Quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis

A technology for small molecule metabolites and microorganisms, which is applied in the field of rapid and high-sensitivity microbial identification, achieving the effects of less sample consumption, rapid microbial identification, and rapid cost.

Inactive Publication Date: 2013-08-14
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods are currently only able to distinguish strains with large differences, mainly because of the influence of the above-mentioned proteins

Method used

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  • Quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis
  • Quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis
  • Quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, identify and distinguish different species-genus microorganisms (bacteria)

[0054] Cultured three mycobacteria (Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium marinum), and two subspecies of Escherichia coli K12 (DH5α and HB101) bacteria grew to greater than OD 600 = 0.8-1.0 after collection. The collected cells were washed with 50-fold phosphate buffer saline with a pH value of 7.4, and then frozen at -20°C or directly used for further processing. The bacteria suspended in the phosphate buffer were replaced with deionized water, centrifuged at 5000 rpm to retain the precipitate, 3 times (ie 30 ul) volume fraction of 80% ethanol aqueous solution was added to the 10 mg precipitate, suspended and allowed to stand for 5 min. After centrifugation at 5000 rpm, the supernatant was taken, or an appropriate amount of sample was taken directly without centrifugation for mass spectrometry analysis.

[0055] Take the sample and matrix (naphthalene eth...

Embodiment 2

[0057] Embodiment 2, identification distinguishes different mutant strain microorganisms (bacteria) of the same species

[0058] Cultured Mycobacterium smegmatis (wild type, strain is mc 2 155) and five single-gene mutant strains (Msmeg2415KO, Msmeg1640KO, Msmeg1641KO, Msmeg1804KO and Msmeg_3312KO) (Msmeg_2415KO is Hemerythrin HHE cation binding region gene knockout (starting site 2497580, end site 2498158), Msmeg_1640pK gene knockout (Start site 1732495, end site 1733076); Msmeg_1641KO is mfpA gene knockout (start site 1733082, end site 1733657); Msmeg_1804KO is Mycobacterium tuberculosis SigF homologous gene knockout (start site 1881660 , ending point 1882412); Msmeg_3312KO is Hemerythrin HHE cation binding domain subfamily gene knockout (starting point 3391207, ending point 3391764). All gene information can be checked through the website after entering the gene name in MSMEG_XXXX format http: / / mycobrowser.epfl.ch / smegmalist.html . ) grow to OD 600 = 0.8-1.0 after colle...

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Abstract

The invention discloses a quick high-sensitivity microbiological identification method based on micromolecular metabolic substance spectral analysis. The micromolecular metabolic substance of microorganism is served as a detection object of the method, and the microorganism is identified through combining a mass spectrometry with a chemometrics method. The method specifically comprises the following steps: 1, culturing a monoclonal growing microorganism strain to be identified; 2, centrifuging an obtained monoclonal microorganism thallus sample to be identified to remove a medium, adding water or a phosphate buffer solution with the pH value of 7.2-7.6 to wash, and centrifuging to remove the residual medium; 3, adding ethanol water which has the volume fraction of 75-95% and is 2-4 times of the volume of the monoclonal microorganism thallus into the monoclonal microorganism thallus to be identified, and thus obtaining an extracting solution sample; 4, carrying out mass spectrometry test on the extracting solution sample; and 5, acquiring 5-20 spectral peaks from a sample mass spectrogram, analyzing the obtained data through utilizing a chemometrics method based on multi-variate statistical analysis, and realizing the thallus identification.

Description

technical field [0001] The invention relates to a rapid and high-sensitivity microorganism identification method based on small molecule metabolite mass spectrometry analysis. Background technique [0002] The rapid identification of microorganisms has important practical significance in the detection of clinical infection strains, food safety, public health early warning, and biological anti-terrorism. Achieving rapid identification can greatly reduce response time and provide more sufficient time for mobilizing treatment and response plans. In addition, rapid microbial identification with high differential sensitivity can more finely distinguish mutant strains below the classification level, which can provide a reference for clinical detection of drug-resistant strains and disease control and safety monitoring of pathogenic microorganism mutants. [0003] There are several ways to achieve the identification of microorganisms. The current reliable identification methods a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62G01N27/626
Inventor 聂宗秀王佳宁陈素明侯剑何清
Owner INST OF CHEM CHINESE ACAD OF SCI
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