利用TaqMan探针实时荧光PCR方法检测香石竹细菌性萎蔫病菌

 根据香石竹细菌性萎蔫病菌基因组16S-23S rRNA保守序列,设计并合成了一对特异性引物和一条具有稳定点突变特异性探针,建立了对香石竹细菌性萎蔫病菌的TaqMan实时荧光PCR检测方法。除香石竹细菌性萎蔫病菌外,还对其他7种病原细菌菌株进行了荧光PCR检测。结果表明,只有香石竹细菌性萎蔫病菌产生荧光,其他病原细菌均没有荧光产生。与常规PCR相比,实时荧光PCR检测特异性强,灵敏度高,能检测到浓度为0.4 pg/μL的DNA,且能直接用于苗木等样品的检测,适合病害的快速诊断和口岸检验检疫应用。      

玉米细菌性枯萎病菌TaqMan探针实时荧光PCR检测方法的建立

成功建立了玉米细菌性枯萎病菌快速检测鉴定的实时荧光PCR方法.该方法根据细菌16S rDNA序列的特异性,设计出对玉米细菌性枯萎病菌具有稳定性点突变特异性探针,并对10种细菌菌株和5种植原体进行了实时荧光PCR.结果表明,只有玉米细菌性枯萎病菌产生荧光信号,而其它参考菌不产生荧光信号,检测的绝对灵敏度是14.2 fg/μl质粒DNA,比常规的PCR电泳检测高约100倍.整个检测过程只需2h,完全闭管,降低了污染的机会,无须PCR后处理.     

植原体TaqMan探针实时荧光PCR检测鉴定方法的建立

 本研究成功建立了植原体分类鉴定和检测的TaqMan探针实时荧光PCR方法,该方法根据植原体16S rDNA保守区设计了1个TaqMan广谱探针和3个植原体组间点突变特异性探针,并对9种植原体和5种细菌以及3个植物样本进行实时荧光PCR。结果表明,用广谱探针可检测到所有植原体产生荧光信号,而细菌不产生荧光信号。当用植原体组间特异性探针检测时,仅能检测到该组植原体产生荧光信号,检测的敏感性比常规的PCR-电泳检测高约100倍、检测速度有较大提高。由于PCR产物是荧光探针检测,本方法特异性强,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法。     

枣疯病植原体TaqMan探针实时荧光定量PCR检测方法的建立

根据枣疯病植原体16S rDNA基因保守区域设计、合成特异性引物和TaqMan探针, 以构建的重组质粒作为阳性标准品, 建立并优化了对枣疯病植原体的TaqMan实时荧光定量PCR检测方法。对优化后的方法进行灵敏度、特异性及稳定性评价, 制作了标准曲线。结果显示, 制作的标准曲线有极好的线性关系, 相关系数( r 2 )达到0.998, 建立的实时荧光定量PCR检测方法能够特异性地检测枣疯病植原体, 能检测到60拷贝的质粒DNA。本研究建立的实时荧光定量PCR检测方法灵敏度、特异性、重复性好, 不仅能够实现对枣疯病植原体的快速检测, 而且为实现从病原定量水平上对枣疯病病情分级奠定了基础。     

大豆茎溃疡病菌TaqMan MGB探针实时荧光PCR检测

 The technique of TaqMan MGB real-time fluorescent PCR was established to detect Diaporthe phaseolorum var.caulivora (DPC) and D. phaseolorum var.meridionalis (DPM). The primers and TaqMan MGB probes were designed based on the ITS of DPC, DPM, D. phaseolorum var. sojae and Phomopsis longicolla. A series of genomic DNA dilution were used to detect sensitivity of the technique, the results showed that the limits of detection for DPM and DPC were 7 fg/μL and 6 fg/μL DNA respectively.     

利用实时荧光PCR技术检测风信子黄腐病菌

风信子黄腐病菌是我国禁止入境的病原细菌之一,我国目前尚无该病的发病报道.本研究根据风信子黄腐病菌基因组16S-23S ribosomal RNA intergenic spacer保守序列,设计并合成了1对特异性引物和1条具有稳定点突变特异性探针进行实时荧光PCR检测,风信子黄腐病菌有很强的荧光信号,供试的其它7种病原细菌菌株均没有荧光产生.与常规PCR相比,实时荧光PCR检测特异性强,灵敏度高,适合病害的快速诊断和口岸检验检疫应用.经优化反应条件,建立了稳定的风信子黄腐病菌实时荧光PCR检测方法.     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

TaqMan探针实时荧光PCR快速检测苹果牛眼果腐病菌方法的建立

苹果牛眼果腐病菌(Neofabraea malicorticis、N.perennans、N.alba和N.kienholzii)是我国检疫性植物病原真菌,为建立该病菌的实时荧光PCR检测法,根据病菌及其近缘种的翻译延伸因子(EF-1α)的保守序列设计了特异性探针,分别以苹果牛眼果腐病菌菌株和本研究构建的EF-1α重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。结果显示探针MAL-P、PER-P、ALB-P和KIE-P分别对N.malicorticis、N.perennans、N.alba和N.kienholzii表现特异性阳性扩增,而与近缘种及其他常见的果腐病菌无交叉反应,单重探针和4种探针混合液的灵敏度分别达1 fg/μL和10 fg/μL DNA。总计从美国、智利、新西兰、法国进境截获的29批可疑病果的分离物中检测到PCR阳性荧光信号,包括20批N.perennans、8批N.alba和1批N.kienholzii。该方法在6 h内即可完成整个检测流程,其特异性强、灵敏度高,适用于实际样品中苹果牛眼果腐病菌的快速检测。     

薄壳山核桃疮痂病菌TaqMan荧光定量PCR检测方法的建立及应用

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源。准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据。本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法。特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号。本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL。该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法。本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具。     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

番茄溃疡病菌的PCR-DHPLC快速检测

根据番茄溃疡病菌ITS序列,设计并合成了PCR-DHPLC检测引物,对番茄溃疡病菌及其他病菌共10个标准菌株进行了PCR-DHPLC检测。结果表明,番茄溃疡病菌的PCR-DHPLC检测图谱出现了特异性吸收峰,而其他病菌均未在相同洗脱时间出现吸收峰,说明这种方法具有检测番茄溃疡病菌的特异性。灵敏度实验结果表明,PCR-DHPLC体系与PCR-琼脂糖凝胶电泳体系的检测灵敏度一致。研究表明,PCR-DHPLC方法是一种特异、灵敏、快速的番茄溃疡病菌检测方法。     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。     

中国北方番茄细菌性溃疡病菌的rep-PCR基因指纹图谱分析

对来自全国若干省市的番茄细菌性溃疡病菌(Clavibacter michiganensis subsp.michiganensis)的33个菌株进行rep-PCR分析。结果表明,用引物BOX分别扩增出6～15条多态性条带,条带大多数集中在500～2800bp之间;用引物ERIC扩增的条带不清晰,可能是反应条件不适合,也可能是其不适合对番茄细菌性溃疡病菌进行多态性分析;BOX-PCR表明番茄细菌性溃疡病菌菌株具有丰富的遗传多态性和较大的遗传变异,对产生的指纹图谱进行分析:在遗传距离为0.18时,测试的33个菌株可划分为Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ和Ⅶ等7个遗传相似组群,其中Ⅵ组群包含的菌株最多。研究还表明番茄细菌性溃疡病菌的遗传分群与菌株的地理来源关系密切,但与该病的发病年份上没有必然联系,这也从另一侧面说明了该病害是土壤带菌引起的。     

基于TaqMan MGB探针的花生矮化病毒检测研究

花生矮化病毒(Peanut stunt virus,PSV)是我国进境检疫性有害生物。本研究根据该病毒不同分离株外壳蛋白基因(coat protein,CP)的保守序列,设计了特异性引物与TaqMan MGB荧光探针,建立了PSV的实时荧光RT-PCR检测方法。方法特异性研究表明,针对PSV的2个不同株系PSV-E和PSV-W,均能够得到典型扩增曲线,Ct值分别为20.10和21.22;而对于黄瓜花叶病毒、番茄不孕病毒、马铃薯Y病毒、菜豆荚斑驳病毒以及烟草环斑病毒等其他毒株则没有典型扩增曲线,也无Ct值。灵敏度比较发现,该方法比普通RT-PCR检测方法的灵敏度提高100倍,具有快速、灵敏和高特异性的优点,适合对PSV的检测。     

Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation

Streptomycin has been used for decades in Chile to control Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker. The aim of this work was to evaluate streptomycin resistance and to analyse the presence of resistance-related genes in Cmm strains from Chile. A collection of 25 Cmm strains isolated from different localities in central Chile between 1996 and 2015 was analysed. Minimum inhibitory concentration (MIC) of streptomycin was determined. A search of streptomycin resistance-related genes was carried out in Cmm genomes, and the presence of these genes was studied in all Chilean strains using PCR and sequencing techniques. MIC results showed that four of 25 strains were highly sensitive to streptomycin, with MIC values <2 μg mL⁻¹. The remaining 21 strains possessed MIC of streptomycin ≥100 μg mL⁻¹. The strB gene, encoding an aminoglycoside 6-phosphotransferase that inactivates streptomycin, was detected in all Chilean strains, including sensitive and resistant strains. In the 21 resistant strains, a mutation in codon 43 of the rpsL gene was determined, conferring high streptomycin resistance. Interestingly, the four streptomycin-sensitive Cmm strains did not possess this mutation. This study proposes that the continuous use of streptomycin leads to emergence of resistant Cmm strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium.     

双重PCR检测马铃薯晚疫病和环腐病方法的建立

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。     

Survival of Clavibacter michiganensis subsp. michiganensis in tomato debris under greenhouse conditions

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is one of the most important diseases of tomato worldwide. Once the pathogen has been introduced into an area, i.e. by contaminated seeds or transplants, it survives mainly on host debris. In different geographic areas the survival time of the pathogen in crop residues under field conditions has been very variable, ranging from 2 months in Morocco to 2 years in Iowa (USA). This study took place in the horticultural belt of Buenos Aires – La Plata, Argentina, where greenhouse production prevails, and monoculture with two production cycles per year is a common practice. The aim was to determine the survival time of this pathogen in plant residues left on the soil surface or buried. During three consecutive years, by the end of both production cycles in July (winter) and December (summer), above‐ (stem, petiole) and belowground (root) tissues were placed into nylon netting bags and left on the soil surface or buried at 10 cm depth. The pathogen population was regularly quantified by dilution plating on semiselective medium. In host debris left on the soil surface, bacteria survived 120–260 days for crop production cycles that ended in winter and 45–75 days for those that ended in summer. In stems or roots buried in winter, this period was 45–75 days. It is concluded that host debris, including roots, might be an important primary inoculum source of the pathogen in greenhouses.     

Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper‐based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable (VBNC) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO₄ and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO₄ and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO₄ retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post‐inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram‐positive phytopathogenic bacteria. Most importantly, because copper‐based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture‐based assays yet present a risk for disease development in the field.     

Clonal populations of Clavibacter michiganensis subsp. michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid‐encoded genes pat‐1 and celA were detected by PCR in all tested strains, except for four Sicilian Cmm strains where the pat‐1 gene was not amplified. Using BOX‐PCR, Cmm strains were differentiated into 13 haplotypes and clonal populations were identified. Cmm strains isolated from different farms in 2008 showed the same BOX‐PCR haplotype. A distinct BOX‐PCR haplotype was obtained from atypical Cmm strains lacking pat‐1 and isolated in 2006/7 from three farms. Cmm strains with two different haplotypes were detected in one farm, whereas the other farms contained strains with only a single haplotype. A new fAFLP protocol based on the amplification of ApaI/MseI fragments was developed and was able to differentiate C. michiganensis subspecies. Different populations were delineated for the multiple outbreaks occurring in Sicily, whereas similar populations were recorded in other Italian regions over a period of 12 years. The results are consistent with previous studies that demonstrate that Cmm outbreaks are associated with propagation material.