PMA-qPCR定量检测青枯菌活菌方法的建立
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摘要
本文将叠氮溴化丙锭(PMA)与荧光实时定量PCR技术相结合,建立了一种适于青枯菌不同小种菌株活细胞精准、快速检测的PMA-qPCR方法。通过单因素变化试验对PMA预处理反应体系中的各参数进行优化,确立了PMA终浓度为15 ng/μL,黑暗孵育时间为10 min,曝光时间为5 min的PMA预处理体系。试验结果表明,当活菌比例大于10%,PMA-qPCR的测定结果均在理论活菌数相对应的95%置信区间内。检测灵敏度测试结果显示,该方法适用于活菌数在5.0×10~2～5.0×10~8 cfu/mL范围内菌悬液的检测。本文建立的PMA-qPCR方法可在一定范围内有效去除青枯菌死菌的干扰,定量检测出活菌数量,研究结果可为植物细菌性青枯病的流行规律研究提供新的技术支撑。
By combining propidium monoazide(PMA) with fluorogenic(TaqMan) PCR, we developed a novel method for specific detection of viable cells of Ralstonia solanacearum at species level. We optimized various parameters of PMA by single factor change test to establish the system of PMA pre-treatment. The optimal conditions for PMA-qPCR were: a final concentration of PMA of 15 ng/mL, a dark incubation time of 10 min, and an exposure time of 5 min. The test results showed that, within the range of 5.0×10~2-5.0×10~8 cfu/mL, when the proportion of viable bacteria was greater than 10%, the results of PMA-qPCR were almost identical with the theoretical values. The method could effectively remove the interference of the dead cells of R.solanacearum and quantitatively detect the number of viable cells, thereby providing a new technical support for the research of epidemiology of bacterial wilt caused by R.solanacearum.
By combining propidium monoazide(PMA) with fluorogenic(TaqMan) PCR, we developed a novel method for specific detection of viable cells of Ralstonia solanacearum at species level. We optimized various parameters of PMA by single factor change test to establish the system of PMA pre-treatment. The optimal conditions for PMA-qPCR were: a final concentration of PMA of 15 ng/mL, a dark incubation time of 10 min, and an exposure time of 5 min. The test results showed that, within the range of 5.0×10~2-5.0×10~8 cfu/mL, when the proportion of viable bacteria was greater than 10%, the results of PMA-qPCR were almost identical with the theoretical values. The method could effectively remove the interference of the dead cells of R.solanacearum and quantitatively detect the number of viable cells, thereby providing a new technical support for the research of epidemiology of bacterial wilt caused by R.solanacearum.
引文
[1] WICKER E, GRASSART L, CORANSON-BEAUDU R, et al. Ralstonia solanacearum strains from Martinique. (French West Indies) exhibiting a new pathogenic potential [J]. Applied and Environmental Microbiology, 2007, 71(21): 6790-6801.
[2] XU J, PAN Z C, PRIOR P, et al. Genetic diversity of Ralstonia solanacearum strains from China [J]. European Journal of Plant Pathology, 2009, 125(4): 641-653.
[3] IMAZAKI I, NAKAHO K. Pyruvate-amended modified SMSA medium: improved sensitivity for detection of Ralstonia solanacearum [J]. Journal of General Plant Pathology, 2010, 76(1): 52-61.
[4] GREY B E, STECK T R.The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection [J]. Applied and Environmental Microbiology, 2001, 67(9): 3866-3872.
[5] ALVAREZ B, LOPEZ M M, BIOSCA E G. Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms [J]. Microbiology, 2008, 154(11): 3590-3598.
[6] IMAZAKI I, NAKAHO K. Temperature-upshift-mediated revival from the sodium-pyruvate-recoverable viable but nonculturable state induced by low temperature in Ralstonia solanacearum: linear regression analysis [J]. Journal of General Plant Pathology, 2009, 75(3): 213-226.
[7] VAN OVERBEEK L S, BERGERVOET J H, JACOBS F H, et al. The low-temperature-induced viable-but-nonculturable state affects the virulence of Ralstonia solanacearum Biovar 2 [J]. Phytopathology, 2004, 94(5): 463-469.
[8] YOUNG U H, KONG H G, JU L H, et al. Altered gene expression and intracellular changes of the viable but nonculturable state in Ralstonia solanacearum by copper treatment [J]. Plant Pathology Journal, 2013, 29(4): 374-385.
[9] BIOSCA E G, CARUSO P, BERTOLINI E, et al. Improved detection of Ralstonia solanacearum in culturable and VBNC state from water samples at low temperatures [M]//ALLEN C, PRIOR P, HAYWARD A C. Bacterial wilt disease and the Ralstonia solanacearum species complex St.Paul, USA: APS Press, 2005: 501-506.
[10] 于小龙, 徐进, 张昊, 等. PMA-PCR方法快速检测VBNC状态青枯菌[J]. 植物保护, 2016, 42(1): 144-149.
[11] 曹梦琪, 王旭东, 王俊, 等. 基于PMA-qPCR检测青枯菌5号生理小种活菌的方法[J]. 蚕业科学, 2015(6): 1004-1010.
[12] 熊书,殷幼平,王芳,等.基于EMA-qPCR的茄科青枯菌活体检测技术的建立[J].微生物学通报,2013,40(9):1723-1732.
[13] KONG H G, BAE J Y, LEE H J, et al. Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase [J/OL]. PLoS ONE, 2014, 9(10): e109792.
[14] WELLER S A, ELPHINSTONE J G, SMITH N C, et al. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay[J]. Applied and Environmental Microbiology, 2000, 66(7): 2853.
[15] HE L Y, SEQUEIRA L, KELMAN A. Characteristics of strains of Pseudomonas solanacearum [J].Plant Disease,1983,67:1357-1361.
[16] 乔俊卿, 陈志谊, 刘邮洲, 等. 茄科作物青枯病研究进展[J]. 植物病理学报, 2013, 43(1): 1-10.
[17] 徐进, 冯洁. 植物青枯菌遗传多样性及致病基因组学研究进展[J]. 中国农业科学, 2013, 46(14): 2902-2909.
[18] NOGVA H K, DR∅MTORP S M, NISSEN H, et al. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR [J]. BioTechniques, 2003, 34(4): 812-813.
[19] LUO L X, WALTERS C, BOLKAN H, et al. Quantification of viable cells of Clavibacter michiganensis, subsp. michiganensis, using a DNA binding dye and a real-time PCR assay[J]. Plant Pathology, 2008, 57(2): 332-337.
[20] NOCKER A, CHEUNG C Y, CAMPER A K. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells [J].Journal of Microbiological Methods,2006,67(2): 310-320.
[21] CAWTHORN D M, WITTHUHN R C. Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide [J]. Journal of Applied Microbiology, 2008, 105(4): 1178.
[22] LI B, CHEN J Q. Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella, spp. in food [J]. BMC Microbiology, 2013, 13(1): 1-13.
[2] XU J, PAN Z C, PRIOR P, et al. Genetic diversity of Ralstonia solanacearum strains from China [J]. European Journal of Plant Pathology, 2009, 125(4): 641-653.
[3] IMAZAKI I, NAKAHO K. Pyruvate-amended modified SMSA medium: improved sensitivity for detection of Ralstonia solanacearum [J]. Journal of General Plant Pathology, 2010, 76(1): 52-61.
[4] GREY B E, STECK T R.The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection [J]. Applied and Environmental Microbiology, 2001, 67(9): 3866-3872.
[5] ALVAREZ B, LOPEZ M M, BIOSCA E G. Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms [J]. Microbiology, 2008, 154(11): 3590-3598.
[6] IMAZAKI I, NAKAHO K. Temperature-upshift-mediated revival from the sodium-pyruvate-recoverable viable but nonculturable state induced by low temperature in Ralstonia solanacearum: linear regression analysis [J]. Journal of General Plant Pathology, 2009, 75(3): 213-226.
[7] VAN OVERBEEK L S, BERGERVOET J H, JACOBS F H, et al. The low-temperature-induced viable-but-nonculturable state affects the virulence of Ralstonia solanacearum Biovar 2 [J]. Phytopathology, 2004, 94(5): 463-469.
[8] YOUNG U H, KONG H G, JU L H, et al. Altered gene expression and intracellular changes of the viable but nonculturable state in Ralstonia solanacearum by copper treatment [J]. Plant Pathology Journal, 2013, 29(4): 374-385.
[9] BIOSCA E G, CARUSO P, BERTOLINI E, et al. Improved detection of Ralstonia solanacearum in culturable and VBNC state from water samples at low temperatures [M]//ALLEN C, PRIOR P, HAYWARD A C. Bacterial wilt disease and the Ralstonia solanacearum species complex St.Paul, USA: APS Press, 2005: 501-506.
[10] 于小龙, 徐进, 张昊, 等. PMA-PCR方法快速检测VBNC状态青枯菌[J]. 植物保护, 2016, 42(1): 144-149.
[11] 曹梦琪, 王旭东, 王俊, 等. 基于PMA-qPCR检测青枯菌5号生理小种活菌的方法[J]. 蚕业科学, 2015(6): 1004-1010.
[12] 熊书,殷幼平,王芳,等.基于EMA-qPCR的茄科青枯菌活体检测技术的建立[J].微生物学通报,2013,40(9):1723-1732.
[13] KONG H G, BAE J Y, LEE H J, et al. Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase [J/OL]. PLoS ONE, 2014, 9(10): e109792.
[14] WELLER S A, ELPHINSTONE J G, SMITH N C, et al. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay[J]. Applied and Environmental Microbiology, 2000, 66(7): 2853.
[15] HE L Y, SEQUEIRA L, KELMAN A. Characteristics of strains of Pseudomonas solanacearum [J].Plant Disease,1983,67:1357-1361.
[16] 乔俊卿, 陈志谊, 刘邮洲, 等. 茄科作物青枯病研究进展[J]. 植物病理学报, 2013, 43(1): 1-10.
[17] 徐进, 冯洁. 植物青枯菌遗传多样性及致病基因组学研究进展[J]. 中国农业科学, 2013, 46(14): 2902-2909.
[18] NOGVA H K, DR∅MTORP S M, NISSEN H, et al. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR [J]. BioTechniques, 2003, 34(4): 812-813.
[19] LUO L X, WALTERS C, BOLKAN H, et al. Quantification of viable cells of Clavibacter michiganensis, subsp. michiganensis, using a DNA binding dye and a real-time PCR assay[J]. Plant Pathology, 2008, 57(2): 332-337.
[20] NOCKER A, CHEUNG C Y, CAMPER A K. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells [J].Journal of Microbiological Methods,2006,67(2): 310-320.
[21] CAWTHORN D M, WITTHUHN R C. Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide [J]. Journal of Applied Microbiology, 2008, 105(4): 1178.
[22] LI B, CHEN J Q. Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella, spp. in food [J]. BMC Microbiology, 2013, 13(1): 1-13.