微生物学通报 MAR 20, 2009, 36(3): 416~426 Microbiology tongbao@im.ac.cn 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 (1. 010018) (2. 100123) 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 010018, China) (2. Chinese Academy of Inspection and Quarantine, Beijing 100123, 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: 86-10-85747382; : shimintian@yahoo.com.cn 收稿日期 :2008-10-22; 接受日期 :2008-12-11
: 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 1.1.1 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 4700 1.1.2 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 1 1.3 样品处理方法 1.3.1 直接分析法 :,,, 0.5 μl,
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 1.3.2 单一溶剂处理 : 1 mg~5 mg 1.5 ml E-cups, 0.2% TFA 2, 10000 r/min 2 min,, 50 μl 0.2% TFA, 10000 r/min 2 min E-cups 1.3.3 混合溶剂处理 : 1 mg~5 mg 1.5 ml E-cups, 300 μl, 900 μl, 10000 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,, 3 1.5 质谱分析, 337 nm, ; 15000 V; 300 ns; 2500; 40 50, m/z 1000 D~20000 D 1000 D~20000 D, (m/z 5734), 6 Data Explorer 4.0 3 2 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 15000 D NBY,, NBY 28 C 24 h 3, 1 2.2 MALDI-TOF 分析结果 2.2.1 棒杆菌属 6 个病原菌的 MALDI-TOF 分析结果 : (Clavibacter) 6 MALDI- TOF ( 1 2) 6 10 30, 6 6, a b c f, (Clavibacter michiganensis) 4, m/z 2884, ; 4 2 15
: MALDI-TOF 419
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 2215 2.16 2216 36.11. 2128 22.83 2180 11.79 2609 11.76 2607 3.54 2726 4.64 2867 13.01 2869 17.93 2883 7.34 2883 15.04 2883 13.44 2882 2.01 2883 19.33 2915 4.11 3003 5.99 3030 12.26 3487 9.02 3246 12.73 3535 2.89 3536 10.19 3549 4.12 3565 22.92 3650 7.84 3640 5.05 3813 9.36 3765 7.94 4268 17.35 4352 12.80 4352 1.86 4350 29.21 4728 41.46 4636 1.42 4492 7.38
: 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 4791 18.36 4776 14.63 4784 3.33 4779 1.49 4775 16.23 4914 8.82 5084 8.08 5081 15.87 5085 4.26 5082 17.97 5218 14.33 5208 100.50 5207 2.60 5201 8.64 5243 28.56 5283 6.47 5410 15.71 5532 7.95 5732 98.00 5742 89.67 5735 98.77 5732 10.14 5726 87.23 5764 89.90 5754 35.11 5763 32.64 5756 82.54 5797 35.61 5807 16.76 5835 19.40 5823 18.89 续表 6049 37.54 6116 18.69 6149 14.66 6193 12.56 6280 9.68 6297 2.79 6291 10.23 6350 14.89 6443 8.68 6488 9.01 6482 19.35 6489 6.36 6482 27.97. 6618 3.43 6712 11.92 6721 4.13 6713 13.75 6918 11.36 6823 9.87 7063 24.20 7055 48.17 7094 95.99 7068 10.88 7100 23.95 7129 22.14 7119 97.75 7225 25.30 7239 45.01 7240 4.89 7286 50.62 7269 34.71 7498 12.75 7536 6.58 7526 4.52 7516 15.53 8489 3.91 8606 14.28 8571 3.66 8558 6.28 8729 14.10 8994 5.96 8965 7.07 9235 10.85 9293 7.69 9307 3.95 9294 5.66 9553 5.54 9574 94.74 9538 15.55 9552 14.49 9553 29.42 9539 34.55 9816 41.62 9930 6.09 9988 3.44 10042 5.81 10059 5.07 10392 6.64 10408 26.75 10392 10.57 10442 15.25
422 微生物学通报 2009, Vol.36, No.3 e f, insidiosus IQCC15 IQCC6 9, m/z 2884 4352 4775 5085 5201 5732 6291 6482 6713, ; e 14 ; f 19, MALDI-TOF-MS 2.2.2 玉米病原菌的 MALDI-TOF 分析结果 :, 6 MALDI-TOF 2 3 a b (Erwinia)
: 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 2170 11.43. 2192 13.59 2182 13.68 2461 10.13 2214 14.79 2214 2.05 2608 3.33 2606 93.23 2604 99.82 2702 9.54 2802 9.04 2793 2.54 2945 5.04 2828 4.30 3089 4.93 3082 6.72 3116 5.49 3122 5.91 3353 9.08 3311 24.91 3707 11.88 3745 4.31 4026 3.81 2660 21.89 2642 4.08 2912 10.27 3587 16.53 3607 8.39 3625 4.73 3646 4.97 3685 67.67 3636 14.31 3867 26.38
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 4171 31.76 4230 12.52 4329 6.93 4309 2.75 4302 7.55 4343 4.61 4348 36.17 4400 13.45 4397 13.15 4494 10.75 4715 8.78 4716 12.23 4713 6.27 4759 14.58 4777 25.45 4774 12.92 4802 13.54 4909 25.74 5165 5.31 5214 100.00 5202 7.32 5201 16.39 5198 6.19 5260 24.86 5277 6.67 5313 5.89 5392 45.78 5400 6.67 5523 17.46 5522 6.19 5815 41.17 5813 48.36 5875 3.51 5868 14.15 5863 7.92 6122 4.16 6153 28.08 6234 28.35 6392 11.18 6412 6.82 6474 14.67 6472 8.41 6697 99.45 6611 27.74 6666 8.61 6664 4.84 6749 16.81 6774 10.44 6821 27.02 6810 10.66 6873 7.25 6935 5.75 7067 10.03 7149 5.73 7163 54.73 7159 19.41 7135 6.27 7203 44.08 7200 10.51 7244 8.98 7260 97.32 7258 45.19 7292 2.77 7283 35.24 7359 92.16 7312 37.57 7750 1.58 7726 1.21 7745 3.77 8337 65.25 8700 11.02 8803 3.46 8891 7.03 8980 5.26 9343 5.41 9508 21.7 9539 83.82 9536 59.03 9591 41.12 9589 12.45 9615 1.08 9673 4.54 9655 21.72 9704 14.91 9832 1.58 9809 7.37 9864 1.25
: MALDI-TOF 425, a 19, b 10, e f (Xanthomonas) 15, m/z 2214 2606 4400 4716 4774 5201 5523 5815 5868 6472 6664 7203 7260 9539 9591,, IQCC5 IQCC4,, e 12 m/z 2660 2912 3607 3635 4802 6810 7159 7312 8700 8891 9655 9704; f 2 m/z 2642 6935; IQCC5 IQCC4 10 MALDI-TOF, Matlab R-2007a (Matlab Com., USA) ( 3), IQCC 4 IQCC 5, 10 3 3 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): 157 171. [2],.., 2005, 39(5): 361 363. [3],,. MALDI-TOF-MS., 2003, 31(2): 47 53. [4] Phan-Thanh L, Mahouin F. A proteomic approach to study the acid response in Listeria monocytogenes. Electrophoresis, 1999, 20(11): 2214 2224. [5] Bernardo K, Pakulat N, Macht M, et al. Identification and discrimination of Staphylococcus aureus strains using
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