Lipiomics-based method for discriminating vibrio alginolyticus wild-type virulent strain and ApphA gene deletion-type attenuated strain

Abstract

The invention discloses a method for distinguishing a vibrio alginolyticus wild type virulent strain and an ApA gene deletion type weak strain based on lipidomics. The ratio of the content of CL (16: 0 / 16: 1 / 16: 1 / 16: 1 / 16: 1) to the content of LPE (20: 0) in vibrio alginolyticus is used for distinguishing a vibrio alginolyticus sRNA molecule AphA virulent strain and a vibrio alginolyticus attenuated strain in a lipidomics research, the vibrio alginolyticus sRNA molecule AphA virulent strain with the ratio of CL to LPE being more than 15 in a sample to be detected is a vibrio alginolyticus wild type virulent strain, and the vibrio alginolyticus AphA gene deleted attenuated strain with the ratio of CL to LPE being less than 1 in the sample to be detected is a vibrio alginolyticus AphA gene deleted The method disclosed by the invention is simple to operate and reliable in detection result, and can provide reference for lipid characteristic analysis of the vibrio alginolyticus sRNA molecule AphA and related research on lipidomics of the vibrio alginolyticus sRNA molecule AphA.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and is a method for distinguishing wild-type strong strains of Vibrio alginolyticus and weak strains of AphA gene deletion type based on lipidomics. Background Art

[0002] Vibrio alginolyticus is a Gram-negative halophilic Vibrio that is widely distributed in marine and freshwater environments. Vibrio alginolyticus is considered to be one of the important epidemic pathogens of marine animals. It is the main pathogenic agent of highly lethal vibriosis outbreaks in a variety of fish, shellfish and shrimp, and is also an important pathogen in the aquaculture industry in southern my country. The key to its pathogenicity depends on virulence factors, and the expression and pathogenicity of virulence factors are tightly regulated by quorum sensing systems, σ factors, non-coding sRNA molecules, etc.

[0003] As one of the important branches of metabolomics, lipidomics mainly uses the theories and techniques of analytical chemistry to qualitatively and quantitatively study the changes in lipid metabolism of organisms after disturbance at the molecular level. It has the advantages of high sensitivity and high accuracy and is widely used in microbial research. Cardiolipin (CL) is an important structural and functional component on the membrane of prokaryotic cells and bacteria. It is not only essential for maintaining the integrity and stability of the cell membrane, but also plays an important role in the defense mechanism of bacteria. Lysophosphatidylethanolamine (LPE) is one of the important components of the cell membrane and may play a certain role in maintaining the integrity and stability of the bacterial cell membrane. It participates in the cell signal transduction process, affects the physiological activities and metabolic pathways of bacteria, and can act as an activator of certain enzymes, participating in the metabolism and growth process of bacteria. So far, no one has proposed to use the content or content ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus for the discrimination of strong and weak strains in the sRNA molecule AphA of Vibrio alginolyticus in lipidomics. Summary of the invention

[0004] The purpose of the present invention is to provide a lipidomics method for distinguishing wild-type strong strains of Vibrio alginolyticus and AphA gene-deficient weak strains, and to distinguish wild-type strong strains of Vibrio alginolyticus and AphA gene-deficient weak strains in lipidomics research by using the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) contents in Vibrio alginolyticus, so as to provide a basis for sample discrimination in lipidomics research.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows: A lipidomics-based method for distinguishing wild-type strong strains of Vibrio alginolyticus and weak strains of AphA gene deletion type, by detecting the contents of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus and calculating their ratios, to distinguish strong strains and weak strains of AphA in lipidomics research: the strains with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) below 1 are weak strains of AphA gene deletion type of Vibrio alginolyticus, and the strains with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) above 15 are strong wild-type strains of Vibrio alginolyticus; The wild-type strong strain of Vibrio alginolyticus is Vibrio alginolyticus EPGS020401, and its strain collection number is CCTCC No: AB 209306.

[0006] The AphA gene-deficient weak strain of Vibrio alginolyticus is an attenuated strain of Vibrio alginolyticus, which lacks the sRNA molecule AphA associated with cell density in the wild-type strong strain of Vibrio alginolyticus.

[0007] The specific method for detecting the content of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus is: Firstly, the extract of Vibrio alginolyticus samples was prepared, and then the lipidomics analysis method using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to perform relative quantitative analysis of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in the Vibrio alginolyticus samples.

[0008] The detection conditions of the liquid chromatography are as follows: the sample is separated by a chromatographic column (model WatersACQUITY™C8, particle size 1.7 μm, 2.1×100 mm, Milford, MA, USA) in the positive and negative ion modes of the chromatography, the column temperature is 55°C, and the flow rate is 0.26 mL / min. Mobile phase A: 10 mM ammonium formate in acetonitrile and water (v / v, 6:4) solution, mobile phase B: 10 mM ammonium formate in isopropanol and acetonitrile (v / v, 9:1) solution. The elution gradient is: the initial mobile phase is 32% B, maintained for 1.5 min; 1.5 min~14 min, 85% B; 14 min~14.1 min, 97% B, stable for 2.4 min, and finally returned to the initial mobile phase 32% B for 1.9 min.

[0009] The mass spectrometry conditions were as follows: using a heated electrospray ion source (ESI) in positive and negative modes, with voltages of 3.5 kV and 3 kV, respectively, a sheath gas flow rate of 45 arb; an auxiliary gas flow rate of 10 arb; an ion transfer capillary temperature of 300 °C; S-lens RF, 50%; the mass resolutions of the full scan mass spectrometry and data-dependent mass spectrometry (ddMS2) were 70,000 and 17,500, respectively. The mass scan range was 150-1,600 m / z.

[0010] The preparation method of the Vibrio alginolyticus sample extract is as follows: weigh 20 mg of Vibrio alginolyticus dry powder sample, add 300 μL of methanol and 1 mL of MTBE respectively, vortex for 1 h, add 300 μL of ultrapure water and vortex for 30 s, and centrifuge (14000 rpm, 4°C, 10 min). Take 500 μL of the upper layer liquid and freeze-dry it in a refrigerated centrifugal concentrator (Labconco, Kansas City, MO, USA) for 10 h to obtain a bacterial powder sample. Before injection, add 100 μL of a mixture of acetonitrile, isopropanol and water (v / v / v, 65:30:5) for resolubilization.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The invention uses the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) contents in the sRNA molecule AphA sample related to the cell density of Vibrio alginolyticus to discriminate between the strong strain and the weak strain of the sRNA molecule AphA of Vibrio alginolyticus. The invention has the following characteristics: simple and rapid operation, accurate and reliable results, and small sample usage. The method can provide reference for the lipid feature analysis of the sRNA molecule AphA related to the cell density of Vibrio alginolyticus and the lipidomics research of the sRNA molecule AphA of related Vibrio alginolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) contents in the example. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below in conjunction with specific examples, but the implementation methods of the present invention include but are not limited to the scope represented by the following examples.

[0014] A lipidomics method for distinguishing a wild-type strong strain of Vibrio alginolyticus from an AphA gene-deficient weak strain, wherein the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) in Vibrio alginolyticus is used to distinguish the wild-type strong strain of Vibrio alginolyticus from the AphA gene-deficient weak strain, wherein a sample to be tested having a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) (i.e., the peak area of ​​a chromatogram) of less than 1 is an AphA gene-deficient weak strain sample, and a sample to be tested having a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) of more than 15 is a sample to be a wild-type strong strain of Vibrio alginolyticus.

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

[0016] Firstly, the extract of Vibrio alginolyticus samples was prepared, and then the lipidomics analysis method based on ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used for relative quantitative analysis of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in the Vibrio alginolyticus samples. The ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) was calculated. The samples with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) below 1 were AphA gene-deficient weak strain samples, and those with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) above 15 were wild-type strong strain samples.

[0017] The preparation method of the Vibrio alginolyticus sample extract is as follows: weigh 20 mg of Vibrio alginolyticus dry powder sample, add 300 μL of methanol and 1 mL of MTBE respectively, vortex for 1 h, add 300 μL of ultrapure water and vortex for 30 s, and centrifuge (14000 rpm, 4°C, 10 min). Take 500 μL of the upper layer liquid and freeze-dry it in a refrigerated centrifugal concentrator (Labconco, Kansas City, MO, USA) for 10 h to obtain a bacterial powder sample. Before injection, add 100 μL of a mixture of acetonitrile, isopropanol and water (v / v / v, 65:30:5) for resolubilization.

[0018] The detection conditions of the liquid chromatography are as follows: the sample is separated by a chromatographic column (model WatersACQUITY™C8, particle size 1.7 μm, 2.1×100 mm, Milford, MA, USA) in the positive and negative ion modes of the chromatography, the column temperature is 55°C, and the flow rate is 0.26 mL / min. Mobile phase A: 10 mM ammonium formate in acetonitrile and water (v / v, 6:4) solution, mobile phase B: 10 mM ammonium formate in isopropanol and acetonitrile (v / v, 9:1) solution. The elution gradient is as follows: the initial mobile phase is 32% B, maintained for 1.5 min; 1.5 min to 14 min, 85% B; 14 min to 14.1 min, 97% B, stable for 2.4 min, and finally returned to the initial mobile phase 32% B for 1.9 min.

[0019] The mass spectrometry conditions were as follows: using a heated electrospray ion source (ESI) in positive and negative modes, with voltages of 3.5 kV and 3 kV, respectively, a sheath gas flow rate of 45 arb; an auxiliary gas flow rate of 10 arb; an ion transfer capillary temperature of 300°C; S-lens RF, 50%; the mass resolutions of the full scan mass spectrometry and data-dependent mass spectrometry (ddMS2) were 70,000 and 17,500, respectively. The mass scan range was 150-1,600 m / z. Example

[0020] The contents of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus samples were determined by lipidomics method based on ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS).

[0021] The samples to be tested include the wild strain WT and the deletion strain of the sRNA molecule AphA.

[0022] The wild strain of Vibrio alginolyticus WT is specifically Vibrio alginolyticus EPGS020401 Vibrio alginolyticus EPGS020401, with strain collection number CCTCC No: AB 209306, deposited in China Center for Type Culture Collection, Wuhan, China, on October 24, 2009. The strain was provided by the Ahua Bioengineering Institute of East China University of Science and Technology.

[0023] The sample analysis steps are as follows: 1. Vibrio alginolyticus sample collection: The wild-type strain and AphA mutant of Vibrio alginolyticus were expanded and cultured, the culture medium in the bacterial sludge was washed away with physiological saline and ultrapure water, and the bacterial suspensions of different genotypes were freeze-dried into powder after centrifugation and stored in a -80 ℃ refrigerator.

[0024] 2. CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) determination method: Weigh 20 mg of Vibrio alginolyticus dry powder sample, add 300 μL of methanol and 1 mL of MTBE, vortex for 1 h, add 300 μL of ultrapure water, vortex for 30 s, and centrifuge (14000 rpm, 4 ℃, 10 min). Take 500 μL of the upper layer liquid and freeze-dry it in a refrigerated centrifugal concentrator (Labconco, Kansas City, MO, USA) for 10 h to obtain the bacterial powder sample. Before injection, add 100 μL of a mixture of acetonitrile, isopropanol, and water (v / v / v, 65:30:5) for reconstitution.

[0025] The detection conditions of the liquid chromatography are as follows: the sample is separated by a chromatographic column (model WatersACQUITY™C8, particle size 1.7 μm, 2.1×100 mm, Milford, MA, USA) in the positive and negative ion modes of the chromatography, the column temperature is 55°C, and the flow rate is 0.26 mL / min. Mobile phase A: 10 mM ammonium formate in acetonitrile and water (v / v, 6:4) solution, mobile phase B: 10 mM ammonium formate in isopropanol and acetonitrile (v / v, 9:1) solution. The elution gradient is: the initial mobile phase is 32% B, maintained for 1.5 min; 1.5 min~14 min, 85% B; 14 min~14.1 min, 97% B, stable for 2.4 min, and finally returned to the initial mobile phase 32% B for 1.9 min.

[0026] The mass spectrometry conditions were as follows: using a heated electrospray ion source (ESI) in positive and negative modes, with voltages of 3.5 kV and 3 kV, respectively, a sheath gas flow rate of 45 arb; an auxiliary gas flow rate of 10 arb; an ion transfer capillary temperature of 300 °C; S-lens RF, 50%; the mass resolutions of the full scan mass spectrometry and data-dependent mass spectrometry (ddMS2) were 70,000 and 17,500, respectively. The mass scan range was 150-1,600 m / z.

[0027] Under the analytical conditions, the contents of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in the above-mentioned Vibrio alginolyticus were obtained. It was found that the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) contents changed significantly, as shown in Table 1; the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) contents was shown in Table 2. Figure 1 (WT means wild type strain, ΔAphA indicates AphA mutant strain).

[0028] Table 1 Experimental material information and ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) used in the examples The present invention uses the ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) content as a standard for distinguishing the lipidomics strong strain and weak strain of the cell density type sRNA molecule AphA of Vibrio alginolyticus. That is, the sample to be tested has a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) of less than 1, which is a weak strain of AphA-deficient Vibrio alginolyticus, and the sample to be tested has a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) to LPE (20:0) of more than 15, which is a wild-type strong strain of Vibrio alginolyticus.

Claims

1. A lipidomics-based method for distinguishing between a wild-type, highly virulent strain of Vibrio alginolyticus and an AphA gene-deficient, weakly virulent strain, characterized in that: By detecting the contents of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus, their ratios were calculated. The strains with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) below 1 were weak strains of AphA gene deletion of Vibrio alginolyticus, and the strains with a ratio of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) above 15 were strong wild-type strains of Vibrio alginolyticus. The wild-type strong strain of Vibrio alginolyticus is Vibrio alginolyticus EPGS020401, and its strain collection number is CCTCC NO: AB209306.

2. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 1, characterized in that: The AphA gene-deficient weak strain of Vibrio alginolyticus is an attenuated strain of Vibrio alginolyticus, specifically a strain lacking the sRNA molecule AphA associated with the cell density type in the wild-type strong strain of Vibrio alginolyticus.

3. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 2, characterized in that: The specific method for detecting the content of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in Vibrio alginolyticus is: Firstly, the extract of Vibrio alginolyticus samples was prepared, and then the lipidomics analysis method using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to perform relative quantitative analysis of CL (16:0 / 16:1 / 16:1 / 16:1) and LPE (20:0) in the Vibrio alginolyticus samples.

4. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 3, characterized in that: The detection conditions of the liquid chromatography are: The samples were separated using a chromatographic column in the positive and negative ion modes. The column temperature was 55 °C and the flow rate was 0.26 mL / min. Mobile phase A: a solution of 10 mM ammonium formate in acetonitrile and water in a volume ratio of 6:4, mobile phase B: a solution of 10 mM ammonium formate in isopropanol and acetonitrile in a volume ratio of 9:

1. The elution gradient was: initial mobile phase 32% B, maintained for 1.5 min; 1.5 min~14 min, 85% B; 14 min~14.1 min, 97% B, stable for 2.4 min, and finally returned to the initial mobile phase 32% B and maintained for 1.9 min.

5. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 4, characterized in that: The mass spectrometry conditions are: A heated electrospray ion source was used in positive and negative modes with voltages of 3.5 kV and 3 kV, respectively, sheath gas flow rate of 45 arb; auxiliary gas flow rate of 10 arb; ion transfer capillary temperature, 300°C; S-lens RF, 50%; the mass resolutions of full scan mass spectrometry and data-dependent mass spectrometry were 70000 and 17500, respectively; and the mass scan range was 150-1600 m / z.

6. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 5, characterized in that: The preparation method of the Vibrio alginolyticus sample extract is as follows: Weigh 20 mg of Vibrio alginolyticus dry powder sample, add 300 μL of methanol and 1 mL of MTBE respectively, vortex for 1 h, add 300 μL of ultrapure water and vortex for 30 s, and centrifuge; take 500 μL of the upper layer liquid and lyophilize it in a refrigerated centrifugal concentrator for 10 h to obtain a bacterial powder sample; add 100 μL of a mixture of acetonitrile, isopropanol and water for reconstitution before injection.

7. The method for distinguishing between a wild-type, virulent strain and an AphA gene-deficient, virulent strain of Vibrio alginolyticus based on lipidomics according to claim 6, characterized in that: In the mixed solution of acetonitrile, isopropanol and water, the volume ratio of acetonitrile, isopropanol and water is 65:30:5.

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