纳米磁珠与PCR联合技术在玉米病原菌快速检测中的应用研究
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摘要
植物病原细菌是一类危害性极大的病原生物,可引起广泛传播,造成巨大经济损失。玉米细菌性枯萎病菌(Pantoea stewartii subsp. stewartii, pstew)和玉米内州萎蔫病菌(Clavibacter michiganensis subsp. nebraskensis, cmn)能引起玉米作物的毁灭性的病害,其中pstew在美国,加拿大,墨西哥、越南、泰国等国家均有报道,cmn因首次爆发于美国内州而得名,我国目前尚未见病原菌爆发的报道。二者的传播方式主要有种媒和虫媒两种传播途径,而且我国每年都要从国外进口大量的玉米种子,因此对玉米种子进行有效快捷的病原菌检疫尤为重要。
植物病原细菌的口岸检疫要求简单、快捷、准确,技术要求比较高,其检测手段一直是口岸植物检疫的难点。因此,有效控制细菌病害的入侵主要是提高口岸病原菌的检测技术水平,研究出一种简便快速且高灵敏度的检测方法十分重要。
采用化学共沉淀等方法合成了纳米裸磁珠和表面修饰的纳米磁珠,并通过TEM和DLS测试对合成磁珠进行表征分析。采用PCR 16S rDNA扩增和测序对玉米细菌性枯萎病菌和玉米内州萎蔫病菌进行初步鉴定。将磁珠和菌液共同孵育,通过磁珠与菌液非特异性结合的方式,选择吸附性能最佳的磁珠,优化孵育反应的条件,比如孵育温度,时间,磁珠用量等,确定最佳孵育条件。在最佳条件下,磁珠和菌液孵育,进行菌体结合和浓缩,磁场分离后,利用特异性引物,通过PCR方法,对目标菌特异性检测。本文还使用扫描电镜对两种病原菌的形态学进行了观察。通过透明圈抑菌实验检测磁珠对目标菌的毒性作用。
通过DLS测得的Zeta电位表明,合成的纳米磁珠表面带有不同的电荷,其中表面修饰PDDA的带有正电荷,共沉淀法合成的裸磁珠,表面带有负电荷,表面修饰有PSS及-COOH,带有很强的负电荷。而且合成磁珠的粒径大小从20 nm到500 nm不等。PCR 16S rDNA扩增和测序比对证实,所用引物正确,能够实现菌株的特异性检测。将四种磁珠和菌液孵育后,初步分析磁珠和菌结合的原因,发现表面带正电荷的磁珠与菌有最大的结合。磁珠大小和形状等也对磁珠与菌的结合有一定的影响,但因本文采用磁珠的电荷差异较大,故无法分析磁珠粒径对磁珠与菌结合的影响。吸附率与磁珠的用量成正相关,在磁珠浓度为4 mg/mL的时候达到平衡。温度对吸附率的影响不大,室温下即可,这与目标菌的最适生长温度为24～28℃一致。孵育时间对吸附率的影响较大,其中玉米细菌性枯萎病菌与磁珠孵育40 min达到饱和,而玉米内州萎蔫病菌与磁珠孵育20 min后就能达到饱和,分析可能的原因为两种病原菌的细胞壁厚度,及菌的大小不一样造成的。磁珠与细菌结合后,通过磁场分离,能达到快速分离的效果并且对溶液中的细菌起到一个浓缩的作用。
在最佳孵育条件下,两种检疫病原菌与纳米磁珠共同孵育,能达到菌体浓缩目的,PCR扩增能够实现简单快速的特异性检测。通过纳米磁珠的吸附浓缩和PCR组合鉴定,可以提高检测灵敏度,满足病原菌检疫的实际检测需要。
The Phytobacteria, which can cause devastating disasters and great loss, are very harmful pathogens. Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis are two typical pathogens parasitized on corn, while neither of them has been detected in china. They are mainly spreaded by seed-borne and insect-borne. Owning to a great deal of imports of corn seeds from aboard, developing effective and efficient methods for quick detection has become gradually significant.
Because of the high technical requirements of the detection for Phytobacteria, methods for the detection have been always the constraints in Port Plant Quarantine. Therefore, in order to effectively control the invasion of bacterial disease and improve detection technology of port pathogen, it is much important to develop a fast, simple and highly sensitive detection method.
In this paper, bear beads and beads with different modifiers on the surface have been synthesized by coprecipitation technique and characterized with TEM and DLS test. PCR 16S rDNA amplification and sequencing have been adopted to preliminarily detect Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis. After incubation, the cause of the combination of beads and pathogens has been analysized. According to the non-specific binding of the beads with pathogens in culture solution, best beads are chosen, and reaction conditions of incubation, such as incubation temperature, time and the amount of beads, are optimized. Beads are incubated with pathogen solution under optimal condition and separated by magnetic power, and specific primers are used to specifically amplificate 16S rDNA products of target pathogens, enriched by magnetic beads, with PCR technique. SEM photographs of the two pathogens have been shot for the study of their morphology. The effect of beads on the toxicity of target pathogens has been detected by bacteriostasis test.
Zeta potentials of the synthesized beads detected by DLS induced that different charges were on the surface of the beads and ones modified by PDDA carried positive charges, ones synthesized by co-precipitation negative charges, and ones modified by PSS and- COOH groups on the surface were with a strong negative charges. Particle size of beads synthesized ranges from 20 nm to 500 nm. PCR 16S rDNA specific amplification and sequencing alignment identified that the designed primers showed better specific. After incubation of four beads with pathogen solution, followed by analyzing the cause of combination of them, it was shown that beads with positive charge on the surface had the strongest power in the adsorption for the pathogens. Size and shape of beads had also an impact on the combination with pathogens. The absorption rate is proportional to the amount of used beads and beads balance at the concentration of 4 mg/mL. Probably because the best growth temperature for the target pathogens in this experiment is 24-28℃, temperature had little effect on absorption rate and so room temperature is advisable. Incubation time had much great effect on the absorption rate, with largest combination of Pantoea stewartii subsp. stewartii and beads at 40min after incubation and largest combination of Clavibacter michiganensis subsp. nebraskensis and beads at 20 min after incubation. The reason may be resulted from the differences in cell wall thickness and size of the two pathogens. After combination of pathogens with beads, they could be separated simply and quickly by magnetic power and pathogens in the solution could be condensed as well.
After pathogens and beads incubation under the optimal condition, pathogens are condensed and the target pathogens can be specifically detected followed by PCR amplification. Thus it is concluded that the combination of magnetic beads absorption and PCR identification together can improve detection technique with simplicity, quickness and sensitivity for the application of pathogens quarantine.
植物病原细菌的口岸检疫要求简单、快捷、准确,技术要求比较高,其检测手段一直是口岸植物检疫的难点。因此,有效控制细菌病害的入侵主要是提高口岸病原菌的检测技术水平,研究出一种简便快速且高灵敏度的检测方法十分重要。
采用化学共沉淀等方法合成了纳米裸磁珠和表面修饰的纳米磁珠,并通过TEM和DLS测试对合成磁珠进行表征分析。采用PCR 16S rDNA扩增和测序对玉米细菌性枯萎病菌和玉米内州萎蔫病菌进行初步鉴定。将磁珠和菌液共同孵育,通过磁珠与菌液非特异性结合的方式,选择吸附性能最佳的磁珠,优化孵育反应的条件,比如孵育温度,时间,磁珠用量等,确定最佳孵育条件。在最佳条件下,磁珠和菌液孵育,进行菌体结合和浓缩,磁场分离后,利用特异性引物,通过PCR方法,对目标菌特异性检测。本文还使用扫描电镜对两种病原菌的形态学进行了观察。通过透明圈抑菌实验检测磁珠对目标菌的毒性作用。
通过DLS测得的Zeta电位表明,合成的纳米磁珠表面带有不同的电荷,其中表面修饰PDDA的带有正电荷,共沉淀法合成的裸磁珠,表面带有负电荷,表面修饰有PSS及-COOH,带有很强的负电荷。而且合成磁珠的粒径大小从20 nm到500 nm不等。PCR 16S rDNA扩增和测序比对证实,所用引物正确,能够实现菌株的特异性检测。将四种磁珠和菌液孵育后,初步分析磁珠和菌结合的原因,发现表面带正电荷的磁珠与菌有最大的结合。磁珠大小和形状等也对磁珠与菌的结合有一定的影响,但因本文采用磁珠的电荷差异较大,故无法分析磁珠粒径对磁珠与菌结合的影响。吸附率与磁珠的用量成正相关,在磁珠浓度为4 mg/mL的时候达到平衡。温度对吸附率的影响不大,室温下即可,这与目标菌的最适生长温度为24～28℃一致。孵育时间对吸附率的影响较大,其中玉米细菌性枯萎病菌与磁珠孵育40 min达到饱和,而玉米内州萎蔫病菌与磁珠孵育20 min后就能达到饱和,分析可能的原因为两种病原菌的细胞壁厚度,及菌的大小不一样造成的。磁珠与细菌结合后,通过磁场分离,能达到快速分离的效果并且对溶液中的细菌起到一个浓缩的作用。
在最佳孵育条件下,两种检疫病原菌与纳米磁珠共同孵育,能达到菌体浓缩目的,PCR扩增能够实现简单快速的特异性检测。通过纳米磁珠的吸附浓缩和PCR组合鉴定,可以提高检测灵敏度,满足病原菌检疫的实际检测需要。
The Phytobacteria, which can cause devastating disasters and great loss, are very harmful pathogens. Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis are two typical pathogens parasitized on corn, while neither of them has been detected in china. They are mainly spreaded by seed-borne and insect-borne. Owning to a great deal of imports of corn seeds from aboard, developing effective and efficient methods for quick detection has become gradually significant.
Because of the high technical requirements of the detection for Phytobacteria, methods for the detection have been always the constraints in Port Plant Quarantine. Therefore, in order to effectively control the invasion of bacterial disease and improve detection technology of port pathogen, it is much important to develop a fast, simple and highly sensitive detection method.
In this paper, bear beads and beads with different modifiers on the surface have been synthesized by coprecipitation technique and characterized with TEM and DLS test. PCR 16S rDNA amplification and sequencing have been adopted to preliminarily detect Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis. After incubation, the cause of the combination of beads and pathogens has been analysized. According to the non-specific binding of the beads with pathogens in culture solution, best beads are chosen, and reaction conditions of incubation, such as incubation temperature, time and the amount of beads, are optimized. Beads are incubated with pathogen solution under optimal condition and separated by magnetic power, and specific primers are used to specifically amplificate 16S rDNA products of target pathogens, enriched by magnetic beads, with PCR technique. SEM photographs of the two pathogens have been shot for the study of their morphology. The effect of beads on the toxicity of target pathogens has been detected by bacteriostasis test.
Zeta potentials of the synthesized beads detected by DLS induced that different charges were on the surface of the beads and ones modified by PDDA carried positive charges, ones synthesized by co-precipitation negative charges, and ones modified by PSS and- COOH groups on the surface were with a strong negative charges. Particle size of beads synthesized ranges from 20 nm to 500 nm. PCR 16S rDNA specific amplification and sequencing alignment identified that the designed primers showed better specific. After incubation of four beads with pathogen solution, followed by analyzing the cause of combination of them, it was shown that beads with positive charge on the surface had the strongest power in the adsorption for the pathogens. Size and shape of beads had also an impact on the combination with pathogens. The absorption rate is proportional to the amount of used beads and beads balance at the concentration of 4 mg/mL. Probably because the best growth temperature for the target pathogens in this experiment is 24-28℃, temperature had little effect on absorption rate and so room temperature is advisable. Incubation time had much great effect on the absorption rate, with largest combination of Pantoea stewartii subsp. stewartii and beads at 40min after incubation and largest combination of Clavibacter michiganensis subsp. nebraskensis and beads at 20 min after incubation. The reason may be resulted from the differences in cell wall thickness and size of the two pathogens. After combination of pathogens with beads, they could be separated simply and quickly by magnetic power and pathogens in the solution could be condensed as well.
After pathogens and beads incubation under the optimal condition, pathogens are condensed and the target pathogens can be specifically detected followed by PCR amplification. Thus it is concluded that the combination of magnetic beads absorption and PCR identification together can improve detection technique with simplicity, quickness and sensitivity for the application of pathogens quarantine.
引文
[1]中国农业年鉴编辑委员会.中国农业年鉴[M].北京：农业出版社,2007(28)：440-442.
[2]Stewart F C. A bacterial disease of sweet corn[J]. New York Agric. Exp. StnBull,1897,130: 422-439.
[3]Aurelien L. Carlier and S. B. von Bodman. The rcsA Promoter of Pantoea stewartii subsp. stewartii Features a Low-Level Constitutive Promoter and an EsaR Quorum-Sensing-Regulated Promoter[J]. Journal of Bacteriology,2006:4581-4584.
[4]White DG. Compendium of corn disease (3rd edition)[M]. USA:APS Press,1999,3.
[5]Pepper, E. H.1967. Stewart's bacterial wilt of corn [M]. Monograph 4. American Phytopathological Society, St. Paul, MN, U.S.A.
[6]Thai Agricultural Standard. TAS 9031-2008. Diagnostic Protocols for Pantoea Stewartii subsp. Stewartii Bacterial Wilt of Maize [S].Thai:Ministry of Agriculture and Cooperatives,2008.
[7]齐玲玲.玉米萎蔫和枯萎病菌胶体金免疫层析技术研究[D].山西：山西大学(硕士),2010：1-10.
[8]Patrick E. Lipps, Anne E. Dorrance, and Dennis R. Mills. Stewart's Bacterial Wilt and Leaf Blight of Corn[J]. The Ohio State University web site,2001, on line at:http://ohioline.osu.edu
[9]Pataky JK,Hawk JA,Weldekidan T, et al. Incidence and severity of Stewart's bacterial wilt on sequential plantings of resistant and susceptible sweet corn hybrids[J]. Plant Dis,1995,79:1202-1207
[10]郭翼奋,梁再群,俞大绂.黑色素选择性培养从进口玉米中分离玉米枯萎病菌[J].科学通报.1982,22(4)：339-344.
[11]Blakemore EJA, Law JR,Reeves JC. PCR identification of Erwinia stwarttii and its comparison with two other methods[J]. Seed Sci&Technol,1999,27:385～396.
[12]Lamka GL, Hill JH, McGee DC, et al. Development of an immunosorbent assay for seedborne Erwinia stewartii in corn seeds[J]. Phytopathology,1991,81:839～846.
[13]王岭,田世民,高海霞,等.玉米细菌性枯萎病菌改良Dot—ELISA检测研究[J]。微生物学通报,2008,35(2),230—234.
[14]玉米细菌性枯萎病菌检疫鉴定方法[S].中华人民共和国出入境检验检疫行业标准.SN/T 1375-2004,2004-12-01实施,中华人民共和国国家质量监督检验检疫总局发布.
[15]齐玲玲,金涌,邹明强,等.胶体金免疫层析试纸条快速检测玉米细菌性枯萎病菌[J].检验检 疫学刊,2010年,20(1)：1-5.
[16]漆艳香,肖启明,朱水芳.玉米细菌性枯萎病菌16S rDNA基因克隆及TaqMan探针实时荧光PCR[J]湖南农业大学学报(自然科学版),2003年6月,29(3)：183-187.
[17]漆艳香,朱水芳,赵文军等.玉米细菌性枯萎病菌TaqMan探针实时荧光PCR检测方法的建立[J].植物保护学报,2004年3月,31(1)：51-56.
[18]王茂华,胡白石,卢玲,等.利用巢式PCR检测玉米细菌性枯萎病菌[J].南京农业大学学报2005,28(2)：37-40.
[19]WU Qiong, CHEN Zhinan, FAN Huaizhong, et al. Identification of Corn Bacterial Wilt Pathogen by Nested-PCR Based on 16S rDNA[J]. Acta Phytopathologica Sin Ica.2005,35 (5): 420-427.
[20]王赢,周国梁,印丽萍,等.玉米细菌性枯萎病菌PCR检测[J].植物病理学报,2009,39(4)368-376.
[21]朱金国,莫瑾,彭梓,等PCR—DHPLC技术快速检测玉米细菌性枯萎病菌[J].植物检疫,2009年,23(5)：15-18.
[22]R. Xu, Q. Chen, Z. Robleh Djama, et al. Tam bong, Miniprimer PCR assay targeting multiple genes:a new rapid and reliable tool for genotyping Pantoea stewartii subsp.stewartii[J]. Letters in Applied Microbiology,2010,50:216-222.
[23]CAB International. Clavibacter michiganensis subsp. nebraskensis, Distribution map][M]. Distribution Maps of Plant Diseases, UK:2000. April(Edition 2), Map 549.
[24]Jackson A T, Harveson R M, Vidaver A K. Goss's bacterial Wilt and Leaf Blight of corn. University of Nebraska—Lincoln Extension publications[J].2007, online at http://www.Extension.unl.edu/publications
[25]Davis M J, Gillaspsie J r, Vidaver A K, et al. Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyll sp. nov., subsp. nov. and Clavibacter xyli subsp. cyrrodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and Bermudagrass stunting disease[J]. International Journal of systematic Bacteriology, 1984,34,107-117.
[26]Kennedy B W, Alcorn S M. Estimates of US crop losses to pro-earyote plant pathogens [J]. Plant Disease,1980,64:674-676.
[27]Pataky J K. Influence of host resistance and growth stage at the time of inoculation on Stewart'S wilt and Goss'wilt development and sweet corn hybrid yield[J]. Plant Disease,1989,73:339-345
[28]Carson M L, Wicks Z W. Relationship between leaf freckles and wilt severity an d yield losses in closely related hybrids[J]. Phytopathology,1991.81:95-98.
[29]陈寔.玉米内州萎焉病菌在中国的适生性和检测方法研究[D].北京：中国农业大学(硕士),2009：1-3.
[30]Biddle J A, Mcgee D C, Braun E J. Seed transmission of Clavibacter michiganensis subsp. nebraskensis in corn[J]. Plant Disease,1990,74:908-911.
[31]Gross D C, Vidaver A k. A selectivemedium forisolation of Corynebacterium nebraskense from soil and plant parts[J]. Phytopathology.1979.62:82～87.
[32]Shepherd, L M. Detection and transmission of Clavibacter michiganesis subsp. nebraskensis of corn[J]. Ms Thesis,1999, Iowa State University, Ames, IA.
[33]Aizawa M, Tsukamoto T, Mizuno A. Studies on the diagnosis of foreign bacterial diseases of quarantine significance Ⅶ. Preparation of selective medium and antiserum for the detection of Clavibacter michiganesis subsp. nebraskensis[J]. Research Bulletin of the Plant Protection Service(Japan),1997,33:7～15.
[34].吴晓巍,金涌,田世民,等.玉米内州萎蔫病菌免疫学检测方法的建立[J].微生物学通报,2010年6月,37(6)：929-934.
[35]Louws F J, Bell J, Medina-Mora C M, et al. Rep-PCR-mediated genomic fingerprinting:A rapid and effective method to identify Clavibacter michiganensis[J]. Phytopathology,1998,88(8): 862～868.
[36]Pastrik K H, Rainey F A. Identification and differentiation of Clavibacter michiganesis subspecies by polymerase chain reaction-based techniques [J]. Journal of Phytopathology,1999,147:687～693.
[37]Ayala L A, Rodriguez H R, Aguilar G C, et al. Dectection of Clavibacter michiganesis subsp. nebraskensis (Schuster, Hoff, Mandel and Lazar) Viadver and Mandel, using the polymerase chain reaction[J]. Revista Mexicana de Fitopatologia,2004,22(2):239～245.
[38]Bach H J, Jessen I, M. Schloter, et al. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies[J]. Journal of Microbiological Methods,2003,52(1):85～91.
[39]周琦,陈寔,高文娜，等.PCR技术快速检测玉米内州萎蔫病菌研究[J].植物检疫,2010年,24(2)：16-18.
[40]操迎梅.磁性纳米材料的发展及应用[J].安庆师范学院学报(自然科学版),2009,15(4),52-55.
[41]赵紫来,卞征云,陈朗星,等.氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用[J].化学进展,2006年10月,18(10),1287-1297.
[42]Schwertmann U, Cornell RM. Iron oxides in the laboratory:preparation and characterization[M], Weinheim, Cambridge:VCH,1991:6-13.
[43]赵慧君,王德平,黄文卫,等.磁性纳米微球的特性及其在生物医学中的应用[J].同济大学学报,2003年8月,24(4),288-291.
[44]张丛云.单分散微米尺寸聚丙烯酸磁珠的制备及其在生物大分子分离技术上的初步应用[D].北京：北京师范大学(博士),2009,1-2.
[45]B Caroccia,A Fassina, TM Seccia, et al. Isolation of Human Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method[J]. Endocrinology.2010,151:1375-1380.
[46]Yoshiaki Nishiya, Takao Hibi, Jun-ichi Oda. A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease[J]. Protein Expression and Purification 2002 (25):426-429.
[47]谢欣,张旭,高华芳,等.基于多功能纳米磁珠的DNA制备与基因分型[J].科学通报,2004年3月,6(49)：541-543.
[48]李文章,李洁,邱克强，等.超顺磁性Fe3O4纳米颗粒的制备及修饰[J].功能材料,2007,38(8)：1279-1286.
[49]Goya, Cerardo F. Handling the particle size and distribution of Fe304 nanoparticles through ball milling[J]. Solid State Commun,2004,130(12):783.
[50]倪颂,严红革,刘芳,等.水溶液中球磨铁粉制备纳米Fe3O4[J].矿冶工程,2007,27(3)：92.
[51]刘飚,官建国,张清杰.多元醇法制备纳米Fe304的研究[J].化学工程,2007,35(7)：56.
[52]王晓斌 黄国林.纳米Fe3O4颗粒的制备及应用[J].化学时刊,2010,24(9)：38-43.
[53]陈明洁,张汉昌,关志荣.化学共沉淀法制备Fe3O4纳米颗粒的结构和磁性能研究[J].材料导报,2008,22(XH)：94.
[54]杨宇翔,张莉苹,梁晓娟,等.纳米Fe3O4及钴离子掺杂Fe3O4：有机碱共沉淀制备和磁性质[J]。无机化学学报,2010年4月,26(4)：668-676
[55]M N Widjojoatmodjo, A C Fluit, R Torensma, et al. The Magnetic Immuno Polymerase Chain Reaction Assay for Direct Detection of Salmonellae in Fecal Samples[J]. Journal Of Clinical Microbiology, Dec.1992,30 (12):3195-3199.
[56]Orjan Olsvik, T Popovic, E Skjerve, et al. Magnetic Separation Techniques in Diagnostic Microbiology[J]. Clinical Microbiology Reviews, Jan.1994,7 (1):43-54.
[57]Hongwei Gu, Pak-Leung Ho, Kenneth WT Tsang, et al. Using biofunctional magnetic nanoparticles to capture Gram-negative bacteria at an ultra-low concentration[J]. Chem Commun,2003:1966-1967.
[58]Hongwei Gu, Pak-Leung Ho, Kenneth W. T. Tsang, et al. Using Biofunctional Magnetic Nanoparticles to Capture Vancomycin-Resistant Enterococci and Other Gram-Positive Bacteria at Ultralow Concentration[J]. J. AM. CHEM. SOC.2003,125:15702-15703.
[59]L. de Leon, F. Siverio, A. Rodriguez. Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation [J]. Journal of Microbiological Methods,2006, 67:141-149.
[60]Madhuka Varshney, Li Y Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples[J]. Biosensors and Bioelectronics,2007,22:2408-2414.
[61]Shu-I Tu, Sue Reed, Andrew Gehring, et al. Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry[J]. Sensors 2009,9: 717-730.
[62]李智洋,何磊,何农跃,等.一种实用的基于化学发光和磁性纳米颗粒的E.coliO157：H7免疫鉴定方法[J].化学学报,2010,68(3),251～256.
[63]毛荣彪,解宇,田菊霞,等.菌源性磁珠在病原菌检测中的应用[J].浙江医学,2010,32(6),846-848.
[64]支援,孟瑾,郑小平,等.一种快速检测阪崎肠杆菌的新方法——免疫磁性分离荧光标记[J].乳业科学与技术,2010,5,231-233.
[65]Uni Ryumae, Kyoko Hibi, Yasutoshi Yoshiura, et al. Rapid and highly sensitive detection of Flavobacterium psychrophilum using high gradient immunomagnetic separation with flow cytometry aquacuhure[J/OL].2010,309:125-130, journal homepage:www.elsevier.com/ locate/aqua-online.
[66]邱晋,樊学军,沈圣,等.自制裸磁珠对常见食源性致病菌吸附性能的研究[J].现代预防学,2006,33(1),4-11.
[67]樊学军,陈恒,孙敏,等.裸磁珠对高菌量细菌吸附性能的初步研究[J].中国卫生检验杂志2006年12月,16(12)：1429-1431
[68]Siamak P. Yazdankhah, Henning Sorum, Hans J. S. Larsen, et al. Use of magnetic beads for Gram staining of bacteria in aqueous suspension[J]. Journal of Microbiological Methods 2001,47:369-371.
[69]陈寔,高文娜,江万春,等.玉米内州萎蔫病菌在中国的适生性[J].植物检疫,2009,23(5)：9-12.
[70]徐进,陈林,许景升,等.玉米细菌性枯萎病菌的适生性风险分析[J].植物保护可见创新与发展,417-419.
[71]刘翔,刘凤权.基于地理信息系统的玉米细菌性枯萎病在中国适宜流行区的初步研究[J].植物检疫,2008,22(1)：9-11.
[72]William G, Barns SM, Pelletier DA, et al.16S Ribosomal DNA Amplification for Phylogenetic Study[J]. Journal of bacteriology, Jan.1991:697-70.
[73]李学贵,袁生.微生物转化过程中利用OD值实时监测测细菌生物量变化的研究[J].南京师大学报(自然科学版)2003年,26(4)：90-93.
[74]孙飞龙,张周梅,马军,等.Fe304纳米磁颗粒的制备及其对水中细菌富集的初步研究[J].化学与生物工程2009,26(10)：90-92.
[2]Stewart F C. A bacterial disease of sweet corn[J]. New York Agric. Exp. StnBull,1897,130: 422-439.
[3]Aurelien L. Carlier and S. B. von Bodman. The rcsA Promoter of Pantoea stewartii subsp. stewartii Features a Low-Level Constitutive Promoter and an EsaR Quorum-Sensing-Regulated Promoter[J]. Journal of Bacteriology,2006:4581-4584.
[4]White DG. Compendium of corn disease (3rd edition)[M]. USA:APS Press,1999,3.
[5]Pepper, E. H.1967. Stewart's bacterial wilt of corn [M]. Monograph 4. American Phytopathological Society, St. Paul, MN, U.S.A.
[6]Thai Agricultural Standard. TAS 9031-2008. Diagnostic Protocols for Pantoea Stewartii subsp. Stewartii Bacterial Wilt of Maize [S].Thai:Ministry of Agriculture and Cooperatives,2008.
[7]齐玲玲.玉米萎蔫和枯萎病菌胶体金免疫层析技术研究[D].山西：山西大学(硕士),2010：1-10.
[8]Patrick E. Lipps, Anne E. Dorrance, and Dennis R. Mills. Stewart's Bacterial Wilt and Leaf Blight of Corn[J]. The Ohio State University web site,2001, on line at:http://ohioline.osu.edu
[9]Pataky JK,Hawk JA,Weldekidan T, et al. Incidence and severity of Stewart's bacterial wilt on sequential plantings of resistant and susceptible sweet corn hybrids[J]. Plant Dis,1995,79:1202-1207
[10]郭翼奋,梁再群,俞大绂.黑色素选择性培养从进口玉米中分离玉米枯萎病菌[J].科学通报.1982,22(4)：339-344.
[11]Blakemore EJA, Law JR,Reeves JC. PCR identification of Erwinia stwarttii and its comparison with two other methods[J]. Seed Sci&Technol,1999,27:385～396.
[12]Lamka GL, Hill JH, McGee DC, et al. Development of an immunosorbent assay for seedborne Erwinia stewartii in corn seeds[J]. Phytopathology,1991,81:839～846.
[13]王岭,田世民,高海霞,等.玉米细菌性枯萎病菌改良Dot—ELISA检测研究[J]。微生物学通报,2008,35(2),230—234.
[14]玉米细菌性枯萎病菌检疫鉴定方法[S].中华人民共和国出入境检验检疫行业标准.SN/T 1375-2004,2004-12-01实施,中华人民共和国国家质量监督检验检疫总局发布.
[15]齐玲玲,金涌,邹明强,等.胶体金免疫层析试纸条快速检测玉米细菌性枯萎病菌[J].检验检 疫学刊,2010年,20(1)：1-5.
[16]漆艳香,肖启明,朱水芳.玉米细菌性枯萎病菌16S rDNA基因克隆及TaqMan探针实时荧光PCR[J]湖南农业大学学报(自然科学版),2003年6月,29(3)：183-187.
[17]漆艳香,朱水芳,赵文军等.玉米细菌性枯萎病菌TaqMan探针实时荧光PCR检测方法的建立[J].植物保护学报,2004年3月,31(1)：51-56.
[18]王茂华,胡白石,卢玲,等.利用巢式PCR检测玉米细菌性枯萎病菌[J].南京农业大学学报2005,28(2)：37-40.
[19]WU Qiong, CHEN Zhinan, FAN Huaizhong, et al. Identification of Corn Bacterial Wilt Pathogen by Nested-PCR Based on 16S rDNA[J]. Acta Phytopathologica Sin Ica.2005,35 (5): 420-427.
[20]王赢,周国梁,印丽萍,等.玉米细菌性枯萎病菌PCR检测[J].植物病理学报,2009,39(4)368-376.
[21]朱金国,莫瑾,彭梓,等PCR—DHPLC技术快速检测玉米细菌性枯萎病菌[J].植物检疫,2009年,23(5)：15-18.
[22]R. Xu, Q. Chen, Z. Robleh Djama, et al. Tam bong, Miniprimer PCR assay targeting multiple genes:a new rapid and reliable tool for genotyping Pantoea stewartii subsp.stewartii[J]. Letters in Applied Microbiology,2010,50:216-222.
[23]CAB International. Clavibacter michiganensis subsp. nebraskensis, Distribution map][M]. Distribution Maps of Plant Diseases, UK:2000. April(Edition 2), Map 549.
[24]Jackson A T, Harveson R M, Vidaver A K. Goss's bacterial Wilt and Leaf Blight of corn. University of Nebraska—Lincoln Extension publications[J].2007, online at http://www.Extension.unl.edu/publications
[25]Davis M J, Gillaspsie J r, Vidaver A K, et al. Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyll sp. nov., subsp. nov. and Clavibacter xyli subsp. cyrrodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and Bermudagrass stunting disease[J]. International Journal of systematic Bacteriology, 1984,34,107-117.
[26]Kennedy B W, Alcorn S M. Estimates of US crop losses to pro-earyote plant pathogens [J]. Plant Disease,1980,64:674-676.
[27]Pataky J K. Influence of host resistance and growth stage at the time of inoculation on Stewart'S wilt and Goss'wilt development and sweet corn hybrid yield[J]. Plant Disease,1989,73:339-345
[28]Carson M L, Wicks Z W. Relationship between leaf freckles and wilt severity an d yield losses in closely related hybrids[J]. Phytopathology,1991.81:95-98.
[29]陈寔.玉米内州萎焉病菌在中国的适生性和检测方法研究[D].北京：中国农业大学(硕士),2009：1-3.
[30]Biddle J A, Mcgee D C, Braun E J. Seed transmission of Clavibacter michiganensis subsp. nebraskensis in corn[J]. Plant Disease,1990,74:908-911.
[31]Gross D C, Vidaver A k. A selectivemedium forisolation of Corynebacterium nebraskense from soil and plant parts[J]. Phytopathology.1979.62:82～87.
[32]Shepherd, L M. Detection and transmission of Clavibacter michiganesis subsp. nebraskensis of corn[J]. Ms Thesis,1999, Iowa State University, Ames, IA.
[33]Aizawa M, Tsukamoto T, Mizuno A. Studies on the diagnosis of foreign bacterial diseases of quarantine significance Ⅶ. Preparation of selective medium and antiserum for the detection of Clavibacter michiganesis subsp. nebraskensis[J]. Research Bulletin of the Plant Protection Service(Japan),1997,33:7～15.
[34].吴晓巍,金涌,田世民,等.玉米内州萎蔫病菌免疫学检测方法的建立[J].微生物学通报,2010年6月,37(6)：929-934.
[35]Louws F J, Bell J, Medina-Mora C M, et al. Rep-PCR-mediated genomic fingerprinting:A rapid and effective method to identify Clavibacter michiganensis[J]. Phytopathology,1998,88(8): 862～868.
[36]Pastrik K H, Rainey F A. Identification and differentiation of Clavibacter michiganesis subspecies by polymerase chain reaction-based techniques [J]. Journal of Phytopathology,1999,147:687～693.
[37]Ayala L A, Rodriguez H R, Aguilar G C, et al. Dectection of Clavibacter michiganesis subsp. nebraskensis (Schuster, Hoff, Mandel and Lazar) Viadver and Mandel, using the polymerase chain reaction[J]. Revista Mexicana de Fitopatologia,2004,22(2):239～245.
[38]Bach H J, Jessen I, M. Schloter, et al. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies[J]. Journal of Microbiological Methods,2003,52(1):85～91.
[39]周琦,陈寔,高文娜，等.PCR技术快速检测玉米内州萎蔫病菌研究[J].植物检疫,2010年,24(2)：16-18.
[40]操迎梅.磁性纳米材料的发展及应用[J].安庆师范学院学报(自然科学版),2009,15(4),52-55.
[41]赵紫来,卞征云,陈朗星,等.氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用[J].化学进展,2006年10月,18(10),1287-1297.
[42]Schwertmann U, Cornell RM. Iron oxides in the laboratory:preparation and characterization[M], Weinheim, Cambridge:VCH,1991:6-13.
[43]赵慧君,王德平,黄文卫,等.磁性纳米微球的特性及其在生物医学中的应用[J].同济大学学报,2003年8月,24(4),288-291.
[44]张丛云.单分散微米尺寸聚丙烯酸磁珠的制备及其在生物大分子分离技术上的初步应用[D].北京：北京师范大学(博士),2009,1-2.
[45]B Caroccia,A Fassina, TM Seccia, et al. Isolation of Human Adrenocortical Aldosterone-Producing Cells by a Novel Immunomagnetic Beads Method[J]. Endocrinology.2010,151:1375-1380.
[46]Yoshiaki Nishiya, Takao Hibi, Jun-ichi Oda. A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease[J]. Protein Expression and Purification 2002 (25):426-429.
[47]谢欣,张旭,高华芳,等.基于多功能纳米磁珠的DNA制备与基因分型[J].科学通报,2004年3月,6(49)：541-543.
[48]李文章,李洁,邱克强，等.超顺磁性Fe3O4纳米颗粒的制备及修饰[J].功能材料,2007,38(8)：1279-1286.
[49]Goya, Cerardo F. Handling the particle size and distribution of Fe304 nanoparticles through ball milling[J]. Solid State Commun,2004,130(12):783.
[50]倪颂,严红革,刘芳,等.水溶液中球磨铁粉制备纳米Fe3O4[J].矿冶工程,2007,27(3)：92.
[51]刘飚,官建国,张清杰.多元醇法制备纳米Fe304的研究[J].化学工程,2007,35(7)：56.
[52]王晓斌 黄国林.纳米Fe3O4颗粒的制备及应用[J].化学时刊,2010,24(9)：38-43.
[53]陈明洁,张汉昌,关志荣.化学共沉淀法制备Fe3O4纳米颗粒的结构和磁性能研究[J].材料导报,2008,22(XH)：94.
[54]杨宇翔,张莉苹,梁晓娟,等.纳米Fe3O4及钴离子掺杂Fe3O4：有机碱共沉淀制备和磁性质[J]。无机化学学报,2010年4月,26(4)：668-676
[55]M N Widjojoatmodjo, A C Fluit, R Torensma, et al. The Magnetic Immuno Polymerase Chain Reaction Assay for Direct Detection of Salmonellae in Fecal Samples[J]. Journal Of Clinical Microbiology, Dec.1992,30 (12):3195-3199.
[56]Orjan Olsvik, T Popovic, E Skjerve, et al. Magnetic Separation Techniques in Diagnostic Microbiology[J]. Clinical Microbiology Reviews, Jan.1994,7 (1):43-54.
[57]Hongwei Gu, Pak-Leung Ho, Kenneth WT Tsang, et al. Using biofunctional magnetic nanoparticles to capture Gram-negative bacteria at an ultra-low concentration[J]. Chem Commun,2003:1966-1967.
[58]Hongwei Gu, Pak-Leung Ho, Kenneth W. T. Tsang, et al. Using Biofunctional Magnetic Nanoparticles to Capture Vancomycin-Resistant Enterococci and Other Gram-Positive Bacteria at Ultralow Concentration[J]. J. AM. CHEM. SOC.2003,125:15702-15703.
[59]L. de Leon, F. Siverio, A. Rodriguez. Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation [J]. Journal of Microbiological Methods,2006, 67:141-149.
[60]Madhuka Varshney, Li Y Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples[J]. Biosensors and Bioelectronics,2007,22:2408-2414.
[61]Shu-I Tu, Sue Reed, Andrew Gehring, et al. Capture of Escherichia coli O157:H7 Using Immunomagnetic Beads of Different Size and Antibody Conjugating Chemistry[J]. Sensors 2009,9: 717-730.
[62]李智洋,何磊,何农跃,等.一种实用的基于化学发光和磁性纳米颗粒的E.coliO157：H7免疫鉴定方法[J].化学学报,2010,68(3),251～256.
[63]毛荣彪,解宇,田菊霞,等.菌源性磁珠在病原菌检测中的应用[J].浙江医学,2010,32(6),846-848.
[64]支援,孟瑾,郑小平,等.一种快速检测阪崎肠杆菌的新方法——免疫磁性分离荧光标记[J].乳业科学与技术,2010,5,231-233.
[65]Uni Ryumae, Kyoko Hibi, Yasutoshi Yoshiura, et al. Rapid and highly sensitive detection of Flavobacterium psychrophilum using high gradient immunomagnetic separation with flow cytometry aquacuhure[J/OL].2010,309:125-130, journal homepage:www.elsevier.com/ locate/aqua-online.
[66]邱晋,樊学军,沈圣,等.自制裸磁珠对常见食源性致病菌吸附性能的研究[J].现代预防学,2006,33(1),4-11.
[67]樊学军,陈恒,孙敏,等.裸磁珠对高菌量细菌吸附性能的初步研究[J].中国卫生检验杂志2006年12月,16(12)：1429-1431
[68]Siamak P. Yazdankhah, Henning Sorum, Hans J. S. Larsen, et al. Use of magnetic beads for Gram staining of bacteria in aqueous suspension[J]. Journal of Microbiological Methods 2001,47:369-371.
[69]陈寔,高文娜,江万春,等.玉米内州萎蔫病菌在中国的适生性[J].植物检疫,2009,23(5)：9-12.
[70]徐进,陈林,许景升,等.玉米细菌性枯萎病菌的适生性风险分析[J].植物保护可见创新与发展,417-419.
[71]刘翔,刘凤权.基于地理信息系统的玉米细菌性枯萎病在中国适宜流行区的初步研究[J].植物检疫,2008,22(1)：9-11.
[72]William G, Barns SM, Pelletier DA, et al.16S Ribosomal DNA Amplification for Phylogenetic Study[J]. Journal of bacteriology, Jan.1991:697-70.
[73]李学贵,袁生.微生物转化过程中利用OD值实时监测测细菌生物量变化的研究[J].南京师大学报(自然科学版)2003年,26(4)：90-93.
[74]孙飞龙,张周梅,马军,等.Fe304纳米磁颗粒的制备及其对水中细菌富集的初步研究[J].化学与生物工程2009,26(10)：90-92.