共查询到17条相似文献，搜索用时 187 毫秒
1.
双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用 总被引：3，自引：0，他引：3
利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。 相似文献
2.
通过克隆马铃薯环腐病菌和晚疫病菌转录间隔区(ITS)序列,并对测序结果进行同源性比较,选取差异位点分别设计了两对引物P.IN1/P.IN2和C.IN1/C.IN2,并检测了引物的特异性及方法的灵敏度。引物P.IN1/P.IN2可扩增出1条363bp马铃薯晚疫病菌的特异性条带,在DNA水平上其灵敏度达18fg/μL;引物C.IN1/C.IN2可扩增出1条218bp马铃薯环腐病菌的特异性条带,在细菌数上检测灵敏度为104 cfu/mL。混合这两对引物构建双重PCR反应体系,能从马铃薯环腐病菌和晚疫病菌的混合DNA及感染这两种菌的马铃薯植株中同时扩增到363bp和218bp的特异片段。实现了同时对马铃薯晚疫病菌和环腐病菌的快速可靠检测。 相似文献
3.
猕猴桃溃疡病菌的分子检测技术研究 总被引：3，自引：0，他引：3
猕猴桃溃疡病是猕猴桃生产上的主要病害,为建立该病的快速诊断技术,本实验通过RAPD分析获得一条1 300 bp左右的致病菌的特异片段,对该片段进行克隆测序,在测序的基础上设计并合成一对特异引物F7/R7,优化特异引物扩增条件,并验证引物的特异性和灵敏性。利用该特异引物对包括猕猴桃溃疡病菌在内的14个菌株基因组DNA进行PCR扩增表明,只有猕猴桃溃疡病菌能扩增出1条约为950 bp的特异条带,其他菌株及对照均未扩增出特异条带。对采自果园的染病枝干组织和接种致病菌的枝干组织的检测表明,该特异引物能特异性地检测到猕猴桃溃疡病菌的存在,其在组织中的检测灵敏度为100 fg/μL。因此,利用设计合成的特异引物F7/R7,参考优化的体系和程序,结合简单的试剂盒法提取猕猴桃溃疡病菌或植物组织DNA,可以在短时间内完成对该病原菌的分子检测。 相似文献
4.
建立马铃薯晚疫病菌抗甲霜灵SCAR(sequenced characterized amplified region,序列特征扩增区域)标记,以马铃薯晚疫病菌对甲霜灵高抗菌株HD01-3和对甲霜灵高感菌株DK98-1为亲本,通过无性单游动孢子分离和有性杂交获得菌株HD01-3无性后代群体、菌株DK98-1无性后代群体以及F1代分离群体,以此为试验材料,利用BSA法(bulked segregant analysis,分离群体分组分析法)构建抗感基因池对后代菌系的甲霜灵抗性进行RAPD(random amplified polymorphic DNA,随机扩增多态性DNA)分析。从178条RAPD随机引物中找到一条特异性引物S2054,其可以扩增出一个与晚疫病菌对甲霜灵抗性相关的遗传标记,将该特异条带回收、克隆、测序,发现此标记大小为457bp,根据测序结果设计特异PCR引物,用于扩增抗感基因池,成功地将特异RAPD标记转化为SCAR标记。初步建立了马铃薯晚疫病菌抗甲霜灵SCAR标记,辅助监测晚疫病菌对甲霜灵的抗性。 相似文献
5.
马铃薯干腐病是马铃薯最重要的贮藏期病害之一,现已成为马铃薯贮藏期烂窖的重要原因。实现病原菌的快速检测对病害诊断和科学防控具有重要的实践意义。本研究基于镰刀菌翻译延伸因子序列设计了一对检测接骨木镰刀菌Fusarium sambucinum的特异引物Fs-F/Fs-R。该特异引物可从接骨木镰刀菌中获得309bp的特异性扩增片段,而其他种类镰刀菌及马铃薯重要病害病原菌中均无此片段,说明该引物具有专一性。该体系的灵敏度检测结果表明,最低能检测到的接骨木镰刀菌基因组DNA浓度为70pg/μL。该引物也适用于发病马铃薯块茎中接骨木镰刀菌的快速检测。 相似文献
6.
寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术,本研究首先采用通用引物 ITS1／ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的 ITS 区进行 PCR 扩增和测序比对。根据该片段与 GenBank 中隐丛赤壳属其他种的 ITS 序列差异,设计了寄生隐丛赤壳菌的特异性引物 ITSP1／ITSP2,片段扩增大小为462 bp。利用该引物对菌株基因组 DNA 进行扩增,可以将寄生隐丛赤壳菌与其他参试菌区分开,检测灵敏度达30 pg。而以引物 ITS1／ITS4和 ITSP1／ITSP2进行的巢氏 PCR,可检测到30 fg 基因组 DNA,其灵敏度较常规 PCR 提高了1000倍。利用巢氏 PCR 检测体系对发病程度不同的组织和携菌组织进行检测,均能快速稳定地检测出寄生隐丛赤壳菌。 相似文献
7.
寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术,本研究首先采用通用引物ITS1/ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的ITS区进行PCR扩增和测序比对。根据该片段与GenBank中隐丛赤壳属其他种的ITS序列差异,设计了寄生隐丛赤壳菌的特异性引物ITSP1/ITSP2,片段扩增大小为462bp。利用该引物对菌株基因组DNA进行扩增,可以将寄生隐丛赤壳菌与其他参试菌区分开,检测灵敏度达30pg。而以引物ITS1/ITS4和ITSP1/ITSP2进行的巢氏PCR,可检测到30fg基因组DNA,其灵敏度较常规PCR提高了1 000倍。利用巢氏PCR检测体系对发病程度不同的组织和携菌组织进行检测,均能快速稳定地检测出寄生隐丛赤壳菌。 相似文献
8.
应用PCR方法快速检测黄瓜细菌性角斑病菌 总被引：1，自引：0，他引：1
黄瓜细菌性角斑病是黄瓜上的一种重要细菌病害,其病原为丁香假单胞菌黄瓜致病变种(Pseudomonas syringae pv.lachrymans),目前未见到该病害特异性PCR检测方法的报道。通过分析丁香假单胞菌(P.syringae)不同致病变种glyceraldehyde-3-phosphate dehydrogenase 1(gap1)基因序列设计得到一对Psl特异性PCR引物。利用该引物对丁香假单胞菌不同致病变种、假单胞菌属其他种及其他属的共46株菌株进行了PCR扩增,结果表明,所有不同来源的12株黄瓜细菌性角斑病菌均得到179bp的目标片段,而所有其他参试菌株均无扩增条带,PCR检测的灵敏度为7.5×103cfu/mL。利用该方法可从接种后发病的黄瓜叶片总DNA中检测到特异条带,而健康叶片无条带。该引物的PCR检测方法可直接用于植株总DNA的检测,无需进行病原菌的分离培养,快速简便,适用于进出境检验检疫及种苗健康检测等。 相似文献
9.
我国植物青枯菌菌株的遗传多样性和组群划分 总被引：14，自引：0，他引：14
采用15条随机引物对我国11个省(市、区)6种不同寄主植物的43个青枯菌代表性菌株和4个国外青枯菌菌株,进行了PCR扩增.引物OPB11、OPA15、OPE1和OPZ10对上述所有菌株扩增获得了相似的产物电泳图谱,分别具1~5条谱带不等;引物OPB7、OPA10和OPF1对马铃薯菌株获得了相同的产物图谱,但对其它寄主菌株的产物间有明显差别;引物OPA14、OPC,6、OPG14、OPF5、OPK14、OPK20和OPK17对于不同菌株的扩增产物多态性很强.供试菌株被聚类为2个组群,即组群A和组群B.组群A中又可分为7个亚组(A1、A2、A3、A4、A5、A6、A7),其中A1含有2个类型(A1-1、A1-2);组群B中也可分为2个亚组(B1、B2),其中B1含有3个类型(B1-1、B1-2、B1-3),B2也含有3个类型(B2-1、B2-2、B2-3).RAPD组群A中包含了27个来自我国不同地区的马铃薯菌株,主要是3号小种、生化变种2;组群B中含有20个来自不同地区、不同寄主的菌株,分属于其它不同的小种和生化变种.研究结果表明,我国青枯菌菌株RAPD组群的划分与菌株的地理来源关系不大,而与寄主来源有明显相关性.此外,通过对我国青枯菌菌株组群进行同源性PCR分析表明,来源自马铃薯的3号小种菌株属于美洲分支"Americanmm",而来自其它寄主的青枯菌1号、5号小种菌株属于亚洲分支"Asiaticum",与本研究RAPD组群A和组群B的划分是一致的. 相似文献
10.
大豆根腐病致病镰孢菌的多重PCR检测技术 总被引：3，自引：0，他引：3
为建立大豆根腐病镰孢菌的多重PCR检测方法,以四川大豆根腐病致病菌包括尖孢镰孢菌、腐皮镰孢菌、禾谷镰刀菌和木贼镰孢菌为对象,设计镰孢菌翻译延伸因子基因EF-1α的种特异引物,建立多重PCR扩增体系,并进行优化与验证。结果表明:25μL体系为最优镰孢菌多重PCR扩增体系,4种镰孢菌等体积混合DNA 4.0μL,各镰孢菌特异正向引物1.0μL,共用反向引物4.0μL,最佳退火温度为54℃,当循环30次时,能清晰地扩增出各镰孢菌EF-1α条带,对4种镰孢菌混合DNA的检测灵敏度可达0.1 ng/μL。室内环境样本验证结果表明,依据EF-1α扩增片段大小,该体系能够特异地检测出大豆黄化苗与致病镰孢菌混合样本中的镰孢菌,但无法从其它真菌的DNA中扩增获得目的片段。表明基于EF-1α基因特异引物建立的镰孢菌多重PCR检测技术可快速、特异地检测大豆根腐病镰孢菌。 相似文献
11.
Rapid and sensitive polymerase chain reaction (PCR) methods are described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R. solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74–97, 455–475, 1454–1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division II (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii , or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum , 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains. 相似文献
12.
13.
12种寄主来源的茄科雷尔氏菌16S-23SrDNA间隔区序列比较 总被引：2，自引：0，他引：2
应用PCR方法,获得了分离自广东番茄、茄子、辣椒、烟草、空心菜、沙姜、姜、马铃薯、花生、菊花、桑树和藿香等12种作物21个茄科雷尔氏菌菌株的16S 23S rDNA 间隔区序列（ITS）。序列分析结果表明,除HZ 1菌株外,其余20个茄科雷尔氏菌菌株ITS序列长均为503 bp,序列间相似性99.2%~100%,序列间差异仅1~4 bp;而HZ 1菌株的ITS序列长为498 bp,与其他菌株的ITS序列相似性为95.4%~95.6%。这些结果说明,这21株来源于12种不同寄主的茄科雷尔氏菌菌株的16S 23S rDNA ITS序列比较保守。系统进化分析显示,仅菌株HZ 1聚类于茄科雷尔氏菌区组2中,其余20个菌株均聚类于茄科雷尔氏菌区组1中。 相似文献
14.
A. Marefat K. Ophel-Keller E. S. Scott M. Sedgley 《European journal of plant pathology / European Foundation for Plant Pathology》2006,116(1):57-68
Pistachio dieback occurs in the main pistachio growing areas of Australia. Xanthomonas strains belonging to the translucens group have been identified as the causal agent of the disease and two distinct groups, A and B, have been recognised within the pathogen population. In this study, specific primers for amplification of DNA of the pathogen were developed by sequencing the Internal Transcribed Spacer (ITS) region of rDNA from strains representing groups A and B, as well as from X. translucens isolated from wheat in Australia and one Xanthomonas
translucens strain from orchard floor grasses. Primers were designed for amplification of DNA sequences specific to each group and a multiplex PCR test was developed that identified and differentiated strains of each group in a single PCR assay. To determine the specificity of the primers, PCR was carried out with DNA from 65 strains of the pistachio pathogen, 31 type and reference strains of Xanthomonas, and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis, which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material. 相似文献
15.
B. P. Borowicz 《EPPO Bulletin》2001,31(4):489-491
A study has been performed to identify Clavibacter michiganensis subsp. insidiosus at the molecular level, using the polymerase chain reaction (PCR) technique with oligonucleotide primers based on specific sequence recognition of the intergenic spacer region between the 16S and 23S rRNA genes. The pair of primers was designed on the basis of available DNA sequence data for that region in C. m. insidiosus and other bacteria. Using this pair of primers, a large amount of an amplified DNA fragment of 218 bp in length was obtained from C. m. insidiosus. The specificity of this amplification was proved by PCR analysis, using the above-mentioned pair of primers and templates from different bacteria, some related to C. m. insidiosus. The PCR products were analysed using agarose gel electrophoresis. 相似文献
16.
A total of 23 Scottish and 14 Dutch potato R-gene differentials as well as five Austrian, two Dutch and two German commercial potato cultivars were screened for the R1 allele conferring resistance to Phytophthora infestans carrying Avr1 , via PCR amplification and sequencing. A single 1400 bp fragment with complete sequence identity to the corresponding part of the R1 allele, was obtained from genomic DNA of all potato R-gene differential clones whose denomination indicates R1 or a combination of R1 and other major resistance factors. The R1 allele was detected, as expected, in all these clones. This fragment also occurred in one Austrian and one German cultivar. Unexpectedly, the same R1 allele also was detected within all R5, R6 and R9 differentials. 相似文献
17.
The diversity of 40 strains of Ralstonia solanacearum causing bacterial wilt of potato in the major potato-growing areas of Iran was assessed. Based on rep-PCR genomic fingerprinting, strains fell into two distinct groups. The first group contained 37 of the 40 strains and the second consisted of three strains from a narrow tropical region in Iran. The three strains from the narrow tropical region were found to be phenotypically and genotypically most similar to R. solanacearum biovar 2T strains, whereas all other strains were phenotypically and genotypically identified as being R. solanacearum biovar 2/race 3. Phylogenetic analysis of endoglucanase gene sequence information of two of the strains from the tropical region revealed that they belonged to phylotype II of the R. solanacearum species complex and had 100% sequence similarity to a biovar 2T strain from potato in Peru. This is the first report of the presence of R. solanacearum phylotype II/biovar 2T in Iran and the first report of the existence of this group of R. solanacearum outside South America. 相似文献