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Pre-treatment method for detecting bacterial metabolomics through gas chromatography-mass spectrometer (GC-MS)

A GC-MS, metabolomics technology, applied in microorganism-based methods, biochemical equipment and methods, treatment of microorganisms with electricity/wave energy, etc. Coverage, failed detection of metabolic compounds, etc., to maximize the number, the best vitality, and the best metabolic state

Inactive Publication Date: 2019-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current bacterial metabolome detection methods have not yet fully met the needs of this type of research. Insufficient fragmentation and extraction in the pretreatment method have seriously affected the number and coverage of bacterial metabolomics products collected. Many metabolic compounds with important markers failed to be detected

Method used

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  • Pre-treatment method for detecting bacterial metabolomics through gas chromatography-mass spectrometer (GC-MS)
  • Pre-treatment method for detecting bacterial metabolomics through gas chromatography-mass spectrometer (GC-MS)
  • Pre-treatment method for detecting bacterial metabolomics through gas chromatography-mass spectrometer (GC-MS)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Pretreatment method for Escherichia coli metabolome analysis:

[0043] (1) Escherichia coli liquid culture:

[0044] Escherichia coli was inoculated on nutrient agar medium, cultured at 37°C for 12-14 hours, stored at 4°C, picked a single colony on the plate with a sterile inoculation loop and inoculated into nutrient broth liquid medium, placed the test tube in Cultivate in a constant temperature shaker (80-120r / min) at 30-37°C for 12-16 hours to obtain an E. coli liquid sample with a McFarland turbidity of 0.4-0.6;

[0045] (2) Ice methanol-sterile water quenching solution quenches E. coli cells:

[0046] ① Put the sample of the bacterial liquid test tube and the ice quenching solution (mixed with methanol and sterile water in a volume ratio of 3:2, and pre-cooled in a -20°C refrigerator) on the ice-water mixture;

[0047] ② Add the methanol-water quenching solution to the E. coli bacterial liquid sample, store in a centrifuge tube, and the volume of the sample and ...

Embodiment 2

[0060] Salmonella metabolome analysis pretreatment method:

[0061] (1) Salmonella liquid culture

[0062] Inoculate Salmonella on nutrient agar medium at 37°C for 12-18 hours, store at 4°C, pick a single colony on the plate with a sterile inoculation loop and inoculate it into nutrient broth liquid medium, place the test tube at 30- Cultivate in a constant temperature shaker (80-120 r / min) at 37°C for 12-18 hours to obtain a Salmonella liquid sample with a McFarland turbidity of 0.4-0.6.

[0063] (2) Ice methanol-sterile water quenching solution quenches Salmonella cells:

[0064] ① Place the sample of the bacterial liquid test tube and the ice quenching solution (mixed with methanol and sterile water in a volume ratio of 2:1, and pre-cooled in a -20°C refrigerator) on the ice-water mixture;

[0065] ② Add the methanol-water quenching solution to the Salmonella liquid sample and store in a centrifuge tube. The volume of the sample and the quenching solution is 1:2;

[0066...

Embodiment 3

[0077] Pretreatment method for metabolome analysis of Staphylococcus aureus

[0078] (1) Liquid culture of Staphylococcus aureus

[0079] Staphylococcus aureus was inoculated on nutrient agar medium, cultured at 37°C for 12-16 hours, picked a single colony on the plate with a sterile inoculation loop and inoculated into nutrient broth liquid medium, and placed the test tube at 30- Cultivate in a constant temperature shaker (80-120 r / min) at 37°C for 12-16 hours to obtain a Staphylococcus aureus liquid sample with a McFarland turbidity of 0.4-0.6.

[0080] (2) ice methanol-sterile water quenching liquid quenches Staphylococcus aureus cells:

[0081] ① Put the sample of the bacterial liquid test tube and the ice quenching solution (mixed with methanol and sterile water in a volume ratio of 3:1, and pre-cooled in a -20°C refrigerator) on the ice-water mixture;

[0082] ② Add the methanol-water quenching solution to the Staphylococcus aureus liquid sample and store in a centrifu...

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Abstract

The invention discloses a pre-treatment method for detecting bacterial metabolomics through a gas chromatography-mass spectrometer (GC-MS), and belongs to the field of chemical analysis and detection.The pre-treatment method comprises the steps: culture of bacteria, quenching of bacterial liquid and extraction of intracellular metabolites; and a bacterial liquid sample is quenched through a mixture of methanol and aseptic water, the intracellular metabolites are extracted through a mixture of acetonitrile, isopropyl alcohol and aseptic water, cells are crushed and extracted repeatedly throughcombination of a tissue crusher and a low-temperature ultrasonic phase to obtain a pre-treatment sample, and detected substances can achieve full coverage and quantity maximization. The pre-treatmentmethod can be used for studying or solving key scientific problems such as bacterial toxin production mechanisms, and drug resistance generation and control, and application prospects are wide.

Description

technical field [0001] The invention specifically relates to a pretreatment method for GC-MS detection of bacterial metabolomics, belonging to the field of chemical analysis and detection. Background technique [0002] Metabolomics targets all metabolites in a specific biological sample under qualitative and quantitative conditions, and interprets all the information reflected in the metabolism of organisms from the perspective of systems biology, complementing proteomics and transcriptomics. Together with genomics, it has become an important means of studying life phenomena. [0003] At present, microbial metabolomics is in the development stage. Microbial metabolomics has attracted extensive attention in the field of microbial research because of its simple system, rich genome data, comprehensive understanding of metabolic network and physiological characteristics, and its pivotal role in biological systems. Microbial metabolomics has been successfully applied in various...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/06C12N13/00G01N30/06C12R1/19C12R1/10C12R1/445
CPCC12N1/06C12N1/20C12N13/00G01N30/06
Inventor 孙秀兰纪剑张怡芸皮付伟邱天宇
Owner JIANGNAN UNIV
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