Sample pretreatment and detection methods in researches of Botrytis cinerea metabolome based on GC-MS

A Botrytis cinerea, sample pretreatment technology, applied in the field of microbiology and instrumental analysis, can solve the problems of high price, influence analysis system error, lack, etc., and achieve fast and simple operation, optimized derivation conditions, and good peak shape quality Effect

Active Publication Date: 2015-03-04
CHINA AGRI UNIV
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Problems solved by technology

[0005] Although there have been many reports on metabolome analysis methods related to medical samples, plant samples and some microbial samples at home and abroad, the metabolome of each type of biological sample requires the establishment of specific pretreatment and detection methods. Otherwise, the detection results obtained are unreliable
Regarding the metabolomics research of Botrytis cinerea, due to the lack of corresponding GC-MS analysis method, it is unclear which substance to use as the internal standard, which seriously affect

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  • Sample pretreatment and detection methods in researches of Botrytis cinerea metabolome based on GC-MS
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  • Sample pretreatment and detection methods in researches of Botrytis cinerea metabolome based on GC-MS

Examples

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Embodiment 1

[0039] Example 1 Detecting and analyzing the metabolome of Botrytis cinerea S28 based on GC-MS

[0040] The process used is as figure 1 As shown, the specific implementation is as follows:

[0041] 1. Sample pretreatment for studying the metabolome of Botrytis cinerea S28 based on GC-MS

[0042] 1) Cultivation of Botrytis cinerea

[0043] Take the PDA medium plate of Botrytis cinerea S28 that has grown for 3 days, and use a 0.5cm puncher to punch out the bacterial cake along the outer edge of the colony in the ultra-clean workbench, pick the bacterial cake with a pick needle and connect it to the cellophane On the solid PDA culture medium plate, cultured at 19.5-20.5°C for 3 days to obtain mycelium culture;

[0044] 2) Botrytis cinerea mycelia collection: Scrape the mycelium culture grown on cellophane with a sterilized scraper, remove the inoculated bacteria cake, and collect it in a 2 mL centrifuge tube.

[0045] 3) Liquid nitrogen inactivation treatment: quickly freeze th...

Embodiment 2

[0069] Example 2 Detecting and analyzing the metabolome of Botrytis cinerea S28 mycelium based on GC-MS

[0070] The process flow and method used are shown in Example 1, wherein the GC-MS instrument model is: gas phase is 6890A, mass spectrometer is 5973C. The optimal experiment was carried out mainly on the dosage of extraction solvent, the temperature and time of derivatization 2 reaction.

[0071] 1. Effect of extraction solvent dosage on metabolome analysis of Botrytis cinerea S28 mycelium

[0072] Using extraction solvent I, the mycelial culture of 100.0 ± 0.5 mg was extracted and detected according to the doses in Table 1-3, and the typical total ion chromatogram obtained was as follows image 3 shown. The peak intensity analysis of metabolites shows that under the levels 1-3 of the extraction solvent I tested, the extraction efficiency increases with the increase of the extraction solvent dose, while there is no significant difference between levels 4 and 5. Therefor...

Embodiment 3

[0083] Example 3 Sclerotia metabolome analysis of Botrytis cinerea S28

[0084] The optimal method described in Examples 1 and 2 was used to analyze the Botrytis cinerea S28 sclerotia metabolome as follows:

[0085] 1) Cultivation of Botrytis cinerea sclerotia: Take the PDA medium plate of Botrytis cinerea S28 that has grown for 3 days, and culture it at 25°C until the mycelia cover the plate, and then culture it at 20°C for 15 days.

[0086] 2) Collection of Botrytis cinerea sclerotia: Pick the sclerotia on the PDA plate, weigh 100 mg into a 2 mL centrifuge tube.

[0087] 3) Liquid nitrogen inactivation treatment: quickly freeze the centrifuge tube containing the sclerotium in liquid nitrogen for 3-5 minutes.

[0088] 4) Preservation of sclerotia samples: the sclerotia samples cultivated, collected and inactivated according to the above method were sealed with a parafilm and stored in a -80°C refrigerator.

[0089] 5) Grinding and crushing: take the sclerotium sample inacti...

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Abstract

The invention discloses a Botrytis cinerea metabolome sample pretreatment method, and a metabolome detection method based on gas chromatography-mass spectrometry (GC-MS). The Botrytis cinerea metabolome sample pretreatment method comprises the following steps: culturing Botrytis cinerea on a PDA plate paved with glass paper for 2-4d (preferably 3d), collecting mycelia, inactivating the mycelia by liquid nitrogen, and preserving the inactivated mycelia in an ultralow temperature freezer; carrying out freeze grinding crushing on the obtained mycelium sample, extracting metabolome, and carrying out enzyme inactivation by adopting a frozen methanol-water mixture; and centrifuging, drying, and carrying out an oximation (or hydrazone formation) and silylation two-step derivatization reaction to obtain the pretreated sample for GC-MS detection. The invention also provides the sample detection method through GC-MS. The detection method is carried out under the following detection conditions: a HP-5MS capillary column (30m*0.25mm*0.25[u]m), a sample size of 1[mu]L, programmed heating, an ion source temperature of 230DEG C and a full scanning range of m/z 20-650. Metabolome detection results with good reappearance can be obtained through the extraction and detection methods.

Description

technical field [0001] The invention provides a sample pretreatment method and a detection method for studying the metabolome of Botrytis cinerea by adopting GC-MS analysis technology, and relates to the technical fields of microbiology and instrument analysis. Background technique [0002] With the completion of the sequencing of the human genome, the study of gene function has become a hot spot, various "omics" have emerged one after another, and metabolomics has emerged after genomics, transcriptomics and proteomics. Different from other omics, metabolomics is a science that studies the changes of all metabolites produced by external stimuli in biological systems (cells, tissues or organisms). Therefore, metabolomics is a downstream product of the central dogma information flow, It often better reflects the dynamic chemical change process in the organism. [0003] Strawberry gray mold is one of the important diseases in strawberry production at present, and it often caus...

Claims

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Application Information

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IPC IPC(8): G01N30/06
Inventor 刘鹏飞胡志宏常旭念李蕾吴嘉纯刘西莉陈晨侯燕华黄中乔
Owner CHINA AGRI UNIV
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