水体中根肿菌孢子实时荧光定量PCR检测体系的建立
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摘要
【目的】十字花科作物根肿病是一种世界性病害,早期准确定量检测病原是实现有效防控的基础,研究旨在建立一种应用于水体中检测根肿菌的实时荧光定量PCR方法。【方法】采用近年已研究并发表的一对引物。建立并优化RT-PCR反应体系,利用大肠杆菌TG-1、大肠杆菌T1-1、大肠杆菌DH5α、肺炎克雷伯菌E3、枯草芽孢杆菌XF-1、解淀粉芽孢杆菌Y2、柑橘黄龙病病原菌、假单胞菌E12、成团泛菌L9、阴沟肠杆菌C6、荧光假单胞菌df等11种病原物进行RT-PCR特异性检测,并对灵敏度、可重复性进行评价。利用该体系分别检测不同接种浓度的水体中的根肿菌。【结果】得出标准曲线的回归方程为Y=-3. 452X+35. 118,其回归系数R2=0. 996,扩增曲线在1×101～1×107拷贝/μl范围内,具有较好的线性关系,扩增效率94. 833%,相邻扩增曲线间距均匀,1×101拷贝/μl为最低检测值。熔解曲线显示,所有产物具有单一峰形,有高度扩增特异性。重复性试验变异系数小于1%。在人工接种的水体中,检测下限至少为102个孢子/m L。【结论】建立的实时荧光定量PCR方法具有快速、特异、敏感和稳定等优点,为检测根肿菌在水体中的含量提供了技术方法。
【Objective】Club root is a kind of worldwide disease in cruciferous plant. The quantification detection in early stage is the basic to control disease efficiently. The purpose of this research was to build aquantification detection method of club root pathogens in water samples.【Method】We built and improved the RT-PCR reaction system with a pair of primers which had been reported in recent years. In the same time we tested specificity by several kind of microbeings,such as E. coil TG-1,E. coil T1-1,E. coil DH5α,Klebsiella pneumoniae E3,Bacillus subtilis XF-1,Bacillus amyloliquefaciens Y2,Candidatus liberibacter,Pseudomonas E12,Pantoea agglomerans L9,Enterobacter cloacae C6 and P. fluorescens df,sensibility and repeatability of this primer. We used this system to detect the water samples with differentconcentration of P. B resting spores. 【Result】The equation of the standard curve was Y =-3. 452 X + 35. 118,R2= 0. 996. The amplification curve was between 1 × 101-1 × 107 copies/μl with good linear relation. The efficiency of reaction was 94. 833 % and the distance of interfacing curve was approximately the same. The melting curve had a single peak which showed higy specificity. The limit of detection was 1 × 101 copies/μl and the c. v. was lower than 1 %. We amplified this technology in artificial water sample which inoculated with different concentration of P. B resting spores which showed that the limit was 1 × 102 Cfu/μl at least.【Conclusion】The quantification detection system we built had many advantages such as fast,high specificity,high sensitivity and steady,which provided a method for quantification detection of club root pathogens in water.
【Objective】Club root is a kind of worldwide disease in cruciferous plant. The quantification detection in early stage is the basic to control disease efficiently. The purpose of this research was to build aquantification detection method of club root pathogens in water samples.【Method】We built and improved the RT-PCR reaction system with a pair of primers which had been reported in recent years. In the same time we tested specificity by several kind of microbeings,such as E. coil TG-1,E. coil T1-1,E. coil DH5α,Klebsiella pneumoniae E3,Bacillus subtilis XF-1,Bacillus amyloliquefaciens Y2,Candidatus liberibacter,Pseudomonas E12,Pantoea agglomerans L9,Enterobacter cloacae C6 and P. fluorescens df,sensibility and repeatability of this primer. We used this system to detect the water samples with differentconcentration of P. B resting spores. 【Result】The equation of the standard curve was Y =-3. 452 X + 35. 118,R2= 0. 996. The amplification curve was between 1 × 101-1 × 107 copies/μl with good linear relation. The efficiency of reaction was 94. 833 % and the distance of interfacing curve was approximately the same. The melting curve had a single peak which showed higy specificity. The limit of detection was 1 × 101 copies/μl and the c. v. was lower than 1 %. We amplified this technology in artificial water sample which inoculated with different concentration of P. B resting spores which showed that the limit was 1 × 102 Cfu/μl at least.【Conclusion】The quantification detection system we built had many advantages such as fast,high specificity,high sensitivity and steady,which provided a method for quantification detection of club root pathogens in water.
引文
[1]陈瑶,王火旭.十字花科根肿病的研究进展[J].天津农业科学,2014,20(3):77-79,85.
[2]Verma S S,Rahman M H,Deyholos M K,et al. Differential expres-sion of miRNA in Brassica napusroot following infection with Plasmo-diophora brassicae[J]. PLo S ONE,2014,9(1):e86648.
[3]索欢,陈龙正,徐海,等.十字花科根肿病研究进展[J].安徽农业科学,2015,43(14):115-117,126.
[4]Plasmodiophora brassicae. Distribution maps of plant diseases[R].Wallingford,UK:CAB International,1977.
[5]孙保亚,沈向群,郭海峰,等.十字花科植物根肿病及抗病育种研究进展[J].中国蔬菜,2005(4):34-37.
[6]于晓坤,吴毅歆,毛自朝,等.水介导十字花科作物根肿病的传播及其化学防治[J].华中农业大学学报,2013,32(1):48-53.
[7]Xu X G,Liu Y T. Application of real-time fluorescence quantitativePCR in plant disease[J]. Chinese Agricultural Science Bulletin,2009,25(7):52-56.
[8]Faggian R,Bulman S R,Lawrie A C,et al. Specific polymerase chainreaction primers for the detection of Plasmodiophora brassicae in soiland water[J]. Phytopathology,1999,89:392-397.
[9]Ito S,Mathara T,Marund E,et al. Development of a PCR-based assayfor the detection of Plasmodiophora brassicaein soil[J]. Journal ofPhytopathology,1999,147:83-88.
[10]Wallenhammar AC,Arwidsson O. Detection of Plasmodiophora bras-sicae by PCR in naturally infested soils[J]. Eur J Plant Pathol,2001,107:313-321.
[11]Cao T,Tewari J,Strelkov SE. Molecular detection of Plasmodiophorabrassicae causal agent of clubroot of crucifers in plant and soil[J].Plant Dis,2007,91:80-87.
[12]李妍.大白菜根肿病病原菌检测与防治技术研究[D].哈尔滨:东北农业大学,2010.
[13]杨佩文,杨勤忠,王群,等.十字花科蔬菜根肿病菌的检测[J].云南农业大学学报,2002,17(2):137-139.
[14]尹全,宋君,刘勇,等.土壤根肿病菌休眠孢子PCR快速检测方法的建立[J].西南农业学报,2010,23(2):390-392.
[15]宋琼,任佐华,杨华,等.应用实时荧光定量PCR技术检测土壤中根肿病菌休眠孢子[J].湖南农业大学学报(自然科学版),2015,41(6):631-635.
[16]李淼,周丽洪,刘雅婷,等.云南省十字花科蔬菜根肿病的实时荧光定量PCR检测[J].云南农业大学学报(自然科学),2016,31(1):43-48.
[17]尚慧,韩树鑫,高艳玲,等.利用qRT-PCR技术对PVY重组株系的鉴定[J].云南农业学报,2018,31(2):238-244.
[18]郭杨,陈世界,郭万柱,等.荧光定量PCR技术及其应用研究进展[J].动物医学进展,2009,30(2):78-82.
[19]朱捷,杨成君,王军.荧光定量PCR技术及其在科研中的应用[J].生物技术通报,2009(2):74-76.
[2]Verma S S,Rahman M H,Deyholos M K,et al. Differential expres-sion of miRNA in Brassica napusroot following infection with Plasmo-diophora brassicae[J]. PLo S ONE,2014,9(1):e86648.
[3]索欢,陈龙正,徐海,等.十字花科根肿病研究进展[J].安徽农业科学,2015,43(14):115-117,126.
[4]Plasmodiophora brassicae. Distribution maps of plant diseases[R].Wallingford,UK:CAB International,1977.
[5]孙保亚,沈向群,郭海峰,等.十字花科植物根肿病及抗病育种研究进展[J].中国蔬菜,2005(4):34-37.
[6]于晓坤,吴毅歆,毛自朝,等.水介导十字花科作物根肿病的传播及其化学防治[J].华中农业大学学报,2013,32(1):48-53.
[7]Xu X G,Liu Y T. Application of real-time fluorescence quantitativePCR in plant disease[J]. Chinese Agricultural Science Bulletin,2009,25(7):52-56.
[8]Faggian R,Bulman S R,Lawrie A C,et al. Specific polymerase chainreaction primers for the detection of Plasmodiophora brassicae in soiland water[J]. Phytopathology,1999,89:392-397.
[9]Ito S,Mathara T,Marund E,et al. Development of a PCR-based assayfor the detection of Plasmodiophora brassicaein soil[J]. Journal ofPhytopathology,1999,147:83-88.
[10]Wallenhammar AC,Arwidsson O. Detection of Plasmodiophora bras-sicae by PCR in naturally infested soils[J]. Eur J Plant Pathol,2001,107:313-321.
[11]Cao T,Tewari J,Strelkov SE. Molecular detection of Plasmodiophorabrassicae causal agent of clubroot of crucifers in plant and soil[J].Plant Dis,2007,91:80-87.
[12]李妍.大白菜根肿病病原菌检测与防治技术研究[D].哈尔滨:东北农业大学,2010.
[13]杨佩文,杨勤忠,王群,等.十字花科蔬菜根肿病菌的检测[J].云南农业大学学报,2002,17(2):137-139.
[14]尹全,宋君,刘勇,等.土壤根肿病菌休眠孢子PCR快速检测方法的建立[J].西南农业学报,2010,23(2):390-392.
[15]宋琼,任佐华,杨华,等.应用实时荧光定量PCR技术检测土壤中根肿病菌休眠孢子[J].湖南农业大学学报(自然科学版),2015,41(6):631-635.
[16]李淼,周丽洪,刘雅婷,等.云南省十字花科蔬菜根肿病的实时荧光定量PCR检测[J].云南农业大学学报(自然科学),2016,31(1):43-48.
[17]尚慧,韩树鑫,高艳玲,等.利用qRT-PCR技术对PVY重组株系的鉴定[J].云南农业学报,2018,31(2):238-244.
[18]郭杨,陈世界,郭万柱,等.荧光定量PCR技术及其应用研究进展[J].动物医学进展,2009,30(2):78-82.
[19]朱捷,杨成君,王军.荧光定量PCR技术及其在科研中的应用[J].生物技术通报,2009(2):74-76.