: MALDI-TOF 417, (m/z), ;, ( ), [1] MALDI-TOF-MS,, ( ) [2], PCR,,, PCR,,, MALDI-TOF-MS, [3],,,, MALDI-TOF-MS, O157:H7, ( BioTyper Mascope ), [4 9] MAL

Size: px

Start display at page:

Download ": MALDI-TOF 417, (m/z), ;, ( ), [1] MALDI-TOF-MS,, ( ) [2], PCR,,, PCR,,, MALDI-TOF-MS, [3],,,, MALDI-TOF-MS, O157:H7, ( BioTyper Mascope ), [4 9] MAL"

道张
5 years ago
Views:

1 微生物学通报 MAR 20, 2009, 36(3): 416~426 Microbiology 研究快报 2009 by Institute of Microbiology, CAS MALDI-TOF 质谱技术分析与鉴定病原细菌研究 1,2 其布勒哈斯 2* 田世民 2 邹明强 1 贺银凤 2 陈艳 2 赵贵明 ( ) ( ) 摘要 : 本文通过基于基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF-MS) 技术分析病原细菌的方法进行研究, 阐明影响分析结果的重要因素, 并建立了 MALDI-TOF-MS 分析病原细菌的标准方法对不同属种和亚种的 12 株植物病原细菌进行全细胞分析结果表明 :MALDI-TOF-MS 能快速而准确的区分和鉴定病原细菌, 分析过程简单灵敏度高此法在细菌属种亚种和菌株水平上, 可快速准确地区分和鉴定关键词 : MALDI-TOF MS, 细菌质量指纹图谱, 鉴定, 病原细菌 Analysis and Identification of Pathogenic Bacteria by MALDI-TOF Mass Spectrometry Qibulehasi 1,2 TIAN Shi-Min 2* ZOU Ming-Qiang 2 HE Yin-Feng 1 CHEN Yan 2 ZHAO Gui-Ming 2 (1. Inner Mongolian Agricultural University, Hohhot, Inner Mongolia , China) (2. Chinese Academy of Inspection and Quarantine, Beijing , China) Abstract: Based on Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF-MS) method for discrimination of pathogenic bacteria was developed. Established the standardization method for MALDI-TOF-MS analysis of bacteria and affecting elements of the analyzing result. The results obtained from twelve plants pathogenic bacteria of different genus, species and subspecies were analyzed by using MALDI-TOF showed that, MALDI-TOF-MS could be rapid and reliable identification of bacteria at the level of genus, species, and strains. Analytical process is simple, high sensitivity are advantages of this strategy. Keywords: MALDI-TOF-MS, Bacteria mass finger-printing, Identification, Pathogenic bacteria (Matrixassisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS) ( ),,,,,,, 基金项目 : (No. 2006BAK10B09); (No. 2007JK011) * 通讯作者 :Tel/Fax: ; : shimintian@yahoo.com.cn 收稿日期 : ; 接受日期 :

2 : MALDI-TOF 417, (m/z), ;, ( ), [1] MALDI-TOF-MS,, ( ) [2], PCR,,, PCR,,, MALDI-TOF-MS, [3],,,, MALDI-TOF-MS, O157:H7, ( BioTyper Mascope ), [4 9] MALDI-TOF-MS,,, Pseudomonas syringae Erwinia carotovora ; Acidovorax avenae 2004 ; Erwinia (Pantoea) stewartii subsp. stewarii ; Clavibacter michiganensis subsp. nebraskensis Clavibacter michiganensis subsp. sepedonicus Clavibacter michiganensis subsp. insidiosus,, 2007 ( 862 ) Clavibacter fangii, 1987 [10],, 1 材料与方法 1.1 材料试剂和仪器 : (Insulin) (TFA) Sigma, ; α- -4- (α-cyano-4-hydroxycinnamic acid, CHCA) ABI ; (NBY) (NA) luria bertani(lb) ; ABI MALDI-TOF/TOF 供试菌株和培养方法 : 1, 4 C (Clavibacter michiganensis) 3 IQCC11 IQCC15 IQCC16, (3 NBY NA LB, 3 24 C 28 C 32 C, 3 24 h 48 h 72 h) MALDI-TOF MS 1.2 基质溶液的配制 - (1 1, V/V), 0.1 μl (TFA) 10 mg α- -4-, 20 C 样品处理方法直接分析法 :,,, 0.5 μl,

3 418 微生物学通报 2009, Vol.36, No.3 Table 1 表 1 供试菌株 Bacterial strains of analyzed No. of strains Name of strains Source of strains IQCC 3 Acidovorax avenae subsp. avenae China General Microbiological Culture Collection Center IQCC 9 Clavibacte fangii China General Microbiological Culture Collection Center IQCC 6 Clavibacter michiganensis subsp. insidiosum Isolated from Nanjing IQCC 15 Clavibacter michiganensis subsp. insidiosum American Type Culture Collection Center IQCC 11 Clavibacter michiganensis subsp. nebraskensis American Type Culture Collection Center IQCC 8 Clavibacter michiganensis subsp. sepedonicus The Institute of Plant Protection Chinese Academy of Agricultural Sciences IQCC 16 Clavibacter michiganensis subsp. tessellarius American Type Culture Collection Center IQCC 1 Erwinia carotovora subsp. carotovora The Institute of Plant Protection Chinese Academy of Agricultural Sciences IQCC 10 Erwinia (Pantoea) stewartii subsp. stewartii Isolated from Nanjing IQCC 2 Pseudomonas syringae pv. syringae The Institute of Plant Protection Chinese Academy of Agricultural Sciences IQCC 4 Xanthomonas campestris pv. holcicola Isolated from Shanghai IQCC 5 Xanthomonas campestris pv. holcicola American Type Culture Collection Center 单一溶剂处理 : 1 mg~5 mg 1.5 ml E-cups, 0.2% TFA 2, r/min 2 min,, 50 μl 0.2% TFA, r/min 2 min E-cups 混合溶剂处理 : 1 mg~5 mg 1.5 ml E-cups, 300 μl, 900 μl, r/min 2 min,, E-cups 50 μl 70% 50 μl,,, E-cups 1.4 点样 0.5 μl MALDI TOF, ( 3 min), 0.5 μl,, 质谱分析, 337 nm, ; V; 300 ns; 2500; 40 50, m/z 1000 D~20000 D 1000 D~20000 D, (m/z 5734), 6 Data Explorer 结果与分析 2.1 细菌培养条件和样品处理方法的标准化 IQCC11 IQCC15 IQCC16, 3 3 ( ), 2000 D D NBY,, NBY 28 C 24 h 3, MALDI-TOF 分析结果棒杆菌属 6 个病原菌的 MALDI-TOF 分析结果 : (Clavibacter) 6 MALDI- TOF ( 1 2) , 6 6, a b c f, (Clavibacter michiganensis) 4, m/z 2884, ;

4 : MALDI-TOF 419

5 420 微生物学通报 2009, Vol.36, No.3 图 1 棒杆菌属 6 个菌株的质量图谱 Fig. 1 Mass spectrometric profiles of six species of genus Clavibacter Note: a: IQCC11; b: IQCC8; c: IQCC16; d: IQCC9; e: IQCC6 ; f: IQCC15. Table 2 表 2 棒杆菌属 6 个菌株的 MALDI-TOF-MS 分析结果 Peaks obtained from the analysis of six species of genus Clavibacter with MALDI-TOF-MS nebraskensis IQCC 11 sepedonicus IQCC 8 tessellarius IQCC 16 C. fangii IQCC 9 insidiosum IQCC 6 insidiosum IQCC 15 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity

6 : MALDI-TOF 421 nebraskensis IQCC 11 sepedonicus IQCC 8 tessellarius IQCC 16 C. fangii IQCC 9 insidiosum IQCC 6 insidiosum IQCC 15 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity 续表

7 422 微生物学通报 2009, Vol.36, No.3 e f, insidiosus IQCC15 IQCC6 9, m/z , ; e 14 ; f 19, MALDI-TOF-MS 玉米病原菌的 MALDI-TOF 分析结果 :, 6 MALDI-TOF 2 3 a b (Erwinia)

8 : MALDI-TOF 423 图 2 六个玉米病原菌的质量图谱 Fig. 2 Mass spectrometric profiles of six species of corn pathogenic bacteria Note: a: IQCC1; b: IQCC10; c: IQCC2; d: IQCC3; e: IQCC4; f: IQCC5. Table 3 表 3 玉米病原菌的 MALDI-TOF-MS 分析结果 Peaks obtained from the analysis of corn pathogenic bacteria with MALDI-TOF-MS E. c. subsp. carotovora IQCC 1 E. s. subsp. stewarii IQCC 10 P. s. subsp. syringae IQCC 2 A. a. subsp. avenae IQCC 3 X. c. subsp. holcicola IQCC 4 X. c. subsp. holcicola IQCC 5 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity

9 424 微生物学通报 2009, Vol.36, No.3 续表 E. c. subsp. carotovora IQCC 1 E. s. subsp. stewarii IQCC 10 P. s. subsp. syringae IQCC 2 A. a. subsp. avenae IQCC 3 X. c. subsp. holcicola IQCC 4 X. c. subsp. holcicola IQCC 5 Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. Avg. m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity m/z intensity

10 : MALDI-TOF 425, a 19, b 10, e f (Xanthomonas) 15, m/z ,, IQCC5 IQCC4,, e 12 m/z ; f 2 m/z ; IQCC5 IQCC4 10 MALDI-TOF, Matlab R-2007a (Matlab Com., USA) ( 3), IQCC 4 IQCC 5, Fig. 3 Three-dimensional scatter plot of the Principal component analysis of two X. c. pv. holcicola strains Note: a: IQCC 4; b: IQCC 5., MALDI-TOF 3 讨论 12 MALDI-TOF,,, MALDI- TOF,, MALDI,,,,,,,, Mazzeo MALDI-TOF [11] IQCC5 IQCC4, 2,, MALDI-TOF MALDI-TOF,, MALDI-TOF, 参考文献 [1] Fenselau C, Demirev PA. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrometry Reviews, 2001, 20(4): [2],.., 2005, 39(5): [3],,. MALDI-TOF-MS., 2003, 31(2): [4] Phan-Thanh L, Mahouin F. A proteomic approach to study the acid response in Listeria monocytogenes. Electrophoresis, 1999, 20(11): [5] Bernardo K, Pakulat N, Macht M, et al. Identification and discrimination of Staphylococcus aureus strains using

11 426 微生物学通报 2009, Vol.36, No.3 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomics, 2002, 2(6): [6] Johanna EC, Faith AH. Discrimination between wild- type and ampicillin-resistant Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analytical and Bioanalytical Chemistry, 2007, 389(5): [7] Demirev PA, Lin JS, Pineda F, et al. Bioinformatics and mass spectrometry for microorganism identification: proteome-wide post-translational modifications and database search algorithms for characterization of intact H. pylori. Analytical Chemistry, 2001, 73(19): [8] Liu YC, Li YB. Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement. Journal of microbiological methods, 2002, 51(3): [9] Wunschel SC, Jarman KH, Petersen CE, et al. Bacterial analysis by MALDI-TOF mass spectrometry: an inter-laboratory comparison. Journal of the American Society for Mass Spectrometry, 2005, 16(4): [10],. ITS PCR., 2003, 33(3): [11] Mazzeo MF, Sorrentino A, Gaita M, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the discrimination of food-borne microorganisms. Applied and Environmental Microbiology, 2006, 72(2): 稿件书写规范高校教改纵横栏目简介及撰稿要求,,,,,,,,,,,,,,,,,,,,,,,,

《中国科学》A、E、G与F小开本版式设计

《中国科学》A、E、G与F小开本版式设计 222 E 2006, 36(2): 222~234 * 1** 1,2 1,2 1,2 1,2 3 3 1 1 1,2 (1., 100080; 2., 100039; 3., 200031),. ;,,.. 20 90 (human genome project, HGP), ; HGP 2003, 21. (proteomics) ; (computational proteomics),,,