水稻细菌性条斑病菌和白叶枯病菌数字PCR检测方法的建立
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摘要
基于水稻细菌性条斑病菌的假定膜蛋白基因和水稻白叶枯病菌的rhs家族基因分别设计引物和探针,建立了这两种病菌的数字PCR检测技术。特异性测试结果显示,两种检测方法均可特异性检测到目标病菌的供试菌株,而其他对照菌和空白对照均为阴性。两种检测方法对目标菌的检测下限分别达到了9个和16个拷贝/反应,且成功检测到人工模拟带菌及自然带菌种子样品中的病菌。
Two digital PCR methods for quantifying Xanthomonas. oryzae pv. oryzicola based on putative membrane protein gene and X. oryzae pv. oryzae based on rhs family gene was developed respectively.The detection results suggest that the dPCR methods established in this study were highly specific for the target strains. The detection threshold of those two d PCR methods were 9 and 16 copies respectively.And bacteria on artificial contaminated and naturally infected seed samples were both successfully detected using those two dPCR methods.
Two digital PCR methods for quantifying Xanthomonas. oryzae pv. oryzicola based on putative membrane protein gene and X. oryzae pv. oryzae based on rhs family gene was developed respectively.The detection results suggest that the dPCR methods established in this study were highly specific for the target strains. The detection threshold of those two d PCR methods were 9 and 16 copies respectively.And bacteria on artificial contaminated and naturally infected seed samples were both successfully detected using those two dPCR methods.
引文
[1]陈功友,邹丽芳,王邢平,等.水稻白叶枯病菌致病性分子遗传学基础.中国农业科学, 2004, 37(9):1301-1307.
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[9] Karlinneumann G, Wang S, Troup C, et al. Abstract 3491:Rapid and ultra-sensitive single-cell transcript profiling with droplet digital PCR(ddPCR):Application to cell cycle analysis. Cancer Research, 2014, 74:3491.
[10] Oxnard G R, Paweletz C P, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clinical Cancer Research, 2014, 20:1698.
[11] Floren C, Wiedemann I, Brenig B, et al. Species identification and quantification in meat and meat products using droplet digital PCR(ddPCR). Food Chemistry, 2014, 173:1054-1058.
[12] Huang J T, Liu Y J, Wang J, et al. Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue. Clinical Chemistry, 2015, 61(1):290-296.
[13] Kang M J, Kim M H, Hwang D J, et al. Quantitative in planta PCR assay for specific detection of Xanthomonas oryzae pv. oryzicola using putative membrane protein based primer set. Crop Protection, 2012, 40:22-27.
[14] Sykes P J, Neoh S H, Brisco M J, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques,1992, 13:444-449.
[15] Kalinina O, Lebedeva I, Brown J, et al. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Research,1997, 25:1999-2004.
[16] Simant D, Jian Q, Ramesh R. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device. Plos One, 2008, 3:e2876.
[17] Bhat S, Herrmann J, Armishaw P, et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Analytical&Bioanalytical Chemistry, 2009, 394:457-467.
[18] Zimmermann B G, Grill S, Holzgreve W, et al. Digital PCR:a powerful new tool for noninvasive prenatal diagnosis. Prenatal Diagnosis, 2010, 28:1087-1093.
[19] Dingle T C, Sedlak R H, Cook L, et al. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clinical Chemistry, 2013, 59:1670.
[20] Dreo T, Pirc M, Ramak■, et al. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria:a case study of fire blight and potato brown rot. Analytical&Bioanalytical Chemistry, 2014, 406:6513-6528.
[21] Zhu P, Fu W, Wang C, et al. Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps. Analytica Chimica Acta, 2016, 916:60-66.
[2] Strange R N, Scott P R. Plant disease:a threat to global food security. Annual Review of Phytopathology, 2005, 43:83.
[3] Jeung J U, Heu S G, Shin M S, et al. Dynamics of Xanthomonas oryzae pv. oryzae Populations in Korea and Their Relationship to Known Bacterial Blight Resistance Genes.Phytopathology, 2006, 96:867-875.
[4] Ni?oliu D O, Ronald P C, Bogdanove A J. Xanthomonas oryzae pathovars:model pathogens of a model crop. Molecular Plant Pathology, 2006, 7:303-324.
[5] Kim H M, Song W Y. Characterization of ribosomal RNA intergenic spacer region of several seedborne bacterial pathogens of rice. Seed Science&Technology, 1996, 24:571-580.
[6] Louws F J, Fulbright D W, Stephens C T, et al. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied&Environmental Microbiology, 1994, 60:2286.
[7] Zhao W J, Zhu S F, Liao X L, et al. Detection of Xanthomonas oryzae pv. oryzae in seeds using a specific TaqMan probe. Molecular Biotechnology, 2007, 35:119-127.
[8] Hsieh S P, Buddenhagen I W, Kauffman H E. An improved method for detecting the presence of Xanthomonas oryzae pv. oryzae in rice seed. Phytopathology, 2001, 64:273-274.
[9] Karlinneumann G, Wang S, Troup C, et al. Abstract 3491:Rapid and ultra-sensitive single-cell transcript profiling with droplet digital PCR(ddPCR):Application to cell cycle analysis. Cancer Research, 2014, 74:3491.
[10] Oxnard G R, Paweletz C P, Kuang Y, et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clinical Cancer Research, 2014, 20:1698.
[11] Floren C, Wiedemann I, Brenig B, et al. Species identification and quantification in meat and meat products using droplet digital PCR(ddPCR). Food Chemistry, 2014, 173:1054-1058.
[12] Huang J T, Liu Y J, Wang J, et al. Next generation digital PCR measurement of hepatitis B virus copy number in formalin-fixed paraffin-embedded hepatocellular carcinoma tissue. Clinical Chemistry, 2015, 61(1):290-296.
[13] Kang M J, Kim M H, Hwang D J, et al. Quantitative in planta PCR assay for specific detection of Xanthomonas oryzae pv. oryzicola using putative membrane protein based primer set. Crop Protection, 2012, 40:22-27.
[14] Sykes P J, Neoh S H, Brisco M J, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques,1992, 13:444-449.
[15] Kalinina O, Lebedeva I, Brown J, et al. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Research,1997, 25:1999-2004.
[16] Simant D, Jian Q, Ramesh R. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device. Plos One, 2008, 3:e2876.
[17] Bhat S, Herrmann J, Armishaw P, et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Analytical&Bioanalytical Chemistry, 2009, 394:457-467.
[18] Zimmermann B G, Grill S, Holzgreve W, et al. Digital PCR:a powerful new tool for noninvasive prenatal diagnosis. Prenatal Diagnosis, 2010, 28:1087-1093.
[19] Dingle T C, Sedlak R H, Cook L, et al. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clinical Chemistry, 2013, 59:1670.
[20] Dreo T, Pirc M, Ramak■, et al. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria:a case study of fire blight and potato brown rot. Analytical&Bioanalytical Chemistry, 2014, 406:6513-6528.
[21] Zhu P, Fu W, Wang C, et al. Development and application of absolute quantitative detection by duplex chamber-based digital PCR of genetically modified maize events without pretreatment steps. Analytica Chimica Acta, 2016, 916:60-66.