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Identification of Shigella sonnei by using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry)

A technology for identification of Shigella sonnei, applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve the problems of no Shigella sonnei identification spectrum, difficulty in identifying diarrhea pathogens, etc., and achieve a cumbersome identification process Effect of improving and shortening identification time

Inactive Publication Date: 2017-08-22
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Because Shigella sonnei is closely related to Escherichia coli, it is considered to be a part of the Escherichia coli strain in the Biotyper database, and there is no identification spectrum of Shigella sonnei in the database, which is undoubtedly Identification of diarrheal pathogens using MALDI-TOF-MS poses difficulties

Method used

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  • Identification of Shigella sonnei by using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry)
  • Identification of Shigella sonnei by using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry)
  • Identification of Shigella sonnei by using MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry)

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, MALDI-TOF-MS identification standard peak and method of Shigella sonnei

[0034] 1. Obtaining total protein of Shigella sonnei

[0035] 1) Strain purification

[0036] Select 24 strains of Shigella sonnei from the establishment of the library, and separate them on the M-H medium for further use.

[0037] 2) fixed inactivation

[0038] Add 300μl of pure water to a 1.5ml Eppendorf tube, pick up 5mg of colony samples with a sterile toothpick and add them to the Eppendorf tube, mix carefully, then add 900μl of absolute ethanol, mix again and centrifuge at high speed (20 000r / min) for 2min , discard the supernatant.

[0039] 3) Protein dissolution

[0040] Add 50 μl of 70% formic acid to the Eppendorf tube where the supernatant was discarded, mix carefully, then add 50 μl pure acetonitrile, mix carefully, centrifuge at high speed (20 000 r / min) for 2 min, absorb the supernatant, and obtain the total protein sample, Used for spotting.

[0041] 2. Identific...

Embodiment 2

[0062] Example 2, MALDI-TOF-MS identification of Shigella sonnei accuracy verification

[0063] 1. Total protein obtained:

[0064] 200 identified strains of Shigella sonnei and 100 strains of Escherichia coli were respectively extracted according to the method of 1 of the above-mentioned Example 1 to obtain the total protein.

[0065] 2. Identification by MALDI-TOF-MS

[0066] 1) Matrix and BTS standard preparation: same as 2 of Example 1;

[0067] 2) Target: use immediate target on the computer.

[0068] 3) Instrument parameter setting: same as 3 of embodiment 1;

[0069] 4) Data collection: Same as in Example 1, 500 points were accumulated for each strain, and the protein peak patterns of 200 strains of Shigella sonnei and 200 strains of Escherichia coli were obtained.

[0070] 5) Analysis results

[0071] If the protein peaks detected by MALDI-TOF-MS of the strain to be tested contain all four characteristic protein peaks with mass-to-charge ratios of 5612.81±8.7, 487...

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Abstract

The invention discloses a novel method for identifying Shigella sonnei by using MALDI-TOF-MS. The invention provides an identification method for distinguishing whether a to-be-detected strain is Shigella sonnei or other batacteria. The invention also discloses application of a set consisting of a MALDI-TOF-MS detection instrument, a reagent for the MALDI-TOF-MS detection instrument and a readable carrier. The readable carrier can record the following conditions: if protein peaks obtained after detection of the to-be-detected strain by using MALDI-TOF-MS contains the four characteristic protein peaks with mass-charge ratios of 5612.81 + / - 8.7, 4871.12 + / - 45.9, 4164.03 + / - 26 and 3247.05 + / - 6.9, respectively, the to-be-detected strain is Shigella sonnei or candidate Shigella sonnei. Experimental results prove that MALDI-TOF-MS has good accuracy and specificity in identification of Shigella sonnei and shortens identification time.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of MALDI-TOF-MS in identifying Shigella sonnei. Background technique [0002] Shigella sonnei belongs to group D of the genus Shigella, and is one of the main pathogenic bacteria causing gastrointestinal tract infection. It occurs frequently in the elderly, children and patients with low immune function. Shigella sonnei is prevalent in developed countries, while Shigella flexneri is prevalent in developing countries for a long time. However, in recent years, Shigella sonnei showed an obvious increasing trend and became the dominant flora. At present, most laboratories identify Shigella sonnei through biochemical metabolic reactions and serum agglutination, which generally take 16-18 hours. [0003] Matrix assisted laser desorption ionization time-of-flight mass spectrometry (matrix assisted laser desorption / ionization-time of flight mass spectrometry, MALDI-TOF-MS) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/64
CPCG01N33/6851
Inventor 曲芬鲍春梅张树永
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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