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

Abstract

The invention discloses a metabonomics-based method for distinguishing a vibrio alginolyticus wild type virulent strain and a crp gene deletion type weak strain. According to the method, a vibrio alginolyticus virulent strain and a crp gene deletion type attenuated strain are distinguished according to the ratio of the content of arabinose to the content of myristic acid in vibrio alginolyticus measured by metabonomics, and the vibrio alginolyticus crp gene deletion type attenuated strain with the ratio of arabinose to myristic acid in a sample to be detected being 5.9 or above is the vibrio alginolyticus crp gene deletion type attenuated strain. The vibrio alginolyticus strain with the ratio of arabinose to myristic acid being less than 1.5 is a vibrio alginolyticus wild type The method is simple to operate and reliable in detection result, and can provide reference for vibrio alginolyticus crp metabolic characteristic analysis and related vibrio alginolyticus crp metabonomics research.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and is a metabolomics-based method for the synthesis of a highly virulent wild-type strain of Vibrio alginolyticus and... crp Methods for identifying gene-deleted attenuated strains. Background Technology

[0002] Vibrio alginolyticus is a Gram-negative bacterium widely distributed in marine environments. It is an opportunistic pathogen that can cause varying degrees of disease in a variety of marine organisms, such as fish, shrimp, and shellfish. Vibrio alginolyticus not only poses a threat to the aquaculture industry but can also be transmitted to humans through contaminated seafood, causing diseases such as gastroenteritis, otitis media, otitis externa, and septicemia. Vibrio alginolyticus has become a major pathogen in Chinese aquaculture, causing vibriosis that can lead to skin hemorrhage, kidney whitening, and internal organ congestion in farmed animals. The pathogenicity rate is significantly increased, especially in the higher temperatures of summer, resulting in severe economic losses.

[0003] crp Genes act as global transcriptional regulators. They regulate the transcriptional expression of various metabolic, stress, and virulence-related genes by binding to cAMP to form a complex. In pathogenic bacteria, crp It plays a central role in pathogen infection by directly or indirectly controlling adhesins, toxin secretion systems, motility-related genes, and anti-host defense mechanisms. crp When genes are deleted, bacteria exhibit a significant decrease in their ability to adapt to the host environment, limited metabolic flexibility, and reduced expression of virulence factors, resulting in a marked reduction in pathogenicity and virulence. For example, in pathogens such as *Escherichia coli*, *Salmonella*, and *Vibrio*, Δ... crp The mutant strain exhibited significantly reduced cell adhesion, invasiveness, and lethality compared to the wild type, and also showed an attenuated phenotype in animal infection models. crp Genes coordinate energy metabolism and virulence expression as global transcriptional regulators, and their absence can significantly weaken bacterial pathogenicity.

[0004] Metabolomics is a science that studies all metabolites in organisms. It reveals the physiological and pathological states of organisms by analyzing changes in metabolites under specific conditions. In the field of microbiology, especially in the study of foodborne Vibrio bacteria, metabolomics plays a crucial role. It can systematically reflect the interactions between microorganisms and their hosts, revealing the pathogenic mechanisms of Vibrio. The arabinose and myristic acid identified in this invention are important metabolites in Vibrio alginolyticus. Bacteria require carbon sources to build cell structures and maintain life activities during metabolism. Arabinose is broken down through a series of metabolic reactions and participates in the energy production and biosynthesis of Vibrio alginolyticus, providing carbon and energy for its growth and maintaining normal growth. Myristic acid, as a signaling molecule, participates in intracellular signal transduction processes, thereby regulating bacterial gene expression, physiological metabolism, and participating in the adaptive regulation of Vibrio alginolyticus to the environment. To date, no one has proposed using the content or ratio of arabinose and myristic acid in Vibrio alginolyticus for metabolomics research on Vibrio alginolyticus. crp Distinguishing between moderately virulent and weakly virulent strains. Summary of the Invention

[0005] The purpose of this invention is to provide a metabolomics-based wild-type virulent strain of Vibrio alginolyticus and... crp A method for identifying gene-deleted attenuated strains was developed, utilizing the ratio of arabinose to myristic acid content in Vibrio alginolyticus to differentiate between wild-type virulent strains and attenuated strains in metabolomics studies. crp The identification of gene-deleted attenuated strains provides a basis for sample identification in metabolomics studies.

[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows: A metabolomics-based study of a highly virulent wild-type Vibrio alginolyticus strain and crp The method for identifying gene-deleted attenuated strains involves metabolomics analysis of the peak areas of arabinose and myristic acid in Vibrio alginolyticus. The peak area ratio of arabinose to myristic acid is calculated, and strains with a peak area ratio of arabinose to myristic acid greater than 5.9 are classified as Vibrio alginolyticus. crp Gene-deleted attenuated strains with a peak area ratio of arabinose to myristic acid below 1.5 are considered wild-type virulent strains of Vibrio alginolyticus. The aforementioned wild-type virulent strain of Vibrio alginolyticus is Vibrio alginolyticus EPGS020401, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: AB 209306 and deposit date of 2009-10-24. The Vibrio alginolyticus crp The gene-deleted attenuated strain is an attenuated strain of Vibrio alginolyticus, lacking the gene encoding a global transcriptional regulatory factor found in the aforementioned virulent wild-type Vibrio alginolyticus strain.crp .

[0007] Furthermore, the specific method for detecting the peak areas of arabinose and myristic acid in Vibrio alginolyticus is as follows: first, prepare an extract of Vibrio alginolyticus sample, and then use triple quadrupole gas chromatography-mass spectrometry (GC-MS) to perform relative quantitative analysis of arabinose and myristic acid in the Vibrio alginolyticus sample.

[0008] Furthermore, the gas chromatography detection conditions for the gas chromatography-mass spectrometry (GC-MS) technique are as follows: separation is performed using an Agilent DB-5MS capillary column; the carrier gas is 99.9995% helium; the flow rate is 1.2 mL / min; the injection volume is 1 μL; the split ratio is 20:1; and the temperature program is as follows: initial temperature 70 °C, maintained for 3 min, then increased 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 280 °C, 280 °C, and 230 °C, respectively.

[0009] Furthermore, the mass spectrometry conditions for the gas chromatography-mass spectrometry (GC-MS) technique are as follows: EI ionization mode, ionization energy of 70 eV, detection voltage of 0.94 kV, full scan mass range of 33-600 m / z, start time of 6.5 min, end time of 56 min, interval of 0.25 sec, solvent cut-off time of 6 min, and full scan mode.

[0010] Further, 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 at 4 ℃ for 10 min, take 500 μL of supernatant and freeze-dry in a new centrifuge tube for 24 hours, store at -80℃ for later use, add 60 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the freeze-dried sample, vortex until no particles remain, incubate at 37 ℃ for 90 min, then add 30 μL of MSTFA, vortex and centrifuge, continue derivatization at 37 ℃ for 60 min, centrifuge the derivatized sample at 12000 rpm for 10 min, take 80 μL of supernatant and place it in a sample vial, let stand for 1 h and then use for GC-MS analysis.

[0011] Compared with the prior art, the beneficial effects of the present invention are: This invention relates to Vibrio alginolyticus cell density crp The correlation between the peak area ratio of arabinose and myristic acid in the sample and different genotype strains for Vibrio alginolyticus. crpThe method for distinguishing between highly virulent and weakly virulent strains is simple, rapid, accurate, reliable, and requires minimal sample volume. Attached Figure Description

[0012] Figure 1 The peak area ratio of arabinose and myristic acid content in the example is shown. Detailed Implementation

[0013] The present invention will be described in further detail below with reference to specific embodiments, but the implementation of the present invention includes, but is not limited to, the scope represented by the following embodiments.

[0014] This invention specifically discloses a metabolomics-based wild-type virulent strain of Vibrio alginolyticus and crp A method for identifying gene-deleted attenuated strains utilizes the ratio of arabinose to myristic acid content in Vibrio alginolyticus to distinguish between wild-type virulent strains and... crp For the identification of gene-deleted attenuated strains, samples with a ratio of arabinose to myristic acid content (i.e., peak area in the chromatogram) of 5.9 or higher were considered... crp Samples of attenuated virulent strains with a gene deletion and a ratio of arabinose to myristic acid below 1.5 are considered virulent wild-type strains of Vibrio alginolyticus.

[0015] The specific method is as follows: Vibrio alginolyticus WT, crp The deletion strains were inoculated into LBS medium and activated at 30°C and 200 rpm for 12 h, then cultured with the initial OD value. 600 =1 was inoculated into a new 250 mL LBS medium and cultured for 9 h. Samples were collected and stored at -80℃ for subsequent metabolomics analysis. During the culture process, depending on the different characteristics of the strains, the medium for all strains needed to be supplemented with an appropriate amount of ampicillin (AMP, 100 ug / mL).

[0016] First, an extract of *Vibrio alginolyticus* was prepared. Then, a metabolomics analysis method based on triple quadrupole gas chromatography-mass spectrometry (GC-MS) was used to perform relative quantitative analysis of arabinose and myristic acid in the *Vibrio alginolyticus* sample. The ratio of arabinose to myristic acid content was calculated. Samples with a ratio of arabinose to myristic acid content greater than 5.9 were considered... crp Samples of attenuated virulent strains with a gene deletion and a ratio of arabinose to myristic acid below 1.5 are considered virulent wild-type strains of Vibrio alginolyticus.

[0017] 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, and vortex for 10 min to extract metabolites. Centrifuge at 14000 rpm at 4 ℃ for 10 min, and take 500 μL of the supernatant into a new centrifuge tube for freeze-drying for 24 hours, storing at -80℃ for later use. Add 60 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the freeze-dried sample, vortex until no particles remain, incubate at 37 ℃ for 90 min, then add 30 μL of MSTFA, vortex, centrifuge, and continue derivatization at 37 ℃ for 60 min. Centrifuge the derivatized sample at 12000 rpm for 10 min. Take 80 μL of the supernatant into a sample vial, let stand for 1 h, and then use it for GC-MS analysis.

[0018] The gas chromatography-mass spectrometry (GC-MS) detection conditions were as follows: separation was performed using an Agilent DB-5MS capillary column with 99.9995% helium as the carrier gas, a flow rate of 1.2 mL / min, an injection volume of 1 μL, and a split ratio of 20:1. The temperature program was as follows: an initial temperature of 70 °C, maintained for 3 min, followed by a temperature increase 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 were 280 °C, 280 °C, and 230 °C, respectively.

[0019] The mass spectrometry conditions were as follows: EI ionization mode was used, ionization energy was 70 eV, detection voltage was 0.94 kV, full scan mass range was set to 33-600 m / z, start time was 6.5 min, end time was 56 min, interval was 0.25 sec, solvent cut-off time was 6 min, and full scan mode was used. Example

[0020] The contents of arabinose and myristic acid in Vibrio alginolyticus samples were determined using a metabolomics method based on triple quadrupole gas chromatography-mass spectrometry (GC-MS).

[0021] The samples to be tested included wild-type WT strains. crp Deletion strain Δ crp.

[0022] The wild strain WT is Vibrio alginolyticus EPGS020401, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: AB 209306. The specific deletion strain Δcrp was constructed and preserved in the laboratory.

[0023] The sample analysis steps are as follows: 1. Collection of Vibrio alginolyticus samples: Wild-type strains of Vibrio alginolyticus and crp The mutant strains were expanded and cultured. The culture medium in the bacterial sludge was washed away with physiological saline and ultrapure water. The bacterial solutions of different genotypes were centrifuged and freeze-dried into powder, and stored in a -80°C refrigerator.

[0024] 2. Determination methods for arabinose and myristic acid: Weigh 20 mg of bacterial powder and add 1.5 mL of 80% methanol / water extraction solution. Vortex for 10 min to extract metabolites. Centrifuge at 14000 rpm at 4 °C for 10 min. Take 500 μL of the supernatant and freeze-dry in a new centrifuge tube for 24 hours. Store at -80 °C for later use. Add 60 μL of methoxyamine hydrochloride-pyridine solution (20 mg / mL) to the freeze-dried sample, vortex until no particles remain, and incubate at 37 °C for 90 min. Then add 30 μL of MSTFA, vortex, centrifuge, and continue derivatization at 37 °C for 60 min. Centrifuge the derivatized sample at 12000 rpm for 10 min. Take 80 μL of the supernatant and place it in a sample vial. After standing for 1 h, use it for GC-MS analysis.

[0025] The gas chromatography-mass spectrometry (GC-MS) detection conditions were as follows: separation was performed using an Agilent DB-5MS capillary column with 99.9995% helium as the carrier gas, a flow rate of 1.2 mL / min, an injection volume of 1 μL, and a split ratio of 20:1. The temperature program was as follows: an initial temperature of 70 °C, maintained for 3 min, followed by a temperature increase 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 were 280 °C, 280 °C, and 230 °C, respectively.

[0026] The mass spectrometry conditions were as follows: EI ionization mode was used, ionization energy was 70 eV, detection voltage was 0.94 kV, full scan mass range was set to 33-600 m / z, start time was 6.5 min, end time was 56 min, interval was 0.25 sec, solvent cut-off time was 6 min, and full scan mode was used.

[0027] The contents of arabinose and myristic acid in the above-mentioned Vibrio alginolyticus were obtained under the analytical conditions. A significant change was found in the ratio of arabinose to myristic acid content (see Table 1); the ratio of arabinose to myristic acid content is shown in... Figure 1 (WT represents wild-type strain, Δ) crp express crp Mutant strain.

[0028] Table 1 Peak areas and ratios of arabinose and myristic acid in the examples This invention uses the ratio of arabinose to myristic acid content as a distinguishing global transcriptional regulatory factor in Vibrio alginolyticus. crp The criteria for classifying virulent and attenuated strains based on metabolomics are as follows: A sample with an arabinose to myristic acid ratio of 5.9 or higher is considered a vibrio alginolyticus. crp Deletion-type attenuated strains: samples with an arabinose to myristic acid ratio below 1.5 are considered wild-type virulent strains of Vibrio alginolyticus.

Claims

1. A method for discriminating between a wild-type virulent strain of Vibrio alginolyticus and a gene-deleted attenuated strain thereof based on metabolomics, characterized by: crp The peak area ratio of arabinose and myristic acid is calculated by detecting the peak area of arabinose and myristic acid in Vibrio alginolyticus through metabolomics, and the Vibrio alginolyticus with the peak area ratio of arabinose and myristic acid above 5.9 is determined crp The peak area ratio of arabinose and myristic acid is calculated by detecting the peak area of arabinose and myristic acid in Vibrio alginolyticus through metabolomics, and the Vibrio alginolyticus with the peak area ratio of arabinose and myristic acid above 5.9 is determined ​ The wild-type virulent strain of Vibrio alginolyticus is Vibrio alginolyticus EPGS020401, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: AB 209306. The said Vibrio alginolyticus crp The gene deletion type attenuated strain is an attenuated strain of Vibrio alginolyticus, which lacks the coding gene related to the global transcriptional regulator in the wild type virulent strain of Vibrio alginolyticus crp .

2. The method according to claim 1, wherein the wild-type virulent strain of Vibrio alginolyticus and the attenuated strain of Vibrio alginolyticus are different strains. crp The method for discriminating between a wild-type virulent strain and an attenuated strain of Vibrio alginolyticus based on metabolomics according to claim 1, characterized in that: The specific method for detecting the peak area of arabinose and myristic acid in Vibrio alginolyticus is as follows: first, prepare the Vibrio alginolyticus sample extract, and then use the triple quadrupole gas chromatography mass spectrometry (GC-MS) technology to perform relative quantitative analysis on arabinose and myristic acid in the Vibrio alginolyticus sample.

3. The method according to claim 2, wherein the wild-type virulent strain of Vibrio alginolyticus and the attenuated strain of Vibrio alginolyticus are distinguished based on metabolomics. crp The method for distinguishing between a wild-type virulent strain of Vibrio alginolyticus and an attenuated strain of Vibrio alginolyticus, characterized in that: The detection conditions of the gas chromatography of the gas chromatography mass spectrometry (GC-MS) technology are as follows: an Agilent DB-5MS capillary chromatographic column is used for separation, the carrier gas is 99.9995% helium, the flow rate is 1.2 mL / min, the injection amount is 1 μL, the split ratio is 20:1, and the programmed temperature conditions are as follows: the initial temperature is 70 ℃, maintained for 3 min, and then increased to 310 ℃ at a rate of 5 ℃ / min, and maintained for 5 min. The temperatures of the injection port, the transfer line and the ion source are 280 ℃, 280 ℃ and 230 ℃, respectively.

4. The method according to claim 3, wherein the wild-type virulent strain of Vibrio alginolyticus and the attenuated strain of Vibrio alginolyticus are different strains. crp The method for discriminating between a wild-type virulent strain and an attenuated strain of Vibrio alginolyticus based on metabolomics according to claim 3, characterized in that: The mass spectrometry conditions of the gas chromatography mass spectrometry (GC-MS) technology are as follows: the EI ionization mode is used, 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 mode is used.

5. The method according to claim 4, wherein the wild-type virulent strain of Vibrio alginolyticus and the attenuated strain of Vibrio alginolyticus are different strains. crp The method for discriminating between a wild-type virulent strain and an attenuated strain of Vibrio alginolyticus based on metabolomics according to claim 4, characterized in that: The preparation method of the Vibrio alginolyticus sample extract is as follows: 20 mg of bacterial powder is weighed, 1.5 mL of 80% methanol / water extraction solution is added, vortexed for 10 min to extract metabolites, centrifuged at 14,000 rpm at 4 ℃ for 10 min, 500 μL of supernatant is taken and freeze-dried in a new centrifuge tube for 24 hours, stored in a-80 ℃ refrigerator for standby, 60 μL of methoxylamine hydrochloride-pyridine solution (20 mg / mL) is added to the freeze-dried sample, vortexed until no particles are left, placed in a 37 ℃ water bath for 90 min, then 30 μL of MSTFA is added, vortexed and centrifuged, and then placed in a 37 ℃ water bath for 60 min, the derivatized sample is centrifuged at 12,000 rpm for 10 min, 80 μL of supernatant is taken and placed in an injection bottle, and after standing for 1 h, it is used for GC-MS analysis.

Images