共查询到18条相似文献，搜索用时 109 毫秒
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一种快速简便的植物病原真菌基因组DNA提取方法 总被引：43，自引：3，他引：40
尿素提取法是最新报道的一种植物拟南芥基因组DNA的提取方法,但是还没有应用于植物病原真菌DNA的提取。本研究采用改进的4种不同的方法(尿素提取法、CTAB提取法、氯化苄提取法以及SDS-CTAB提取法),分别对5种分类地位不同的植物病原真菌(草莓疫霉病菌、西瓜蔓枯病菌、草莓炭疽病菌、西瓜枯萎病菌和水稻纹枯病菌)的基因组DNA进行提取和比较。结果表明,尿素提取法和SDS-CTAB法均能提取到所有5种真菌的基因组DNA,而尿素提取法提取DNA的效率更高、质量更好,操作步骤更为快速简单。 相似文献
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小麦白粉病菌基因组DNA的微量提取及ISSR-PCR反应体系的优化 总被引：4，自引：0，他引：4
为了寻找适合小麦白粉菌基因组DNA微量提取的方法,本试验利用改进的CTAB法、电钻微量研磨一管法、FastPrep DNA试剂盒法和Chelex-100法分别提取小麦白粉菌基因组DNA。比较得出,在微量提取时,CTAB法的提取率较高;在分生孢子量较多时,FastPrep DNA试剂盒方法提取的DNA质量较高,这两种方法提取的DNA均适用于ISSR-PCR。采用正交设计L16(45)法优化了适合于小麦白粉病菌群体的ISSR体系,确定了25μL时优化的反应体系:1×buffer、模板DNA0.5ng、dNTP0.14mmol/L、TaqDNA聚合酶1U、引物1.4μmol/L、Mg2+1.8mmol/L。利用该体系筛选出了一批多态性较好的ISSR引物,为小麦白粉病菌遗传多样性研究奠定了基础。 相似文献
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以亚洲小车蝗为研究材料,提出了使用液氮保存蝗虫样本的有效方法,并用基因组DNA提取试剂盒分别对液氮速冻后保存、直接冷冻、无水乙醇保存和干制蝗虫标本进行了基因组DNA的提取和电泳检测。结果表明,在保存3个月后,检测蝗虫样本直接冷冻法和干标本提取的总DNA浓度较低,因而琼脂糖电泳检测亮度低;液氮速冻后保存和无水乙醇保存的蝗虫样本提取的基因组DNA浓度大,琼脂糖电泳检测亮度高。表明液氮速冻后保存和无水乙醇保存的标本适合用于基因组学研究。通过对比,直接冷冻保存与液氮速冻保存结果得出,蝗虫在冷冻胁迫死亡的过程中有DNA降解发生。 相似文献
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土壤真菌分子生态学研究需要大量和高效地提取真菌的DNA。应用市售的一般试剂盒提取土壤虫生真菌DNA,常常存在得率和质量低的问题,甚至根本提取不到土壤真菌的DNA样品。针对这一问题,本研究对细胞破碎方式及后续DNA提取参数进行了一系列的改进和优化,建立了一套针对性的提取方法。该提取法对虫生真菌—蜡蚧轮枝菌纯培养物的灵敏度极高,可在10个孢子的条件下提取到DNA,而市售的试剂盒则不能提取到(试剂盒在107个孢子条件仍提不到DNA);该提取法对人工投菌土样的DNA提取灵敏度达到102个孢子/克土,所得的DNA纯度高,无明显抑制后续PCR扩增的物质;施用过蜡蚧轮枝菌的田间土样所得DNA样品可成功扩增出蜡蚧轮枝菌特异片段,而未施用过蜡蚧轮枝菌的田间土样所得DNA样品不能扩增出蜡蚧轮枝菌特异片段。本提取方法具有灵敏度和纯度高的特点,适用于土壤虫生真菌的分子生态学样品制备。 相似文献
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随着大豆进口量的日益增加,大豆携带病毒传入我国的风险在不断增大,高效、快速地进行植物病毒检测,防止外来有害生物入侵,是目前各口岸植物检疫工作的重点。本文介绍了一种植物病毒检测的新方法—纳米上转换荧光技术。该技术是一种利用磁性纳米颗粒(magnetic nanoparticles, MNP)进行病毒分离、富集和定位;同时引入上转换荧光(up converting phosphor, UCP)材料作为标记物的免疫检测方法,具有高度的抗干扰性、多元性、稳定性和安全性等特点。 相似文献
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matK序列作为DNA条形码在苍耳属中的应用 总被引：1，自引：0，他引：1
以matK序列作为DNA条形码,对从国外截获的7种苍耳属植物进行物种区分鉴定研究,采用DNeasyOPlantMiniKit试剂盒进行总DNA的提取,应用通用引物对其matK基因进行扩增,测序得到7种苍耳属植物的marK序列,利用MEGA5．1软件对这7种苍耳属植物的matK序列进行比对和分析并构建系统树,结果显示：matK序列能从基因位点层面对苍耳属植物进行区分鉴定。 相似文献
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为了找出一种更适于河西走廊地区盐碱土壤微生物总DNA的提取方法,分别运用方法 1(Omega试剂盒法)、方法 2(Mo Bio试剂盒法)、方法 3(CTAB-SDS法)和方法 4(Ca Cl2-SDS-酶解法),对河西走廊盐碱土壤微生物总DNA的提取方法进行比较。4种方法所提取总DNA片段分子量大约在23.1 kb,完整性较好,无明显降解现象。但4种方法所提取DNA的量差异很大,其中方法 1和方法 2提取的DNA目的条带不清晰,但纯度较高;方法 3和方法 4所提取的DNA产量高于方法 1和方法 2,但方法 3提取的总DNA杂质较多,影响后续分析的准确性;而方法4所提取得DNA纯度基本与试剂盒法接近,DNA提取量可达到452.875μg·g-1,远远高于其他3种方法,能满足后续实验,是一种较理想的且适合于河西走廊盐碱土壤微生物多样性和种群结构的总DNA提取方法。 相似文献
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P. Parikka A. Lemmetty 《European journal of plant pathology / European Foundation for Plant Pathology》2004,110(4):393-398
Colletotrichum acutatum, a quarantine organism on strawberries in the EU, was found in Finland for the first time in 2000. Concern about rapid, unnoticeable spread of this pathogen has necessitated studies to find methods with which the quiescent fungus infection can be detected in imported, cold-stored strawberry plant material. Successful detection of C. acutatum in strawberry tissues by polymerase chain reaction (PCR) is dependent on the method of DNA extraction used. Good-quality nucleic acid, free of PCR inhibitors, was successfully prepared by slightly modifying the DNA extraction method of a commercially available kit. Species-specific primers, previously described in the literature, were successfully used in the PCR reaction. C. acutatum was detected by PCR both on symptomatic and asymptomatic plant parts and in artificially and naturally infected strawberry tissues. Positive PCR results were obtained from ripe and unripe berries, runners, petioles and different parts of crowns. The data demonstrate that the PCR technique can be used to detect C. acutatum in strawberry tissue even in plant parts that do not show visible symptoms. 相似文献
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Baswaraj Raigond Ambika Verma Tarvinder Kochhar Shivani Roach Sanjeev Sharma S. K. Chakrabarti 《Phytoparasitica》2018,46(2):255-262
A simple, cost-effective and rapid viral nucleic acid release (NAR) buffer suitable for RT-PCR based diagnostic assay was developed for the detection of potato viruses. The NAR buffer and commercially available RNA isolation kit were compared for RT-PCR based assay, where an amplicon of expected size (~380 bp) targeting PVY was observed in both isolations indicating that it can be used in RT-PCR based diagnostic assays. The same was further validated for its repeatability by running across more than hundred suspected potato leaf samples collected from different sources where, it showed consistent results for the presence of PVY indicating its reliability. The NAR buffer assay was examined for its sensitivity in comparison with the kit based isolation where both the assays were able to detect even up to 10?5 dilution without affecting the sensitivity. NAR buffer was found stable up to 28 days at -20 °C and for 14 days at 4 °C without losing PCR sensitivity. The assay was also found effective to release the nucleic acid from potato leaves, thrips and aphids for PCR and RT-PCR based detection of DNA viruses like ToLCNDV-potato and other RNA viruses. The developed protocol is simple, less laborious, time-saving (10-15 min) and economical (1/100th of kit) as compared to kit based protocol. The assay can be adopted in diagnostic laboratories for detection of RNA/DNA viruses from potato plants and in thrips as well. 相似文献
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A rapid method for direct detection of Polymyxa DNA in soil 总被引：1，自引：0，他引：1
Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis- infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae- infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil. 相似文献
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Asymmetric PCR ELISA: Increased Sensitivity and Reduced Costs for the Detection of Plant Viruses 总被引：2，自引：0，他引：2
Gustavo Nolasco Zita Sequeira Claudia Soares Ana Mansinho Ana M. Bailey Charles L. Niblett 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(4):293-298
PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan, a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest. 相似文献
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构建了固相微萃取(SPME)富集与功能磁性材料净化相结合的一体化协同提取技术,并应用于茶饮料中三唑类杀菌剂的残留分析。以磁性材料四氧化三铁-N-丙基乙二胺 (Fe3O4-PSA)为净化吸附剂,采用SPME萃取针富集样品溶液中的目标农药后直接进气相色谱-质谱联用仪 (GC-MS)进样口解吸附、分析,无需其他样品转移等操作,方法简便、灵敏。本研究系统优化了SPME条件 (如萃取时间、萃取温度、氯化钠用量及pH值),并采用正交试验设计,探明了磁性材料用量与SPME条件的交互影响,以确定最佳试验条件。对优化的分析方法进行方法确证,结果表明:在0.002～0.2 mg/L范围内,19种三唑类农药在茶饮料中均表现出很好的线性关系,相关系数 (r)均大于0.99,检出限 (LOD)在0.00012~0.0089 mg/L之间,定量限 (LOQ)均为0.002 mg/L。采用基质匹配标准溶液校正时,回收率在71%～98%之间,RSD不高于16% (n=3)。此方法较常规法简单、操作便捷、可靠,且环境友好,适用于茶饮料中三唑类农药残留的分析。 相似文献
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A. B. Sahana H. Nagaraja P. K. Maheshwar M. Y. Sreenivasa M. N. Nagendra Prasad C. R. Adkar-Purushothama 《Phytoparasitica》2014,42(4):519-527
Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples. 相似文献
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