样本保存方法及冷冻胁迫对蝗虫基因组DNA提取的影响

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。     

检疫性真菌DNA提取方法的比较

为获得一种简便、快速、较低费用的DNA提取方法,用于口岸检疫性真菌后续识别检测工作。本文系统比较了8种不同提取方法对DNA浓度、纯度、DNA中蛋白质含量、以及后续PCR扩增、测序的影响,同时对不同方法的提取时间、价格、原理等进行了比较。结果表明,基于磁珠法提取的DNA得率较高,耗时较少,但较易蛋白质污染;CTAB法得率与磁珠法相似,但耗时最长,人力成本高;膜吸附法得率较低,但纯度较高。     

油菜籽中油菜茎基溃疡病菌基因组DNA提取方法对比

比较磁珠法、离心柱法、CTAB法及快速DNA裂解检测试剂对油菜籽中油菜茎基溃疡病菌的检测结果,综合考虑各提取方法的效率及成本。结果显示离心柱法性价比最高,快速DNA裂解检测试剂对油菜籽中油菜茎基溃疡病菌快速筛查具有更佳的应用价值。     

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

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。     

柑橘黄龙病病原DNA微量提取方法比较

对比分析了3种黄龙病病原DNA微量提取方法,方法1和方法2分别采用Sandrine等人和Hung等人的黄龙病病原DNA提取方法,但取样量分别由100mg和250mg减少为20mg和10mg;方法3采用周常勇等人的微量核酸提取方法,取样量为10mg.实验结果表明:方法1提取的病原DNA浓度低、杂质多,PCR效果差;方法2提取的病原DNA浓度较高、杂质少,PCR效果好,但提取周期较长,不适合大批量样品的PCR快速检测;方法3提取的病原DNA浓度较高、杂质少,PCR效果好,且提取速度快,适宜大批量样品的PCR快速检测.     

蚜虫内共生菌基因组DNA提取方法的比较和优化

为探究蚜虫共生菌基因组DNA的提取方案,以桃蚜为试验材料,比较了目前较为常用的4种蚜虫基因组DNA提取方法,从DNA纯度、完整性、PCR扩增效率及稳定性等方面进行了比较和评价,并通过调整蛋白酶K用量和水浴温度及时间对STE法进行了优化。结果表明,4种方法提取的蚜虫共生菌基因组DNA均可用于共生菌的PCR扩增检测;CTAB法和SDS法提取的DNA纯度较高,条带较完整,稳定性相对较高,不易降解,而STE法和PCR缓冲液法操作简便,适于快速提取单头蚜虫共生菌的基因组DNA,但纯度相对较低;可根据试验条件和要求进行选择。STE法优化条件为:用30 μL STE缓冲液将蚜虫匀浆,加入1.5 μL 20 mg/mL蛋白酶K,于56 ℃水浴1.5 h;再加入0.1 μL 10 mg/L RNA酶,于37 ℃培养1 h,95 ℃下处理5 min,5 000 r/min离心3 min,将提取到的DNA于-20 ℃保存或直接用于PCR扩增。优化后的STE法可作为提取蚜虫共生菌基因组DNA经济而快捷有效的方法。     

啤酒花潜隐类病毒RNA提取方法及检测技术的研究

本研究采用纳米磁珠提取法(Magnetic Nanoparticles,MNP)、改良的Li Cl沉淀法、CTAB法、TRIzol法和RNeasy Plant M ini Kit 5种方法提取感染啤酒花潜隐类病毒(Hop latent viroid,HLVd)的啤酒花叶片总RNA,结果显示Li Cl沉淀法、CTAB法和M NP法提取的RNA的质量较好,而M NP法具有提取时间短、操作简便,环境友好和批量提取的优势,适用于啤酒花潜隐类病毒RNA的快速提取。体外转录制备HLVd RNA标准品,利用实时荧光定量RT-PCR绘制标准曲线,并对其特异性、灵敏度进行评估。应用纳米磁珠提取啤酒花总RNA,结合实时荧光RT-PCR技术,建立了HLVd的快速、高效的M NP-RT-q PCR定量检测方法。     

一种快速简便的植物病原真菌基因组DNA提取方法

 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。     

河西走廊地区盐碱土壤微生物总DNA提取方法的比较

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。     

苜蓿假盘菌基因组DNA提取方法

以保定苜蓿上分离的苜蓿假盘菌为试验材料,采用SDS、SDS-CTAB及氯化苄3种方法提取基因组DNA,并进行比较。结果表明:3种方法均能提取到DNA,其中氯化苄法效果最佳,获得的DNA纯度高,此法操作简单、经济;其次是SDS-CTAB法,但DNA有所降解;而SDS法效果最差。此外还较详细地研究了采用氯化苄法提取缓冲液pH值、液氮和蛋白酶K对DNA提取效果的影响。     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

A rapid method for detecting and quantifying bacterial DNA in rust fungal DNA samples

Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at > or = 1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was approximately 70 pg/mul or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.     

Development of a PCR-based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium-aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small-subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii , a plasmodiophorid pathogen of watercress ( Rorippa nasturtium-aquaticum ). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration-based method for obtaining pathogen DNA for PCR from watercress-bed water was developed.     

Nuclear DNA analysis of maize seedlings treated with the triazole fungicide,triticonazole

Maize kernels were treated with the fungicide triticonazole (RP-727), belonging to the triazole family. The nuclear DNA content was analyzed in various maize plant organs by using flow cytometry. Two fluorochromes with different DNA binding mechanisms were used for probing nuclear DNA and chromatin structure. Results indicated nuclear alterations with the use of triticonazole and the alterations observed were different in the various tissues analyzed. Active meristematic plant organs, such as root tip, did not show any significant change in nuclear DNA, but chromatin structure was altered with increasing concentrations of fungicide. Stem and mesocotyl showed both DNA and chromatin condensation changes with triticonazole concentration, but the trends observed in these two plant organs were opposite. The magnitude of the alterations was minute in the different plant organs studied. These alterations do not appear to be deleterious for normal plant development.     

Early brown rot infections in sweet cherry fruit are detected by monilinia-specific DNA primers

ABSTRACT Visible and nonvisible quiescent infections of immature and mature fruit are an integral component of the disease cycle of brown rot of sweet cherry in California. Detection of these infections is critical for developing efficient and efficacious fungicide management programs. The previously published DNA amplification primers mfs3 and NS5 for the identification of Monilinia fructicola were very specific in amplifying DNA of M. fructicola only and not M. laxa. This primer set, however, only detected DNA from some of the California isolates of M. fructicola. This genetic diversity was supported by random amplified polymorphic DNA (RAPD) analysis. Using eight 10-mer primers, seven M. fructicola isolates from California were all identified as genetically distinct. Using the same primers, only one polymorphism was detected among seven isolates of M. laxa. The multiple genotypes identified within the small population sample of M. fructicola, but not of M. laxa, using RAPD analysis could be indicative of genetic recombination within M. fructicola but not within M. laxa. To detect early brown rot infections in fruit, two primer sets that were developed from DNA sequences of either ribosomal DNA (MF5/ITS4/ITS3) or a RAPD fragment (X-09intF3/X-09R) specifically amplified DNA from isolates of M. fructicola and Monilinia species, respectively. No amplification products were present when using DNA from Botrytis cinerea or from other fungi commonly found on sweet cherry fruit. Primers X-09intF3 and X-09R were more sensitive and reliable for detecting small amounts of target DNA either extracted from conidia or from laboratory-inoculated cherry fruit with early brown rot infections that showed no visual symptoms or with visible quiescent infections. Furthermore, these primers also were effective for detecting visible quiescent infections in cherry fruit that were collected in the field.     

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.     

Development of Contamination-Free Restriction Fragment Length Polymorphism Probes for the Obligate Biotroph Peronospora tabacina, an Oomycete Causing Blue Mold of Tobacco

ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism.     

禾谷镰刀菌和稻瘟病菌基因组中的微卫星序列比较

利用禾谷镰刀菌Fusarium graminearum和稻瘟病菌Magnaporthe grisea基因组测序结果,对这两种植物病原真菌基因组中的微卫星(SSR)序列进行了系统地分析和比较.结果表明,在禾谷镰刀菌基因组中,共发现4679个SSR序列,总长度为96.2kb,占基因组全长的0.27%.平均7.7kb碱基中有一个大于15 bp的SSR序列.在稻瘟病菌基因组中共发现16398个SSR系列,其总长度达到330kb,约占整个基因全长的0.85%,平均2.36kb碱基中就分布有1个SSR序列.在禾谷镰刀菌基因组中,数量最多的是五碱基重复序列,其次是六碱基重复序列;稻瘟病菌基因组中数量最多的是单碱基重复序列,其次为三碱基重复序列和五碱基重复序列.两基因组中数量最少的都是二碱基重复序列.尽管这两种植物病原真菌都属子囊菌,基因组大小也十分接近,但无论是在SSR的总体数量上,还是在各类SSR的分布上,两种植物病原真菌都存在十分显著的差别.