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Microbial analysis

A technology for microbial and mass spectrometry analysis, applied in the field of analysing microorganisms, can solve the problems of lack of, no successful analysis of lipid MALDI-MS, preventing widespread application, etc., to achieve the effect of simple method

Active Publication Date: 2016-11-16
KRATOS ANALYTICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lack of reliable sample preparation steps and instrumentation techniques have prevented the widespread use of these methods
Routine bacterial identification by phospholipid analysis using MALDI-MS is not possible for closely related bacteria, hindering their use in taxonomy
Furthermore, no successful MALDI-MS analysis of lipids has been reported so far in other types of microorganisms (such as yeast and fungi), which must still be observed by light microscopy for cell morphology, genotyping, and / or protein fingerprinting. to identify
[0018] Furthermore, the use of MALDI-MS-based phospholipid analysis to differentiate microbial strains (within a species) has not been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] Embodiment 1-comparison of extraction solvents

[0264] figure 1 The extraction efficiency of charged phospholipids (PLs) and neutral lipids (NLs) by 6 different organic solvents is shown. Extraction efficiencies were calculated based on the signal intensities of the corresponding lipid species in the MALDI-MS spectra.

[0265] It can be seen that Folch, MTBE and MeOH solvents preferentially allow the detection of PLs, while acetone favors the detection of NLs. Almost equal efficiencies were observed for both lipids when using DIPE / BuOH, with hexane having the worst results.

[0266] Based on these preliminary results, the use of Folch, MeOH and acetone in the lipid extraction step and in the MALDI-MS analysis of phospholipids from different bacterial, yeast and fungal species was further evaluated.

[0267] These three solvents were applied to Gram-positive bacteria (Staphylococcus aureus). From figure 2 It can be seen that methanol gives lipid MALDI-MS spectra w...

Embodiment 2

[0268] Example 2 - Comparison of matrix materials

[0269] image 3 Three different mass spectra of the same sample containing mixtures of different lipid standards are shown. Mass spectra were obtained using different matrix substances according to the method described above. It can be seen that in the negative ion mode, 9AA-GUA allows detection of a large number of peaks. These correspond to anionic phospholipids.

[0270] It can also be seen that both ATT and THAP can return clear spectra in positive ion mode. Peaks in the ATT-DC spectrum correspond to cationic phospholipids, and THAP-Na peaks correspond to neutral lipids. It can be seen that in positive ion mode, fewer peaks were detected.

[0271] The inventors of the present application evaluated the selectivity of three different matrix materials for ionizing the different main PL species present in biofilms. The results are shown in Figure 4 .

[0272] The data show that in positive ion mode, cationic phosphol...

Embodiment 3

[0275] Example 3 - Effect of Culture Medium and Ionization Mode

[0276] Figure 5 Mass spectra from blood agar in the presence of methanolic lipid extraction solvent are shown. Several background peaks can be seen in the lipid profile mass range (m / z 700-1500) when measured in (+) MALDI mode but not (-) MALDL mode.

[0277] This is because blood agar contains some plasma lipids (e.g., derived from blood cells and lipoproteins), which are the main cationic PL- species (mainly LPC, PC and SM), and they are therefore preferentially in the (+) ion mode down is detected.

[0278] This problem can be circumvented in (+)MALDI mode by using minimal medium (without exogenous lipids) instead of blood agar. The resulting lipid mass profile is essentially the same, but contains fewer media contaminants. However, the use of minimal media often leads to less favorable culture conditions and to longer incubation times to obtain sufficient numbers of cells for analysis. This (-) mode al...

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Abstract

The invention relates to microbial analysis. The present invention is concerned with a method of identifying microbial strains (e.g. from a cell culture), the method comprising; i) a lipid extraction step, comprising extraction of phospholipids from the microbe, suitably with an extraction composition comprising more than 50vol% MeOH; ii) a sample preparation step, comprising preparation of a MALDI sample incorporating the extracted lipids; iii) a data gathering step, comprising performing MALDI-based mass spectrometry on the MALDI sample, and iv) a microbe identification step, comprising analysis of the mass spectrometry data to characterise or identify the microbial strain. Suitably the method also includes extracting proteins from the microbes and analysing the extracted proteins using MALDI-based mass spectrometry so as to obtain not only lipid m / z data but also protein m / z data.

Description

technical field [0001] The present invention relates to methods of analyzing microorganisms. The analysis is based on lipid profiling and can be used for microbial identification. Background technique [0002] It would be desirable to identify microorganisms quickly and easily and reliably. Reliable identification allows pathogenicity and / or other properties of a microbial sample to be identified. Determining the biomolecular composition of microorganisms can aid in identification and thus diagnosis. [0003] Mass spectrometry can be used to analyze biomolecules, typically using soft ionization techniques. Matrix-assisted laser desorption / ionization (MALDI)-MS is one such technique and has been widely used in the analysis of large biomolecules including proteins. Protein fingerprinting enables MALDI-MS to be applied to microbial identification and diagnosis. [0004] However, protein fingerprinting cannot reliably distinguish different strains within the same species. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04G01N33/68G01N33/92
CPCC12Q1/04G01N33/6851G01N33/92G01N33/68H01J49/0418
Inventor G·施蒂比格O·贝勒卡塞姆
Owner KRATOS ANALYTICAL
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