切换至 "中华医学电子期刊资源库"
高级检索
中华临床实验室管理电子杂志

中华临床实验室管理电子杂志 ›› 2018, Vol. 06 ›› Issue (01) : 27 -31. doi: 10.3877/cma.j.issn.2095-5820.2018.01.008

所属专题： 文献；

实验研究

两种菌体前处理方法对MALDI-TOF MS鉴定临床分离菌效果的影响
曹韪凡1, 宋召2, 周凤丽2, 刘昕超2, 马庆伟2, 曹峰林3,†()   
  1. 1. 150040　哈尔滨　东北林业大学生命科学院
    2. 102206　北京毅新博创生物科技有限公司
    3. 150001　哈尔滨医科大学附属第一医院中心实验室
  • 收稿日期:2017-11-30 出版日期:2018-02-28
  • 通信作者: 曹峰林

Effect of two kinds of bacterial pretreatment methods on the clinical isolate strains identification by MALDI-TOF MS

Weifan Cao1, Zhao Song2, Fengli Zhou2, Xinchao Liu2, Qingwei Ma2, Fenglin Cao3,†()   

  1. 1. College of Life Science, Northeast Forestry University, 150040 Harbin, China
    2. Bioyong Technologies Inc., 102206 Beijing, China
    3. The First Affiliated Hospital of Harbin Medical University, 150001 Harbin, China
  • Received:2017-11-30 Published:2018-02-28
  • Corresponding author: Fenglin Cao
  • About author:
    Corresponding author: Cao Fenglin, Email:
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

曹韪凡, 宋召, 周凤丽, 刘昕超, 马庆伟, 曹峰林. 两种菌体前处理方法对MALDI-TOF MS鉴定临床分离菌效果的影响[J]. 中华临床实验室管理电子杂志, 2018, 06(01): 27-31.

Weifan Cao, Zhao Song, Fengli Zhou, Xinchao Liu, Qingwei Ma, Fenglin Cao. Effect of two kinds of bacterial pretreatment methods on the clinical isolate strains identification by MALDI-TOF MS[J]. Chinese Journal of Clinical Laboratory Management(Electronic Edition), 2018, 06(01): 27-31.

目的

评价2种不同前处理方法对基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ ionization time of flight mass spectrometry，MALDI-TOF MS)鉴定临床分离菌效果的影响。

方法

收集临床菌株43株，划线传代后，分别使用提取法和抹板法对微生物样本进行前处理，然后用MALDI-TOF MS鉴定比较两种处理方法的准确率和蛋白指纹图谱特征。

结果

以16S rRNA/ITS RNA测序结果为参考标准，待测菌株经提取法处理后，MALDI-TOF MS鉴定革兰阳性菌、革兰阴性菌和真菌的种水平准确率分别为93.8%、100.0%和30.8%，待测菌株经抹板法处理后，MALDI-TOF MS鉴定革兰阳性菌、革兰阴性菌和真菌的种水平准确率分别为75.0%、100%和7.7%。提取法处理菌株后得到的蛋白指纹图谱中特征峰更多，信噪比更高，蛋白指纹图谱重现性更好。

结论

推荐采用提取法为MALDI-TOF MS鉴定的常规前处理方法，其对建立标准的微生物检测流程，提高质谱鉴定的正确率，有重要价值。

关键词: 基质辅助激光解吸电离飞行时间质谱, 蛋白指纹图谱, 前处理, 提取法, 抹板法
Objective

To evaluate the effect of different pretreatment methods on the identification of clinical bacterial isolates by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).

Methods

Forty-three clinical isolates were collected and subcultured before treated by two different sample preparation methods including off-plate extraction method and direct on-plate method. And then the treated strains were identified by MALDI-TOF MS. The identification accuracy of the two methods and the peak of protein fingerprint were compared.

Results

Using the results of 16S rRNA or ITS RNA as reference standard, the identification rates of gram positive bacteria, gram negative bacteria and fungus treated by off-plate extraction method were 93.8%, 100% and 30.8%, respectively. And the identification rates of gram positive bacteria, gram negative bacteria and fungus treated by direct on-plate extraction method were 75.0%, 100% and 7.7%, respectively. The characteristic peak in the protein fingerprint obtained after extraction was more; the signal-to-noise ratio was higher and the reproducibility of protein fingerprint was better.

Conclusion

It is recommended that the off-plate extraction method for MALDI-TOF identification should be used, which is of great clinical significance for the establishment of a standard microbial detection process and the improvement of the accuracy of mass spectrometry identification.

Key words: Matrix-assisted laser desorption/ ionization time of flight mass spectrometry, Protein fingerprint, Pretreatment, Off-plate extraction method, Direct on-plate method
表1 提取法和抹板法处理临床分离菌株后，MALDI-TOF MS鉴定准确率结果
菌株名称 株数 鉴定准确率（%）
提取法 抹板法
革兰阳性细菌 ? ? ?
? 粪肠球菌、表皮葡萄球菌、人葡萄球菌、小肠肠球菌、溶血葡萄球菌、屎肠球菌、铅黄肠球菌、解没食子酸链球菌、耐久肠球菌 12 100 100
? 鸟肠球菌 1 100 90
? 咽峡炎链球菌、金黄色葡萄球菌 2 100 80
? 头葡萄球菌 1 20 40
革兰阴性细菌 ? ? ?
? 克氏柠檬酸杆菌、阴沟肠杆菌、铜绿假单胞菌、产酸克雷伯氏菌、产气肠杆菌、醋酸钙不动杆菌、醋酸钙不动杆菌－鲍曼不动杆菌复合体、弗劳地柠檬酸杆菌、大肠埃希氏菌、肺炎克雷伯氏菌、豚鼠/嗜水/简氏气单胞复合体、卡塔莫拉氏菌 14 100 100
真菌 ? ? ?
? 近平滑念珠菌 1 100 90
? 粉状毕赤酵母 1 100 80
? 产朊假丝酵母 1 100 30
? 季也蒙毕赤酵母 1 100 10
? 季也蒙毕赤酵母 1 90 90
? 异常毕赤酵母 1 90 30
? 酿酒酵母 1 90 10
? 费比恩毕赤酵母 1 90 0
? 东方伊萨酵母 1 70 30
? 解脂耶氏酵母 1 70 0
? 白色念珠菌 1 60 100
? 白色念珠菌 1 60 80
? 东方伊萨酵母 1 40 30
图1 提取法处理咽颊炎链球菌的蛋白指纹图谱
图2 抹板法处理咽颊炎链球菌的蛋白指纹图谱
图3 提取法处理白色念珠菌的蛋白指纹图谱
图4 抹板法处理白色念珠菌的蛋白指纹图谱
1
赵苏瑛，李岷. MALDI-TOF质谱分析在临床微生物实验室的应用[J]. 现代检验医学杂志, 2013,28(5):110-112.
2
Hsieh SY, Chen RK, Pan YH, et al. Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling[J]. Proteomics, 2006,6(10):3189-3198.
3
Williams TL, Andrzejewski D, Lay JO, et al. Experimental factors affecting the quality and reproducibility of MALDI TOF mass spectra obtained from whole bacteria cells[J]. J Am Soc Mass Spectrom, 2003,14(4):342-351.
4
黄迎波，周慧平，莫瑾，等. MALDI-TOF-MS鉴定水稻细菌性叶鞘褐腐病菌的方法研究[J]. 植物检疫, 2014,28(6):54-58.
5
Ghosh AK, Paul S, Sood P, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections[J]. Clin Microbiol Infect, 2015,21(4):372-378.
6
Krishnamurthy T, Ross PL. Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells[J]. Rapid Commun Mass Spectrom, 1996,10(15):1992-1996.
7
Spanu T, Posteraro B, Fiori B, et al. Direct maldi-tof mass spectrometry assay of blood culture broths for rapid identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories[J]. J Clin Microbiol, 2012,50(1):176-179.
8
Chen WH, Hsu IH, Sun YC, et al. Immunocapture couples with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid detection of type 1 dengue virus[J]. J Chromatogr A, 2013,1288:21-27.
9
Zhang Y, Liu Y, Ma Q, et al. Identification of Lactobacillus from the saliva of adult patients with caries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[J]. PLoS One, 2014,9(8):e106185.
10
Marín M, Arroyo R, Espinosa-Martos I, et al. Identification of emerging human mastitis pathogens by MALDI-TOF and assessment of their antibiotic resistance patterns[J]. Front Microbiol, 2017,8:1258.
11
Croxatto A, Prod′ Hom G, Greub G. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology[J]. FEMS Microbiol Rev, 2012,36(2):380-407.
12
任雯，陈峰，张翼飞，等. MALDI-TOF质谱技术鉴定龋病患者唾液中的变异链球菌[J]. 北京大学学报(医学版), 2014,46(1):25-29.
13
陈飞，杨峰，张景皓，等. 基质辅助激光解吸电离飞行时间质谱在幽门螺杆菌鉴定中的应用[J]. 中华检验医学杂志, 2017,40(1):31-35.
14
卫军营，蔡耘，钱小红，等. 基质辅助激光解析电离质谱靶上样品前处理方法研究进展[J]. 质谱学报, 2007,28(2):122-128.
15
杜美红，赵瑞雪，李静雯. 基质辅助激光解吸电离飞行时间质谱分析技术在微生物检测与鉴定中的应用[J]. 食品安全质量检测学报, 2017,8(2):457-461.
16
鲍春梅，宋新爱，崔恩博，等. MALDI-TOF-MS鉴定宋内志贺菌的临床应用[J]. 传染病信息, 2014,27(3):152-155.
17
陈秀金，尹红红，匡华，等. 沙门氏菌MALDI-TOF-MS蛋白质指纹图谱分析方法的研究[J]. 食品与生物技术学报, 2012,31(11):1189-1197.
18
Alcamo IE. 微生物学[M]. 林雅兰，宋怡玲，红龙等，译. 北京：科学出版社，2002.
19
Schrödl W, Heydel T, Schwartze VU, et al. Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry[J]. J Clin Microbiol, 2012,50(2):419-427.
20
赵贵明，刘洋，陈颖，等. 克罗诺杆菌MALDI-TOF-MS数据库的建立及应用[J]. 食品科学, 2014,35(8):105-110.
[1] 曹敬荣, 王岩, 谢威, 李文军, 陈典典, 段园园, 刘云屹, 闵嵘, 王培昌. 质谱技术快速鉴定临床分离丝状真菌的应用[J]. 中华实验和临床感染病杂志(电子版), 2020, 14(05): 374-379.
[2] 刘晔华, 穆红, 刘萍, 江雁, 张坚磊. MALDI-TOF MS鉴定一株小菌落变异型金黄色葡萄球菌[J]. 中华临床实验室管理电子杂志, 2018, 06(01): 32-38.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号