基于锁核苷酸(LNA)增敏的植烟土壤青枯雷尔氏菌定量PCR检测
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摘要
为了定量检测植烟土壤中青枯雷尔氏菌的数量,有效防控烟草青枯病,构建了一种锁核苷酸(LNA)荧光定量PCR检测方法。以青枯雷尔氏菌细胞色素c基因(Cyt c)(NCBI Genebank序列号:WP_011002214.1)为靶标,采用LNA技术合成覆盖目标片段的引物序列,结合荧光定量PCR技术,进行了植烟土壤中烟草青枯雷尔氏菌数量的快速检测。结果表明,所构建的Cyt c基因标准曲线的R~2>0.99,相关性较好。与普通DNA引物相比,采用Cyt c基因LNA引物进行的PCR适用退火温度更高,扩增效率也更高。通过对水稻土发病烟田18个土壤样品的检测,表明青枯雷尔氏菌数量在2.52×10~4~1.88×10~7个/g土壤之间。因此,构建的一种基于Cyt c基因的LNA增敏定量PCR检测体系,适用于水稻土等植烟土壤中青枯雷尔氏菌的定量检测,对烟田土壤的提前检测有助于烟草青枯病发生的预警。
To detect the amount of Ralstonia solanacearum in tobacco planting soil before bacterial wilt occurrence and effectively control tobacco bacterial wilt, locked nucleic acid-based(LNA-based) quantitative real-time PCR assay was established and used in this study. Taking cytochrome c gene(NCBI Genbank accession number: WP_011002214.1) from Ralstonia solanacearum as the target, its primer sequence covering the target fragment was synthesized with LNA technique. Quantitative real-time PCR was used to quickly detect the amount of Ralstonia solanacearum in tobacco planting soil. The results showed that the standard curve of cytochrome c gene had a good correlation with R~2> 0.99. Comparing with conventional DNA primers, LNA primer of cytochrome c gene could increase annealing temperature of PCR reaction and amplification efficiency.The DNA of 18 soil samples from tobacco fields(paddy soil) infected with bacterial wilt were tested and the amount of Ralstonia solanacearum in per gram of soil was 2.52 × 10~4-1.88 × 10~7. Therefore, LNA-based quantitative real-time PCR assay is suitable for the quantitative determination of Ralstonia solanacearum in paddy soil, and the early detection of tobacco soil will contribute to the forecast of tobacco bacterial wilt.
To detect the amount of Ralstonia solanacearum in tobacco planting soil before bacterial wilt occurrence and effectively control tobacco bacterial wilt, locked nucleic acid-based(LNA-based) quantitative real-time PCR assay was established and used in this study. Taking cytochrome c gene(NCBI Genbank accession number: WP_011002214.1) from Ralstonia solanacearum as the target, its primer sequence covering the target fragment was synthesized with LNA technique. Quantitative real-time PCR was used to quickly detect the amount of Ralstonia solanacearum in tobacco planting soil. The results showed that the standard curve of cytochrome c gene had a good correlation with R~2> 0.99. Comparing with conventional DNA primers, LNA primer of cytochrome c gene could increase annealing temperature of PCR reaction and amplification efficiency.The DNA of 18 soil samples from tobacco fields(paddy soil) infected with bacterial wilt were tested and the amount of Ralstonia solanacearum in per gram of soil was 2.52 × 10~4-1.88 × 10~7. Therefore, LNA-based quantitative real-time PCR assay is suitable for the quantitative determination of Ralstonia solanacearum in paddy soil, and the early detection of tobacco soil will contribute to the forecast of tobacco bacterial wilt.
引文
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[5]陈志敏,彭业敏,张晓阳,等.烟草青枯病田间防治药剂的筛选[J].湖南农业科学,2012(7):79-81.CHEN Zhimin,PENG Yemin,ZHANG Xiaoyang,et al.Screening of control medicaments against tobacco bacterial wilt in the field[J].Hunan Agricultural Sciences,2012(7):79-81.
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[8]昌珩.腐植酸钾对土壤微生物种类和数量的作用初探[J].广东化工,2013,40(13):66-67.CHANG Heng.A preliminary study of the impact of potassium humate of the microbial type and quantity on soil[J].Guangdong Chemical Industry,2013,40(13):66-67.
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[11]Ramesh R,Anthony J,Jaxon T C D,et al.PCR-based sensitive detection of Ralstonia solanacearum from soil,eggplant,seeds and weeds[J].Archives of Phytopathology and Plant Protection,2011,44(19):1908-1919.
[12]Thammakijjawat P,Thaveechai N,Kositratana W,et al.Detection of Ralstonia solanacearum in ginger rhizomes by real-time PCR[J].Canadian Journal of Plant Pathology,2006,28(3):391-400.
[13]Ozakman M,Schaad N W.A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3,biovar2,in asymptomatic potato tubers[J].Canadian Journal of Plant Pathology,2003,25(3):232-239.
[14]Chen Y,Zhang W Z,Liu X,et al.A real-time PCRassay for the quantitative detection of Ralstonia solanacearum in the horticultural soil and plant tissues[J].Journal of Microbiology and Biotechnology,2010,20(1):193-201.
[15]Koshkin A A,Singh S K,Nielsen P,et al.LNA(Locked Nucleic Acids):synthesis of the adenine,cytosine,guanine,5-methylcytosine,thymine and uracil bicyclonucleoside monomers,oligomerisation,and unprecedented nucleic acid recognition[J].Tetrahedron,1998,54(14):3607-3630.
[16]Latorra D,Campbell K,Wolter A,et al.Enhanced allele-specific PCR discrimination in SNP genotyping using 3′locked nucleic acid(LNA)primers[J].Human Mutation,2003,22(1):79-85.
[17]Simeonov A,Nikiforov T T.Single nucleotide polymorphism genotyping using short,fluorescently labeled locked nucleic acid(LNA)probes and fluorescence polarization detection[J].Nucleic Acids Research,2002,30(17):e91.
[18]Wang L,Yang C J,Medley C D,et al.Locked nucleic acid molecular beacons[J].Journal of the American Chemical Society,2005,127(45):15664-15665.
[19]Burbano C S,Reinhold-Hurek B,Hurek T.LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples[J].Environmental Microbiology Reports,2010,2(2):251-257.
[20]Ikenaga M,Tabuchi M,Kawauchi T,et al.Application of locked nucleic acid(LNA)primer and PCR clamping by LNA oligonucleotide to enhance the amplification of internal transcribed spacer(ITS)regions in investigating the community structures of plant-associated fungi[J].Microbes and Environments,2016,31(3):339-348.
[21]Owczarzy R,You Y,Groth C L,et al.Stability and mismatch discrimination of locked nucleic acid-DNAduplexes[J].Biochemistry,2011,50(43):9352-9367.
[22]Hunt E A,Goulding A M,Deo S K.Direct detection and quantification of micro RNAs[J].Analytical Biochemistry,2009,387(1):1-12.
[23]Robertson K L,Vora G J.Locked nucleic acid flow cytometry-fluorescence in situ hybridization(LNAflow-FISH):a method for bacterial small RNA detection[J].Journal of Visualized Experiments,2012(59):e3655.
[24]Ambler R P,Bartsch R G,Daniel M,et al.Amino acid sequences of bacterial cytochromes c'and c-556[J].Proceedings of the National Academy of Sciences of the United States of America,1981,78(11):6854-6857.
[25]Meyer T E,Cusanovich M A,Kamen M D.Evidence against use of bacterial amino acid sequence data for construction of all-inclusive phylogenetic trees[J].Proceedings of the National Academy of Sciences of the United States of America,1986,83(2):217-220.
[26]Ambler R P,Kamen M D,Bartsch R G,et al.Amino acid sequences of Euglena viridis ferredoxin and cytochromes C[J].Biochemical Journal,1991,276:47-52.
[27]Latorra D,Arar K,Hurley J M.Design considerations and effects of LNA in PCR primers[J].Molecular and Cellular Probes,2003,17(5):253-259.
[28]Ballantyne K N,van Oorschot R A H,Mitchell R J.Locked nucleic acids in PCR primers increase sensitivity and performance[J].Genomics,2008,91(3):301-305.
[29]Ballantyne K N,van Oorschot R A H,Mitchell R J.Locked nucleic acids:increased trace DNAamplification success with improved primers[J].Forensic Science International:Genetics Supplement Series,2008,1(1):4-6.
[30]彭怀俊,顾钢,纪成灿,等.烤烟根系土壤中青枯病菌动态与田间病害发生发展的关系[J].湖南农业大学学报(自然科学版),2005,31(4):384-387.PENG Huaijun,GU Gang,JI Chengcan,et al.Relationship between state of a disease in field and dynamic monitoring of Ralstonia solanacearum in tobacco root soil[J].Journal of Hunan Agricultural University(Natural Sciences),2005,31(4):384-387.
[31]赖荣泉,王刚,赖成连,等.烟株青枯病的动态监测技术研究[J].中国农学通报,2012,28(25):129-133.LAI Rongquan,WANG Gang,LAI Chenglian,et al.Study on dynamic monitoring of tobacco bacterial wilt in flue-cured tobacco fields[J].Chinese Agricultural Science Bulletin,2012,28(25):129-133.
[32]李生茂,徐祥,梁华平,等.锁核酸研究进展[J].生理科学进展,2003,34(4):319-323.LI Shengmao,XU Xiang,LIANG Huaping,et al.Progress in locked nucleic acid research[J].Progress in Physiological Sciences,2003,34(4):319-323.
[33]刘芳,宋纪真,范艺宽,等.基于锁核苷酸(LNA)增敏的植烟土壤中烟草黑胫病菌定量PCR检测方法[J].烟草科技,2015,48(12):14-19.LIU Fang,SONG Jizhen,FAN Yikuan,et al.Aquantitative PCR method for detecting Phytophthora parasitica var.nicotianae in tobacco planting soil based on LNA sensitization[J].Tobacco Science&Technology,2015,48(12):14-19.
[2]朱贤朝,王彦亭,王智发,等.中国烟草病害[M].北京:中国农业出版社,2002.ZHU Xianchao,WANG Yanting,WANG Zhifa,et al.Tobacco diseases of China[M].Beijing:China Agriculture Press,2002.
[3]任欣正,潘小玫,蒋晓芳,等.间接血凝抑制技术检测植物青枯病[J].南京农业大学学报,1984,7(3):45-53.REN Xinzheng,PAN Xiaomei,JIANG Xiaofang,et al.Detection of bacteria wilt disease(P.solanacearum)by indirect hemagglutinative inhibition technique[J].Journal of Nanjing Agricultural University,1984,7(3):45-53.
[4]蒋承耿,刘琼,陈鹏.烟草青枯病防治药剂室内筛选及田间药效研究[J].安徽农业科学,2013,41(3):1098-1099,1102.JIANG Chenggeng,LIU Qiong,CHEN Peng.Indoor screening and field trial of bactericides against tobacco bacterial wilt caused by Pseudomonas solanacearum[J].Journal of Anhui Agricultural Sciences,2013,41(3):1098-1099,1102.
[5]陈志敏,彭业敏,张晓阳,等.烟草青枯病田间防治药剂的筛选[J].湖南农业科学,2012(7):79-81.CHEN Zhimin,PENG Yemin,ZHANG Xiaoyang,et al.Screening of control medicaments against tobacco bacterial wilt in the field[J].Hunan Agricultural Sciences,2012(7):79-81.
[6]陈江华.中国烟叶生产技术指南[G].北京:中国烟叶公司,2016.CHEN Jianghua.Guide to leaf tobacco production technique in China[G].Beijing:China National Leaf Tobacco Corporation,2016.
[7]车海彦,罗大全.植原体病害的检测方法研究进展[J].华南热带农业大学学报,2006,12(3):69-73.CHE Haiyan,LUO Daquan.Advances in detection of phytoplasma diseases[J].Journal of South China University of Tropical Agriculture,2006,12(3):69-73.
[8]昌珩.腐植酸钾对土壤微生物种类和数量的作用初探[J].广东化工,2013,40(13):66-67.CHANG Heng.A preliminary study of the impact of potassium humate of the microbial type and quantity on soil[J].Guangdong Chemical Industry,2013,40(13):66-67.
[9]Rajeshwari N,Shylaja M D,Krishnappa M,et al.Development of ELISA for the detection of Ralstonia solanacearum in tomato:its application in seed health testing[J].World Journal of Microbiology and Biotechnology,1998,14(5):697-704.
[10]Huang J J,Wu J M,Li C,et al.Specific and sensitive detection of Ralstonia solanacearum in soil with quantitative,real-time PCR assays[J].Journal of Applied Microbiology,2009,107(5):1729-1739.
[11]Ramesh R,Anthony J,Jaxon T C D,et al.PCR-based sensitive detection of Ralstonia solanacearum from soil,eggplant,seeds and weeds[J].Archives of Phytopathology and Plant Protection,2011,44(19):1908-1919.
[12]Thammakijjawat P,Thaveechai N,Kositratana W,et al.Detection of Ralstonia solanacearum in ginger rhizomes by real-time PCR[J].Canadian Journal of Plant Pathology,2006,28(3):391-400.
[13]Ozakman M,Schaad N W.A real-time BIO-PCR assay for detection of Ralstonia solanacearum race 3,biovar2,in asymptomatic potato tubers[J].Canadian Journal of Plant Pathology,2003,25(3):232-239.
[14]Chen Y,Zhang W Z,Liu X,et al.A real-time PCRassay for the quantitative detection of Ralstonia solanacearum in the horticultural soil and plant tissues[J].Journal of Microbiology and Biotechnology,2010,20(1):193-201.
[15]Koshkin A A,Singh S K,Nielsen P,et al.LNA(Locked Nucleic Acids):synthesis of the adenine,cytosine,guanine,5-methylcytosine,thymine and uracil bicyclonucleoside monomers,oligomerisation,and unprecedented nucleic acid recognition[J].Tetrahedron,1998,54(14):3607-3630.
[16]Latorra D,Campbell K,Wolter A,et al.Enhanced allele-specific PCR discrimination in SNP genotyping using 3′locked nucleic acid(LNA)primers[J].Human Mutation,2003,22(1):79-85.
[17]Simeonov A,Nikiforov T T.Single nucleotide polymorphism genotyping using short,fluorescently labeled locked nucleic acid(LNA)probes and fluorescence polarization detection[J].Nucleic Acids Research,2002,30(17):e91.
[18]Wang L,Yang C J,Medley C D,et al.Locked nucleic acid molecular beacons[J].Journal of the American Chemical Society,2005,127(45):15664-15665.
[19]Burbano C S,Reinhold-Hurek B,Hurek T.LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples[J].Environmental Microbiology Reports,2010,2(2):251-257.
[20]Ikenaga M,Tabuchi M,Kawauchi T,et al.Application of locked nucleic acid(LNA)primer and PCR clamping by LNA oligonucleotide to enhance the amplification of internal transcribed spacer(ITS)regions in investigating the community structures of plant-associated fungi[J].Microbes and Environments,2016,31(3):339-348.
[21]Owczarzy R,You Y,Groth C L,et al.Stability and mismatch discrimination of locked nucleic acid-DNAduplexes[J].Biochemistry,2011,50(43):9352-9367.
[22]Hunt E A,Goulding A M,Deo S K.Direct detection and quantification of micro RNAs[J].Analytical Biochemistry,2009,387(1):1-12.
[23]Robertson K L,Vora G J.Locked nucleic acid flow cytometry-fluorescence in situ hybridization(LNAflow-FISH):a method for bacterial small RNA detection[J].Journal of Visualized Experiments,2012(59):e3655.
[24]Ambler R P,Bartsch R G,Daniel M,et al.Amino acid sequences of bacterial cytochromes c'and c-556[J].Proceedings of the National Academy of Sciences of the United States of America,1981,78(11):6854-6857.
[25]Meyer T E,Cusanovich M A,Kamen M D.Evidence against use of bacterial amino acid sequence data for construction of all-inclusive phylogenetic trees[J].Proceedings of the National Academy of Sciences of the United States of America,1986,83(2):217-220.
[26]Ambler R P,Kamen M D,Bartsch R G,et al.Amino acid sequences of Euglena viridis ferredoxin and cytochromes C[J].Biochemical Journal,1991,276:47-52.
[27]Latorra D,Arar K,Hurley J M.Design considerations and effects of LNA in PCR primers[J].Molecular and Cellular Probes,2003,17(5):253-259.
[28]Ballantyne K N,van Oorschot R A H,Mitchell R J.Locked nucleic acids in PCR primers increase sensitivity and performance[J].Genomics,2008,91(3):301-305.
[29]Ballantyne K N,van Oorschot R A H,Mitchell R J.Locked nucleic acids:increased trace DNAamplification success with improved primers[J].Forensic Science International:Genetics Supplement Series,2008,1(1):4-6.
[30]彭怀俊,顾钢,纪成灿,等.烤烟根系土壤中青枯病菌动态与田间病害发生发展的关系[J].湖南农业大学学报(自然科学版),2005,31(4):384-387.PENG Huaijun,GU Gang,JI Chengcan,et al.Relationship between state of a disease in field and dynamic monitoring of Ralstonia solanacearum in tobacco root soil[J].Journal of Hunan Agricultural University(Natural Sciences),2005,31(4):384-387.
[31]赖荣泉,王刚,赖成连,等.烟株青枯病的动态监测技术研究[J].中国农学通报,2012,28(25):129-133.LAI Rongquan,WANG Gang,LAI Chenglian,et al.Study on dynamic monitoring of tobacco bacterial wilt in flue-cured tobacco fields[J].Chinese Agricultural Science Bulletin,2012,28(25):129-133.
[32]李生茂,徐祥,梁华平,等.锁核酸研究进展[J].生理科学进展,2003,34(4):319-323.LI Shengmao,XU Xiang,LIANG Huaping,et al.Progress in locked nucleic acid research[J].Progress in Physiological Sciences,2003,34(4):319-323.
[33]刘芳,宋纪真,范艺宽,等.基于锁核苷酸(LNA)增敏的植烟土壤中烟草黑胫病菌定量PCR检测方法[J].烟草科技,2015,48(12):14-19.LIU Fang,SONG Jizhen,FAN Yikuan,et al.Aquantitative PCR method for detecting Phytophthora parasitica var.nicotianae in tobacco planting soil based on LNA sensitization[J].Tobacco Science&Technology,2015,48(12):14-19.