利用环介导等温扩增(LAMP)技术快速检测辣椒疫霉菌

<正>辣椒疫霉(Phytophthora capsici)是一种重要植物病原菌,能造成植株坏死、果实腐烂,严重影响产量[1]。辣椒疫霉侵染植物的早期病症并不明显,易被忽视。因此,对辣椒疫霉早期快速、准确检测显得尤为重要。聚合酶链式反应(PCR)为动植物病原物检测的重要方法,但需要较昂贵的仪器、试剂与耗材,后期的电泳检测也费时费力,致使这一技术很难在生产一线普及推广。Notomi等2000年研发了环介     

水稻细菌性条斑病菌LAMP快速检测方法的建立

以SYBR Green I荧光染料为指示剂,针对水稻细菌性条斑病菌糖基转移酶,设计了4条普通LAMP引物,并在此基础上设计了2条LAMP环引物,建立了水稻细菌性条斑病菌的环介导等温扩增可视化快速检测方法。结果表明:水稻细菌性条斑病菌呈荧光的阳性反应,近缘菌株则呈橙色的阴性反应;检测灵敏度DNA为100 fg/μL,菌悬液为3×103 cfu/m L。LAMP技术的建立为现场检疫和大规模检测提供了新的方法。     

基于环介导等温扩增技术快速检测美澳型核果褐腐病菌

本文建立了美澳型核果褐腐病菌(Monilinia fructicola)环介导等温扩增技术,并利用此技术开展了M.fructicola的检测和鉴定。采用M.fructicola SCAR分子标记的MO368特异片段为靶标序列,设计并筛选出M.fructicola的特异性LAMP引物,建立了该病菌快速诊断方法。试验结果表明,仅M.fructicola的菌株DNA扩增后呈天蓝色的阳性反应,而在其他真菌的供试菌株中均呈紫色的阴性反应。检测灵敏度可达到100 pg/μL。利用该方法对无锡机场截获的7份疑似李子褐腐病样本进行检测,结果有3份样品呈LAMP阳性反应。本文建立的M.fructicola LAMP检测方法具有灵敏度高、特异性好,简便易行等优点,为美澳型核果褐腐病菌的快速检测提供了一种新技术。     

逆转录环介导等温扩增技术检测花生条纹病毒

花生条纹病毒病(Peanut stripe virus,PStV)是我国花生上流行最广[1]、田间发病率最高的一种花生病毒病,严重影响花生的产量和品质.由于PStV存在不同症状类型株系,在中国占优势的轻斑驳株系,引起花生症状较轻,并且与其他花生病毒病症状类似,不易分辨,目前主要利用PCR方法对PStV进行检测[2],但该方法检测时间长,并且需要专业仪器,较难普及.     

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) of plum pox potyvirus Turkey (PPV-T) strain

Journal of Plant Diseases and Protection - Plum pox potyvirus (PPV) is one of the most important viral pathogens of stone fruit trees. PPV-T (Turkey) is important, unique and dominated strain in...     

逆转录环介导等温扩增技术检测南方菜豆花叶病毒

 根据南方菜豆花叶病毒外壳蛋白基因序列设计并合成了4组RT-LAMP引物,通过引物筛选试验,确定SB1组引物为最佳引物,并进行了引物特异性与灵敏度检测试验,最终建立了南方菜豆花叶病毒的RT-LAMP检测方法。灵敏度检测试验显示,RT-LAMP方法比普通RT-PCR法灵敏度高10倍,而检测时间明显缩短,整个反应过程只需40 min。此外,体系中加入钙黄绿素,反应结束后,可裸眼观察颜色变化来判定结果。本研究所建立的SBMV RT-LAMP方法具有快速、稳定、灵敏、特异、操作简单的特点,适合于SBMV的现场快速检测。     

木薯细菌性萎蔫病菌的LAMP快速检测方法

建立了一种木薯细菌性萎蔫病菌的环介导恒温扩增快速检测方法,为木薯细菌性萎蔫病的快速检测提供有力的技术支持。针对木薯细菌性萎蔫病菌TAL效应器蛋白质(pthBXam)靶序列的6个位点设计4条特异性引物,并对反应温度和内引物浓度等参数进行了优化,设计的引物与试验中提供的其他黄单胞近缘种都没有扩增反应,表现了较好的特异性。LAMP方法对木薯细菌性萎蔫病菌菌株DNA的检测下限为1pg/μL,比常规PCR灵敏度高100倍。该方法采用SYBR Green I染料法对扩增产物闭管检测,裸眼观察颜色变化判断反应结果,能快速、准确地对田间样品进行检测,没有出现假阳性和假阴性。与其他检测方法相比,LAMP方法检测时间短,效率高,降低了设备投入,易于操作,适合木薯细菌性萎蔫病菌的现场检疫和大规模监测。     

Rapid diagnosis of soybean anthracnose caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> using a loop-mediated isothermal amplification (LAMP) assay

A loop-mediated isothermal amplification (LAMP) assay that directly detects Colletotrichum truncatum in diseased soybean tissues is described, thus allowing rapid diagnosis of soybean anthracnose. Using the target gene Rpb1 (that codes for the large subunit of RNA polymerase II), we designed and screened a set of species-specific primers allowing amplification at 62 °C over 70 min. After addition of SYBR Green I to the LAMP reaction products, a yellow-green color (visible to the unaided eye) developed only in the presence of C. truncatum. The detection limit of the LAMP assay was 100 pg (per μL genomic DNA). The Rpb1-Ct-LAMP assay has been successfully used to diagnose soybean anthracnose in field samples collected from Jiangsu, Anhui and Hubei provinces of China, and to detect C. truncatum in soybean seeds from farmers’ markets. Our results show that the Rpb1-Ct-LAMP assay is a useful and convenient method for detecting C. truncatum, and thus for diagnosis of soybean anthracnose.     

Population dynamics of Pythium aphanidermatum and response of tomato plants as affected by root-zone temperature

Journal of Plant Diseases and Protection - Tomato plants were cultivated in a climate chamber in 12-L-containers with aerated nutrient solution at root-zone temperatures of 20, 25 and 30 °C....