Method for distinguishing wild virulent strain of vibrio alginolyticus and cya gene deletion type attenuated strain based on metabonomics

Abstract

The invention discloses a metabonomics-based method for distinguishing a vibrio alginolyticus wild type virulent strain and a cya gene deletion type low virulent strain. According to the method, the ratio of peak areas of xylitol and stearic acid in vibrio alginolyticus measured by metabonomics is used for distinguishing an adenylate cyclase coding gene cya-deleted attenuated strain and a wild virulent strain, and the vibrio alginolyticus cya gene deleted attenuated strain with the ratio of xylitol to stearic acid in a to-be-detected sample being more than 8 is the vibrio alginolyticus cya gene deleted attenuated strain; the vibrio alginolyticus strain with the ratio of xylitol to stearic acid being less than 4 is a vibrio alginolyticus wild type virulent strain. The method is simple to operate, low in sample consumption, high in detection speed and reliable in result.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and is a method for discriminating between a wild-type virulent strain and a cya gene deletion attenuated strain of Vibrio alginolyticus based on metabolomics. Background Art

[0002] Vibrio alginolyticus is one of the most common, large in number and severely harmful opportunistic pathogens during the aquaculture of aquatic products. It is widely distributed in coastal areas, rivers and aquatic ecological environments around the world. Its characteristic virulence factors include motility, biofilm, extracellular protease, adhesin, secretion system, quorum sensing system, etc. Marine aquaculture animals such as fish, shellfish, crabs, and shrimps are easily infected by Vibrio alginolyticus, causing septicemia, anemia, gill rot, and even death, bringing huge economic losses to the marine aquaculture industry.

[0003] cya is the coding gene of adenylate cyclase. When the bacterium cya is deleted, it will lose the ability to generate cAMP and to a certain extent affect various physiological activities and material metabolisms of the bacterium. For example, the cya deletion strain of Salmonella choleraesuis not only has pleiotropic defects in carbohydrate metabolism, but also has pleiotropic defects in other inducible enzyme systems. cya also has a regulatory effect on the synthesis of specific capsular polysaccharides, biofilms, flagella and pathogenicity of bacteria. For example, when Salmonella typhimurium deletes cya , it will affect the expression of fimbriae and flagella, and significantly reduce its adhesion and invasion abilities; the cya deletion strain of Salmonella choleraesuis and the Salmonella typhimurium Δ cya / crp double mutant attenuated strain also has good immunogenicity and low toxicity.

[0004] As an important part of systems biology, metabolomics can systematically reflect the physiological functions of the whole organism or tissue cells and their interactions with internal or external factors, and has been widely used in the research on the pathogenic mechanism of foodborne Vibrio. The xylitol and stearic acid determined in the present invention are relatively important intermediate metabolites in Vibrio alginolyticus. Xylitol can provide energy for the growth of the bacterial cells through the gluconeogenesis pathway, and its accumulation in the cya deletion strain may imply the obstruction of the xylitol gluconeogenesis pathway; in bacteria, fatty acids mainly serve as components of the phospholipid bilayer. These membrane phospholipids maintain the homeostasis within the membrane through synthesis, modification, recycling and degradation, and respond to environmental stress. The significant down-regulation of stearic acid in the cya deletion strain is one of the important signs of its lipid metabolism rearrangement. So far, no one has proposed to discriminate the adenylate cyclase coding gene of Vibrio alginolyticus by the peak area ratio of xylitol and stearic acid determined by metabolomicscya Strong virulent strains and attenuated virulent strains. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide a method for discriminating wild-type strong virulent strains and cya gene-deleted attenuated virulent strains of Vibrio alginolyticus based on metabolomics, and to use metabolomics to detect the peak area ratio of xylitol and stearic acid in Vibrio alginolyticus for discriminating strong virulent strains and cya attenuated virulent strains of the adenylate cyclase-encoding gene of Vibrio alginolyticus, so as to provide a basis for sample discrimination in metabolomics research.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: A method for discriminating wild-type strong virulent strains and cya gene-deleted attenuated virulent strains of Vibrio alginolyticus based on metabolomics, which detects the peak areas of xylitol and stearic acid in Vibrio alginolyticus through metabolomics, calculates the peak area of xylitol / peak area of stearic acid, and when the ratio of xylitol to stearic acid is above 8, it is a cya gene-deleted attenuated virulent strain of Vibrio alginolyticus, and when the ratio of xylitol to stearic acid is below 4, it is a wild-type strong virulent strain of Vibrio alginolyticus; The wild-type strong virulent strain of Vibrio alginolyticus is the EPGS wild type, and the strain preservation number is CCTCC No. AB209306; The cya gene-deleted attenuated virulent strain of Vibrio alginolyticus is an attenuated strain of Vibrio alginolyticus, and the coding gene related to adenylate cyclase synthesis in the wild-type strong virulent strain of Vibrio alginolyticus is deleted cya .

[0007] The specific method for detecting the peak areas of xylitol and stearic acid in Vibrio alginolyticus is as follows: Prepare an extraction solution of Vibrio alginolyticus metabolic samples, and use triple quadrupole gas chromatography-mass spectrometry (GC-MS) hyphenated technology to perform relative quantitative analysis of xylitol and stearic acid in Vibrio alginolyticus samples.

[0008] The detection conditions of the gas chromatography in the gas chromatography-mass spectrometry (GC-MS) hyphenated technology are as follows: Separation is carried out using an Agilent DB-5MS capillary chromatographic column, the carrier gas is 99.9995% helium, the flow rate is 1.2 mL / min, the injection volume is 1 μL, and the split ratio is 10:1. The programmed temperature rise conditions are as follows: the initial temperature is maintained at 70 °C for 3 min and then heated to 310 °C at a rate of 5 °C / min and held for 5 min. The temperatures of the injection port, transfer line, and ion source are 300 °C, 280 °C, and 240 °C, respectively.

[0009] The mass spectrometry conditions of the gas chromatography-mass spectrometry (GC-MS) hyphenated technology are as follows: The EI ionization mode was adopted, with an ionization energy of 70 eV, a detection voltage of 0.94 kV, a full-scan mass range set at 33 - 600 m / z, a start time of 6.5 min, an end time of 56 min, an interval of 0.25 sec, a solvent cutting time of 6 min, and the full-scan mode was used.

[0010] The preparation method of the sample extract of Vibrio alginolyticus is as follows: Weigh 20 mg of bacterial powder, add 1.5 mL of methanol / water extraction solution with a volume fraction of 80% and vortex for 10 min to extract metabolites. Centrifuge at 14000 rpm for 10 min at 4 °C, take 500 μL of the supernatant and freeze-dry it. Add 60 μL of methoxyamine hydrochloride-pyridine solution to the freeze-dried sample, vortex until there is no particle residue, incubate in a water bath at 37 °C for 90 min, then add 30 μL of MSTFA, vortex and centrifuge, continue to derivatize in a water bath at 37 °C for 60 min, and centrifuge the derivatized sample at 14000 rpm for 10 min. Take 80 μL of the supernatant and place it in a sample vial, and let it stand for 1 h for GC-MS analysis.

[0011] The said silylation reaction is a GC-MS derivatization method, which is used to improve the volatility of polar metabolites and expand the coverage of detected metabolites.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention utilizes the correlation between the peak area ratio of xylitol and stearic acid in the Vibrio alginolyticus sample and different genotype strains to discriminate between the virulent strains and avirulent strains of the Vibrio alginolyticus adenylate cyclase encoding gene cya The discrimination method of the present invention has the following characteristics: simple and rapid operation, accurate and reliable results, and less sample consumption. Description of the Drawings

[0013] Figure 1 It is the peak area ratio of xylitol and stearic acid detected in the examples. Detailed Embodiments

[0014] The present invention will be further described in detail below in combination with specific embodiments, but the embodiments of the present invention include but are not limited to the scope represented by the following embodiments.

[0015] The present invention utilizes the correlation between the peak area ratio of xylitol and stearic acid in the Vibrio alginolyticus sample and different genotype strains to discriminate between the virulent strains and avirulent strains of the Vibrio alginolyticus adenylate cyclase encoding gene cya The specific technical solution is as follows.

[0016] Prepare the extraction solution of Vibrio alginolyticus metabolic samples, and relatively quantify xylitol and stearic acid in Vibrio alginolyticus samples by using the triple quadrupole gas chromatography-mass spectrometry (GC-MS) technique.

[0017] The preparation method of the extraction solution of Vibrio alginolyticus samples is as follows: Inoculate Vibrio alginolyticus WT, cya the deletion strain into LBS medium respectively, activate and culture at 30 °C and 200 rpm for 12 h, and then inoculate with an initial OD 600 = 1 into a new 250 mL LBS medium for enlarged culture for 9 h, collect the samples and store them at -80 °C for metabolomics analysis; Weigh 20 mg of bacterial powder, add 1.5 mL of methanol / water extraction solution with a volume fraction of 80% and vortex for 10 min to extract metabolites. Centrifuge at 14000 rpm for 10 min at 4 °C, and take 500 μL of the supernatant for freeze-drying. Add 60 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the freeze-dried sample, vortex until there is no particle residue, incubate in a water bath at 37 °C for 90 min, then add 30 μL of MSTFA (N-methyl-trimethylsilyl-trifluoroacetamide), vortex and centrifuge, continue to derivatize in a water bath at 37 °C for 60 min, and centrifuge the derivatized sample at 14000 rpm for 10 min. Take 80 μL of the supernatant and place it in an injection vial, and let it stand for 1 h for GC-MS analysis.

[0018] The detection conditions of gas chromatography in the gas chromatography-mass spectrometry (GC-MS) technique are as follows: Use an Agilent DB-5MS capillary column for separation, the carrier gas is 99.9995% helium, the flow rate is 1.2 mL / min, the injection volume is 1 μL, and the split ratio is 10:1. The programmed temperature rise conditions are as follows: the initial temperature is 70 °C and is maintained for 3 min, then it is heated at a rate of 5 °C / min to 310 °C and held for 5 min. The temperatures of the injection port, transfer line and ion source are 300 °C, 280 °C and 240 °C respectively.

[0019] The mass spectrometry conditions of the gas chromatography-mass spectrometry (GC-MS) technique are as follows: Adopt the EI ionization mode, the ionization energy is 70 eV, the detection voltage is 0.94 kV, the full scan mass range is set to 33 - 600 m / z, the start time is 6.5 min, the end time is 56 min, the interval is 0.25 sec, the solvent cutting time is 6 min, and the full scan (SCAN) mode is adopted. Example

[0020] A Vibrio alginolyticus wild-type virulent strain based on metabolomics andcya Discrimination method for gene deletion attenuated strains, by the peak area ratio of xylitol and stearic acid in Vibrio alginolyticus, to discriminate between cya related virulent strains and attenuated strains: when the ratio of xylitol and stearic acid in the test sample is above 8, it is Vibrio alginolyticus cya gene deletion attenuated strain, and when the ratio of xylitol and stearic acid is below 4, it is Vibrio alginolyticus wild-type virulent strain.

[0021] The specific method is as follows: Adopt a metabolomics method based on triple quadrupole gas chromatography-mass spectrometry (GC-MS) to determine the contents of xylitol and stearic acid in Vibrio alginolyticus samples.

[0022] The test samples include wild strain WT, cya deletion strain Δ cya .

[0023] The specific Vibrio alginolyticus wild strain WT is Vibrio alginolyticus ( Vibrio alginolyticus ) EPGS020401, with the strain preservation number CCTCC No. AB209306, preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on October 24, 2009.

[0024] The deletion strain Δ cya is specifically constructed and preserved in the laboratory.

[0025] The sample analysis steps are as follows: 1. Collection of Vibrio alginolyticus samples: Vibrio alginolyticus WT, cya deletion strains are respectively inoculated into LBS medium, activated and cultured at 30 °C and 200 rpm for 12 h, and then inoculated into a new 250 mL LBS medium at an initial OD 600 =1 for enlarged culture for 9 h, and the samples are collected and stored at -80 °C for metabolomics analysis. During the culture process, according to the different characteristics of the strains, the culture media of all strains need to be supplemented with an appropriate amount of ampicillin (AMP, 100 μg / mL).

[0026] Weigh 20 mg of bacterial powder, add 1.5 mL of methanol / water extraction solution with a volume fraction of 80%, and vortex for 10 min to extract metabolites. Centrifuge at 14000 rpm for 10 min at 4 °C, and take 500 μL of the supernatant for freeze-drying. Add 60 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the freeze-dried sample, vortex until there is no particle residue, incubate in a water bath at 37 °C for 90 min, then add 30 μL of MSTFA (N-methyl-trimethylsilyl-trifluoroacetamide), vortex and centrifuge, continue derivatization in a water bath at 37 °C for 60 min, and centrifuge the derivatized sample at 14000 rpm for 10 min. Take 80 μL of the supernatant and place it in an injection vial, and let it stand for 1 h for GC-MS analysis.

[0027] 2. Determination method of xylitol and stearic acid: The detection conditions of gas chromatography are as follows: Separation is carried out using an Agilent DB-5MS capillary column (30 m × 250 μm × 0.25 μm), the carrier gas is 99.9995% helium, the flow rate is 1.2 mL / min, the injection volume is 1 μL, and the split ratio is 10:1. The programmed temperature conditions are as follows: The initial temperature is maintained at 70 °C for 3 min and then heated to 310 °C at a rate of 5 °C / min and held for 5 min. The temperatures of the injection port, transfer line, and ion source are 300 °C, 280 °C, and 240 °C, respectively.

[0028] The mass spectrometry conditions are as follows: The EI ionization mode is adopted, the ionization energy is 70 eV, the detection voltage is 0.94 kV, the full scan mass range is set to 33 - 600 m / z, the start time is 6.5 min, the end time is 56 min, the interval is 0.25 sec, the solvent cutting time is 6 min, and the full scan (SCAN) mode is adopted.

[0029] Under these analysis conditions, the peak areas of xylitol and stearic acid in the above-mentioned Vibrio alginolyticus were obtained, and it was found that the ratio of the peak areas of xylitol and stearic acid changed significantly, as shown in Table 1; the ratio of the peak areas of xylitol and stearic acid is shown in Figure 1 (WT represents the wild strain, Δ cya represents cya the mutant strain).

[0030] Table 1 Ratio of peak areas of xylitol and stearic acid The present invention uses the ratio of the peak areas of xylitol and stearic acid as the discrimination criterion for differentiating the adenylate cyclase-encoding gene cya deletion strains, weak strains, and wild-type strong strains of Vibrio alginolyticus. That is, when the ratio of xylitol and stearic acid in the test sample is above 8, it is Vibrio alginolyticus cyaThe gene deletion attenuated strain, and the Vibrio alginolyticus wild-type virulent strain has a ratio of xylitol to stearic acid below 4.

Claims

1. A metabolomics-based analysis of the characteristics of a virulent wild-type strain of Vibrio alginolyticus cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The peak areas of xylitol and stearic acid in Vibrio alginolyticus were detected by metabolomics, and the peak area of ​​xylitol / stearic acid was calculated. The ratio of xylitol to stearic acid was above 8, which was Vibrio alginolyticus. cya The gene-deficient weak strains and the ones with a xylitol-stearic acid ratio below 4 are the wild-type strong strains of Vibrio alginolyticus; The wild-type highly virulent strain of Vibrio alginolyticus is the EPGS wild-type, with a strain collection number of CCTCC No. AB209306; Vibrio alginolyticus cya The gene-deficient attenuated strain is an attenuated strain of Vibrio alginolyticus, which lacks the coding gene related to the synthesis of adenylate cyclase in the wild-type virulent strain of Vibrio alginolyticus. cya .

2. A metabolomics-based method for detecting the virulent wild-type strain of Vibrio alginolyticus according to claim 1 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The specific method for detecting the peak areas of xylitol and stearic acid in Vibrio alginolyticus is: The metabolic sample extract of Vibrio alginolyticus was prepared, and the xylitol and stearic acid in the Vibrio alginolyticus sample were relatively quantitatively analyzed using triple quadrupole gas chromatography-mass spectrometry (GC-MS).

3. A metabolomics-based method for detecting the virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The detection conditions of the gas chromatography of the gas chromatography-mass spectrometry GC-MS coupling technology are: An Agilent DB-5MS capillary column was used for separation. The carrier gas was 99.9995% helium, the flow rate was 1.2 mL / min, the injection volume was 1 μL, and the split ratio was 10:

1. The programmed temperature conditions were as follows: the initial temperature was maintained at 70°C for 3 min, then increased to 310°C at a rate of 5°C / min and maintained for 5 min. The temperatures of the injection port, transfer line, and ion source were 300°C, 280°C, and 240°C, respectively.

4. A metabolomics-based method for detecting the presence of a virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The mass spectrometry conditions of the gas chromatography-mass spectrometry GC-MS coupling technology are: The EI ionization mode was used, the ionization energy was 70 eV, the detection voltage was 0.94 kV, the full scan mass range was set to 33-600 m / z, the start time was 6.5 min, the end time was 56 min, the interval was 0.25 sec, the solvent cutting time was 6 min, and the full scan mode was used.

5. A metabolomics-based method for detecting the virulent wild-type strain of Vibrio alginolyticus according to claim 2 cya The method for distinguishing a gene-deficient attenuated strain is characterized by: The preparation method of the Vibrio alginolyticus sample extract is as follows: Weigh 20 mg of bacterial powder, add 1.5 mL of 80% methanol / water extraction solution, vortex for 10 min to extract metabolites; centrifuge at 14000 rpm for 10 min at 4 °C, take 500 μL of supernatant and freeze-dry; add 60 μL of methoxyamine hydrochloride-pyridine solution to the freeze-dried sample, vortex until no particles remain, incubate in a 37 °C water bath for 90 min, then add 30 μL of MSTFA, vortex and centrifuge, continue to derivatize in a 37 °C water bath for 60 min, and the derivatized sample is centrifuged at 14000 rpm for 10 min; take 80 μL of supernatant and place it in an injection bottle, let it stand for 1 h before using for GC-MS analysis.

6. The method for preparing the Vibrio alginolyticus sample extract according to claim 5, characterized in that: The silylation reaction is a GC-MS derivatization method used to increase the volatility of polar metabolites and expand the coverage of detected metabolites.

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