葡萄霜霉病菌实时荧光定量PCR检测体系的建立和应用
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摘要
【目的】葡萄霜霉病是葡萄生产上重要的单年流行病害,研究旨在构建葡萄霜霉病菌(Plasmopara viticola)的实时荧光定量PCR(real-time PCR)检测体系,为葡萄霜霉病的早期诊断和预测预报提供依据。【方法】依据GenBank中葡萄霜霉病菌cox2基因序列设计1对特异性引物(F-cox-Pv/R-Pv),建立并优化常规PCR和real-time PCR反应体系,利用葡萄霜霉病菌、葡萄炭疽病菌(Colletotrichum gloeosporioides)、葡萄白粉病菌(Uncinula necator)、葡萄灰霉病菌(Botrytis cinerea)、葡萄白腐病菌(Coniella diplodiella)、葡萄溃疡病菌(Botryosphaeria dothidea)、白菜黑斑病菌(Alternaria brassicae)、辣椒炭疽病菌(C.capsica)、甘草根腐病菌(Fusarium solani)、西葫芦白粉病菌(Sphaerotheca fuliginea)、番茄菌核病菌(Sclerotinia sclerotiorum)、马铃薯干腐病菌(F.equiseti)、西瓜枯萎病菌(F.oxysporum)、哈茨木霉(Trichoderma harzianum)等14种葡萄及其他作物病原菌和拮抗菌的菌丝DNA进行常规PCR和real-time PCR特异性检测,并对灵敏度和可重复性进行评价。运用已构建的real-time PCR体系对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行定量检测,利用SPSS 19.0软件分析接种时间与叶片内葡萄霜霉病菌潜伏侵染量的关系。【结果】研究设计的引物特异性高,常规PCR仅对葡萄霜霉病菌DNA有扩增条带,为139 bp;real-time PCR检测结果表明该对引物对葡萄霜霉病菌有唯一的产物吸收峰,对其他供试菌株均未检测到产物吸收峰。常规PCR检测的灵敏度为10 pg·μL~(-1),real-time PCR的灵敏度可达到0.1 pg·μL~(-1),是常规PCR检测灵敏度的100倍。以携带目的基因片段的重组质粒为标准品,构建real-time PCR循环阈值(Ct)与模板浓度的线性关系,标准曲线y=42.27-3.36x,相关系数R~2=0.997,扩增效率为98.50%,线性范围达7个数量级,在2.4×10~3—2.4×10~9 copies/μL呈现良好的线性关系。对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行real-time PCR检测,结果表明叶片内病原菌潜伏侵染量随接种时间的变化呈指数关系增长,y=6.34×10~4·e~(0.084x),相关系数R~2=0.936。该real-time PCR检测体系在接种6 h后就可以检测到葡萄霜霉病菌DNA,检测量为5.68×10~4 copies/μL病原菌DNA。【结论】构建的葡萄霜霉病菌real-time PCR检测体系的灵敏度远高于常规PCR,且特异性强、重复性好,其Ct值与模板浓度呈较好的线性关系,扩增效率高,可用于定量检测葡萄霜霉病菌的潜伏侵染量。
【Objective】Grape downy mildew caused by Plasmopara viticola is one of the most important monoetic diseases on Vitis vinifera.The objective of this study is to establish a real-time PCR detection system of P.viticola based on the pathogen sequence information,and to provide a scientific basis for early diagnosis and prediction of grape downy mildew.【Method】According to the cox2 gene sequence of P.viticola in GenBank,a pair of specific primers,F-cox-Pv/R-Pv,was designed to establish and optimize the conventional PCR and real-time PCR reaction system.The mycelium DNA of 14 species of grape and other crop pathogens and antagonistic fungus(including P.viticola,Colletotrichum gloeosporioides,Uncinula necator,Botrytis cinerea,Coniella diplodiella,Botryosphaeria dothidea,Alternaria brassicae,C.capsica,Fusarium solani,Sphaerotheca fuliginea,Sclerotinia sclerotiorum,F.equiseti,F.oxysporum,Trichoderma harzianum)were used to detect the specificity of conventional PCR and real-time PCR.The sensitivity and repeatability of the system were also evaluated.The pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P.viticola was quantitatively detected by the established real-time PCR system.The relationship between inoculation time and latent infection amount of P.viticola in the grape leaves was analyzed by SPSS 19.0 software.【Result】The primers designed in this study had high specificity.A 139 bp fragment of genome DNA was only amplified from P.viticola by conventional PCR.The detection results of real-time PCR assays showed that the primers had only one absorption peak for P.viticola,and no product absorption peak was detected for other test strains.The sensitivity of conventional PCR was 10 pg·μL~(-1) genomic DNA,while the sensitivity of real-time PCR was 0.1 pg·μL~(-1),which was 100 times higher than that of the conventional PCR.The linear relationship between real-time PCR cycle threshold(Ct)and template concentration was constructed by using recombinant plasmid containing cox2 gene fragment as standard material.The standard curve was y=42.27-3.36x,with the correlation coefficient of 0.997 and amplification efficiency of 98.50%.The linear range was up to 7 orders of magnitude,showing a good linear relationship at 2.4×10~3-2.4×10~9 copies/μL.The real-time PCR system was used to detect the pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P.viticola.The results showed that the latent infection of P.viticola in the grape leaves increased exponentially with the change of inoculation time,the curve equation was y=6.34×10~4·e~(0.084x),with the correlation coefficient of 0.936.The real-time PCR system could detect the P.viticola DNA 6 h after inoculation,and the DNA content was 5.68×10~(4 )copies/μL.【Conclusion】The sensitivity of the established real-time PCR system for detection of grape downy mildew is much higher than that of conventional PCR,and the specificity and reproducibility of this real-time PCR system are good.There was a good linear relationship between Ct value and template concentration,and the amplification efficiency is high.This method can be used to quantitatively detect the latent infection of P.viticola.
【Objective】Grape downy mildew caused by Plasmopara viticola is one of the most important monoetic diseases on Vitis vinifera.The objective of this study is to establish a real-time PCR detection system of P.viticola based on the pathogen sequence information,and to provide a scientific basis for early diagnosis and prediction of grape downy mildew.【Method】According to the cox2 gene sequence of P.viticola in GenBank,a pair of specific primers,F-cox-Pv/R-Pv,was designed to establish and optimize the conventional PCR and real-time PCR reaction system.The mycelium DNA of 14 species of grape and other crop pathogens and antagonistic fungus(including P.viticola,Colletotrichum gloeosporioides,Uncinula necator,Botrytis cinerea,Coniella diplodiella,Botryosphaeria dothidea,Alternaria brassicae,C.capsica,Fusarium solani,Sphaerotheca fuliginea,Sclerotinia sclerotiorum,F.equiseti,F.oxysporum,Trichoderma harzianum)were used to detect the specificity of conventional PCR and real-time PCR.The sensitivity and repeatability of the system were also evaluated.The pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P.viticola was quantitatively detected by the established real-time PCR system.The relationship between inoculation time and latent infection amount of P.viticola in the grape leaves was analyzed by SPSS 19.0 software.【Result】The primers designed in this study had high specificity.A 139 bp fragment of genome DNA was only amplified from P.viticola by conventional PCR.The detection results of real-time PCR assays showed that the primers had only one absorption peak for P.viticola,and no product absorption peak was detected for other test strains.The sensitivity of conventional PCR was 10 pg·μL~(-1) genomic DNA,while the sensitivity of real-time PCR was 0.1 pg·μL~(-1),which was 100 times higher than that of the conventional PCR.The linear relationship between real-time PCR cycle threshold(Ct)and template concentration was constructed by using recombinant plasmid containing cox2 gene fragment as standard material.The standard curve was y=42.27-3.36x,with the correlation coefficient of 0.997 and amplification efficiency of 98.50%.The linear range was up to 7 orders of magnitude,showing a good linear relationship at 2.4×10~3-2.4×10~9 copies/μL.The real-time PCR system was used to detect the pathogen DNA in the grape leaves during the latent infection period of artificial inoculation with P.viticola.The results showed that the latent infection of P.viticola in the grape leaves increased exponentially with the change of inoculation time,the curve equation was y=6.34×10~4·e~(0.084x),with the correlation coefficient of 0.936.The real-time PCR system could detect the P.viticola DNA 6 h after inoculation,and the DNA content was 5.68×10~(4 )copies/μL.【Conclusion】The sensitivity of the established real-time PCR system for detection of grape downy mildew is much higher than that of conventional PCR,and the specificity and reproducibility of this real-time PCR system are good.There was a good linear relationship between Ct value and template concentration,and the amplification efficiency is high.This method can be used to quantitatively detect the latent infection of P.viticola.
引文
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