Extraction and enrichment method of coral symbiont virus

Abstract

The invention discloses a coral symbiont virus extraction and enrichment method which comprises the following steps: acquiring coral tissues, and grinding the coral tissues in sterile seawater; carrying out vortex treatment on the ground coral tissue suspension; performing low-temperature centrifugation on the coral tissue suspension subjected to vortex treatment; performing graded filtration and purification on the centrifuged supernate; the step of step-by-step filtration and purification comprises filter membrane preliminary screening filtration and tangential flow ultrafiltration; and carrying out ultracentrifugal concentration on the coral tissue filtrate subjected to graded filtration and purification. The extraction and enrichment method provided by the invention can efficiently capture various known and unknown viruses in the coral symbiotic system, is suitable for low-abundance viruses and covers known and unknown virus groups, and is simple and convenient to operate, free of complex professional equipment and controllable in cost.

Description

Technical Field

[0001] This invention belongs to the field of marine biotechnology, and particularly relates to a method for extracting and enriching viruses from coral symbionts. Background Technology

[0002] Coral reef ecosystems are among the most biodiverse ecosystems in the ocean, playing an irreplaceable role in maintaining marine ecological balance. In recent years, global coral reefs have faced serious threats such as bleaching and diseases. Among these threats, viruses, as an important microbial group in coral symbiosis systems, have become a research hotspot due to their infection mechanisms and their correlation with coral health.

[0003] However, the enrichment of viruses in coral symbionts faces three major challenges: First, the viral content is extremely low. The biomass of host cells (such as nematocysts and interstitial cells), symbiotic algae (such as zooxanthellae), and symbiotic microorganisms (bacteria and archaea) in coral tissues accounts for more than 99%, and virus particles are encapsulated by a large number of impurities. Second, existing enrichment methods are highly targeted but lack broad-spectrum application. For example, the immunoaffinity method relies on specific antibodies and can only enrich known viruses, but cannot cover unknown viruses. Although ultracentrifugation can separate viruses, it requires specialized equipment (such as an ultracentrifuge), is time-consuming (a single centrifugation takes 4 hours), and is prone to causing virus particles to break down due to excessive centrifugal force. Third, impurities cause serious interference. Polysaccharides, proteins, and free nucleic acids (host DNA / RNA) released by coral tissues can adsorb virus particles or produce false positive signals in subsequent detection (such as virus sequencing and electron microscopy).

[0004] Therefore, developing a simple, broad-spectrum, efficient, and low-cost method for the extraction and enrichment of coral symbiotic viruses is key to overcoming the technical bottleneck in coral virus research. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for extracting and enriching viruses in coral symbionts. This method is applicable to the broad-spectrum enrichment of various known and unknown viruses in coral symbiotic systems, providing key technical support for coral virus diversity analysis, virus-host interaction research, and coral disease diagnosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for extracting and enriching viruses from coral symbionts, comprising the following steps: (1) Obtain coral tissue and grind it in sterile seawater; (2) The ground coral tissue suspension was subjected to vortex treatment; (3) The coral tissue suspension after vortex treatment was centrifuged at low temperature; (4) The supernatant after centrifugation is purified by fractional filtration; the fractional filtration purification includes primary filtration through a filter membrane and tangential flow ultrafiltration; (5) The coral tissue filtrate after graded filtration and purification was concentrated by ultracentrifugation.

[0007] In a preferred embodiment, in step (1), the salinity of the sterile seawater is consistent with the native environment of the coral; Preferably, the sterile seawater undergoes a pre-cooling treatment at 3-4°C; In some specific embodiments, the sterile seawater is seawater that has been filtered and sterilized through a filter membrane of less than 0.22 μm; Preferably, the grinding is performed until a homogeneous slurry is formed; Preferably, during the grinding process, the container holding the coral tissue is placed on ice to prevent the virus from being inactivated due to high temperature; Preferably, the container holding the coral tissue is a sterile container; In some specific embodiments, the coral tissue is taken from the branch tissue of healthy corals without obvious disease, and has a diameter of 2-3 cm.

[0008] In a preferred embodiment, in step (2), sterilized glass beads are added in the vortex; Preferably, the diameter of the sterilizing glass beads is 1-3 mm; Preferably, the vortex is repeated 3 to 5 times at a frequency of 2500 to 3500 rpm for a duration of 30 to 60 seconds each time.

[0009] In a preferred embodiment, in step (3), the temperature of the low-temperature centrifugation is 4~6℃; the rotation speed of the low-temperature centrifugation is 3500~5000 g; and the time of the low-temperature centrifugation is 15~20 min.

[0010] In a preferred embodiment, in step (4), the initial filtration of the filter membrane is carried out by sequentially using filter membranes of 8~10 μm, 3~5 μm, and 0.45~1 μm. Preferably, the tangential flow ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100-150 kDa; Preferably, the transmembrane pressure of the tangential flow ultrafiltration is 0.1~0.2 MPa; Preferably, the flow rate of the tangential flow ultrafiltration is 10~15 mL / min.

[0011] In a preferred embodiment, in step (5), the molecular weight cutoff of the ultracentrifugation concentration is 100~150kDa; Preferably, the rotation speed of the ultracentrifugation concentration is 3500~5000 g; the ultracentrifugation concentration time is 15~20 min; Preferably, the temperature of the ultracentrifugation concentration is 4~6℃.

[0012] The above technical solution has the following advantages or beneficial effects: The extraction and enrichment method provided by this invention can efficiently capture a variety of known and unknown viruses in coral symbiosis systems. It is applicable to low-abundance viruses, covers known and unknown virus groups, and the enrichment method is simple to operate, requires no complex professional equipment, and has controllable costs.

[0013] The extraction and enrichment method provided by this invention introduces a multi-mechanism synergistic capture module to achieve efficient adsorption and separation of low-abundance viruses in coral samples. At the same time, it optimizes the impurity removal process to accurately remove interfering substances such as polysaccharides, proteins and free nucleic acids. This effectively overcomes the technical bottlenecks of low virus content, poor broad-spectrum application of existing methods and severe impurity interference, providing a stable and reliable enrichment scheme for coral virus research. It has significant scientific research application value and promotion prospects. Attached Figure Description

[0014] Figure 1 This document describes the flowchart for the extraction and enrichment of viruses from coral symbionts in this invention, as well as the reagents and instruments used.

[0015] Figure 2 This is a comparison chart of the effects of the virus enrichment group and the non-enrichment group in coral symbionts in an embodiment of the present invention.

[0016] Figure 3 Electron micrographs of the virus captured in an embodiment of the present invention. Detailed Implementation The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0017] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0018] Example This embodiment provides a method for extracting and enriching viruses from coral symbionts. The instruments, apparatus, and specific procedures used are detailed in [link to documentation]. Figure 1 This includes the following steps: I. Coral Sample Pretreatment Healthy coral tissue without obvious disease was selected as a sample. Coral branch tissue with a diameter of about 2 cm was cut with a sterile scalpel, quickly transferred to a sterile centrifuge tube, and placed in an ice box for low-temperature preservation to maintain the biological activity of the virus in the sample.

[0019] II. Sterile seawater grinding and secondary vortexing of glass beads This step effectively breaks down coral tissue and releases virus particles.

[0020] Sterile seawater grinding: Add 10 mL of pre-cooled sterile seawater at 4°C (filtered through a 0.22 μm filter membrane for sterilization, with salinity consistent with the coral's native environment) to a centrifuge tube containing coral tissue; grind the coral tissue thoroughly into a homogenous paste using a sterile mortar; keep the mortar on ice during grinding to prevent the virus from being inactivated by high temperature.

[0021] Secondary vortexing with glass beads: The ground coral tissue suspension was transferred to a sterile centrifuge tube containing sterile glass beads (1 mm in diameter) and subjected to secondary vortexing using a vortex mixer. The vortexing frequency was set to 2500 rpm, each vortex lasted 30 seconds, and was repeated 3 times to ensure that the coral cells were fully lysed and to release virus particles to the maximum extent.

[0022] III. Centrifugation to remove large particulate impurities Transfer the vortexed suspension to a new sterile centrifuge tube and centrifuge at low temperature: set the temperature to 4℃ and the rotation speed to 3500× g Centrifugation time is 15 min. After centrifugation, carefully aspirate the supernatant and transfer it to a new container, removing any large particles such as unbroken tissue fragments and cell debris.

[0023] IV. Staged Filtration and Purification This step achieves the initial enrichment and purification of the virus by "gradually intercepting impurities of different particle sizes and separating small molecule interfering substances," including two processes: primary screening filtration and tangential flow ultrafiltration.

[0024] Primary filtration: The supernatant was filtered sequentially using 8 μm, 5 μm, and 1 μm sterile filter membranes (using a sterile syringe or a vacuum filtration device) to gradually remove impurities such as plankton and large cell fragments, resulting in a preliminarily purified virus-containing filtrate.

[0025] Tangential flow ultrafiltration: The primary filtrate is introduced into a tangential flow ultrafiltration system, using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The transmembrane pressure is set to 0.1 MPa and the flow rate to 10 mL / min for tangential flow filtration. During this process, viruses and biomacromolecules >100 kDa are retained, while interfering substances such as polysaccharides, free nucleic acids, and small protein molecules are discharged with the permeate, achieving preliminary enrichment of viruses and removal of impurities.

[0026] V. High-speed centrifugation to concentrate viruses The retentate from tangential flow ultrafiltration was transferred to an ultracentrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4°C and 5000 × 10⁻⁶ rpm. g Centrifuge for 15 minutes. After centrifugation, discard the supernatant; the precipitate at the bottom of the centrifuge tube is the high-concentration virus enrichment.

[0027] Effect test 1. Enrichment effect verification To visually verify the enrichment effect, this embodiment performs fluorescent labeling and confocal microscopy observation on the virus enrichment: Resuspend the virus precipitate in an appropriate amount of sterile seawater, add virus-specific fluorescent probes (such as SYBR Green I dye against viral nucleic acid, or fluorescent antibodies against specific viral capsid proteins), and incubate at 37°C for 30 min. The labeled sample was dropped onto a confocal microscope slide, and the distribution and enrichment of the virus were observed using a laser confocal microscope (excitation wavelength selected according to the probe, such as 488nm excitation light for SYBR Green I). Figure 2 The visualization results shown in "fluorescence confocal imaging" Figure 2 In the middle, a small amount of virus was roughly extracted, specifically 10. 5 -10 6 CFU / mL; for non-enriched samples, direct observation showed a concentration less than 10 CFU / mL.

[0028] 2. Virus images captured by transmission electron microscopy (TEM) To further observe the morphological characteristics of the enriched virus, clarify the integrity of the virus particles, and verify the enrichment effect at the ultrastructural level, this embodiment uses transmission electron microscopy to photograph the samples, and the steps are as follows: Sample fixation: Take 200 μL of virus enrichment suspension, add 2.5% glutaraldehyde solution (final concentration 1%), and fix at 4℃ for 2h to maintain the morphology and structure of virus particles; then wash 3 times with 0.1 mol / L phosphate buffer (PBS, pH 7.4) for 15 min each time to remove residual fixative. Gradient dehydration: The fixed sample was dehydrated sequentially with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, for 10 minutes at each concentration (100% ethanol was repeated twice) to gradually remove water from the sample in preparation for subsequent embedding. Negative staining: Take 10 μL of dehydrated sample and drop it onto a copper grid (200 mesh, pre-treated with hydrophilicity). Let it stand at room temperature for 5 min to allow the virus particles to be fully adsorbed. Gently blot away the excess liquid with filter paper, and immediately add 2% phosphotungstic acid solution (pH 6.8). Negative stain for 1-2 min, blot away the stain again with filter paper, and let the copper grid air dry at room temperature (avoid direct sunlight to prevent damage to the virus structure). TEM observation and imaging: The dried copper mesh was placed in the sample chamber of the transmission electron microscope (Hitachi H-7650). The accelerating voltage was set to 80 kV. Under low magnification (1000×), a region with uniform sample distribution was found. The microscope was then gradually switched to medium magnification (5000×) and high magnification (20000×-50000×) to observe the morphology (such as spherical, rod-shaped, tadpole-shaped, etc.), size, and surface structure of the virus particles. A typical virus particle field of view was selected, and the focal length and exposure parameters were adjusted to capture clear electron microscope images. Key morphological data such as the diameter and envelope integrity of the virus particles were recorded. Results Analysis: TEM images of unenriched raw seawater samples obtained using the same fixation, negative staining, and observation procedures were used as a control. By comparing the number density and morphological integrity of virus particles in the experimental group (enriched samples) and the control group, the recovery rate and structural preservation effect of the enrichment method on the virus were quantitatively evaluated. Transmission electron micrographs of the enriched virus in this embodiment are shown below. Figure 3 This also provides ultrastructural basis for subsequent virus classification and identification (such as preliminary judgment of virus family and genus based on morphological characteristics).

[0029] The above steps confirm that the enrichment method provided by this invention can efficiently capture low-abundance viruses up to 10 in coral samples. 5 ~10 6 The concentration of CFU / mL was reduced while removing interfering substances such as polysaccharides and proteins, providing high-purity virus-enriched samples for subsequent work such as coral virology research and electron microscopy observation.

[0030] 3. Quantitative analysis (nucleic acid test report) Table 1 shows the quality control report of the DNA metavitomym sample of coral virus after enrichment in this embodiment. It is used to verify the concentration, purity and integrity of viral nucleic acid, and to provide quality basis for subsequent metavitomym sequencing and other studies.

[0031] ① Description of detection methods and indicators DNA purity testing: OD was detected using Nanodrop. 260 / 280 (Reflects the ratio of nucleic acid to protein impurities, ideal value ~1.8), OD 260 / 230(Reflects the ratio of nucleic acids to impurities such as polysaccharides and phenols, ideal value ~2.0), assessing the residual status of interfering substances in the sample.

[0032] DNA concentration detection: The Qubit (quantitative PCR) method is used to accurately determine the nucleic acid concentration, avoiding interference from impurities and reflecting the enrichment efficiency of viral nucleic acid.

[0033] ② Analysis of quality inspection results The table contains 9 coral virus enriched samples (sample1~sample9), and the indicators are as follows: Nucleic acid concentration and total amount: The nucleic acid concentration ranged from 23.4 to 60.8 ng / μL, and the total nucleic acid amount (concentration × volume, assuming a uniform volume) ranged from 1.87 to 4.58 μg. The results show that the method of this invention effectively concentrated the originally "low-abundance" viral nucleic acid in corals, solving the problem of insufficient nucleic acid quantity caused by low viral content.

[0034] Nucleic acid purity (OD value): OD of all samples 260 / 280 The value is between 1.82 and 1.97, reaching the ideal value of 1.8, indicating that protein impurities in the sample have been effectively removed. OD 260 / 230 The value was between 2.02 and 2.19, reaching the ideal value of 2.0, indicating that interfering substances such as polysaccharides and free nucleic acids in the sample were accurately removed.

[0035] This result verifies the effectiveness of the "optimized impurity removal process" of the present invention, ensuring that the purity of viral nucleic acid meets the needs of subsequent metaviromography research.

[0036] Detection qualification and extraction stability: All samples had a "test result" of "A" (representing qualified nucleic acid quality) and a "remaining extraction times" of 1, indicating that the viral nucleic acid integrity was good and the extraction process was highly stable, which can provide a reliable nucleic acid template for subsequent experiments (such as metagenomic sequencing, viral gene amplification, etc.).

[0037] Table 1

[0038] In summary, the method for extracting and enriching coral symbiotic viruses provided by this invention can efficiently concentrate the nucleic acids of low-abundance viruses, accurately remove impurities, and ensure the purity and integrity of nucleic acids. This provides high-quality nucleic acid samples for metagenomic analysis and sequence determination of coral viruses, further verifying the reliability and practical value of the method.

[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting and enriching viruses from coral symbionts, characterized in that, Includes the following steps: (1) Obtain coral tissue and grind it in sterile seawater; (2) The ground coral tissue suspension was subjected to vortex treatment; (3) The coral tissue suspension after vortex treatment was centrifuged at low temperature; (4) The supernatant after centrifugation is purified by fractional filtration; the fractional filtration purification includes primary filtration through a filter membrane and tangential flow ultrafiltration; (5) The coral tissue filtrate after graded filtration and purification was concentrated by ultracentrifugation.

2. The extraction and enrichment method according to claim 1, characterized in that, In step (1), the salinity of the sterile seawater is consistent with the native environment of the coral; Preferably, the sterile seawater undergoes a pre-cooling treatment at 3-4°C; Preferably, the sterile seawater is seawater that has been filtered and sterilized through a filter membrane with a diameter of less than 0.22 μm.

3. The extraction and enrichment method according to claim 1, characterized in that, In step (2), the grinding is to grind until a homogeneous slurry is formed.

4. The extraction and enrichment method according to claim 1, characterized in that, In step (2), during the grinding process, the container holding the coral tissue is placed on ice; Preferably, the container holding the coral tissue is a sterile container.

5. The extraction and enrichment method according to claim 1, characterized in that, The coral tissue was taken from the branch tissue of healthy corals without obvious disease, and had a diameter of 2-3 cm.

6. The extraction and enrichment method according to claim 1, characterized in that, In step (2), sterilized glass beads are added in the vortex; Preferably, the diameter of the sterilizing glass beads is 1-3 mm; Preferably, the vortex is repeated 3 to 5 times at a frequency of 2500 to 3500 rpm for a duration of 30 to 60 seconds each time.

7. The extraction and enrichment method according to claim 1, characterized in that, In step (3), the temperature of the low-temperature centrifugation is 4~6℃; the rotation speed of the low-temperature centrifugation is 3500~5000 g; and the time of the low-temperature centrifugation is 15~20 min.

8. The extraction and enrichment method according to claim 1, characterized in that, In step (4), the initial filtration of the filter membrane is carried out by sequentially using filter membranes of 8~10 μm, 3~5 μm, and 0.45~1 μm.

9. The extraction and enrichment method according to claim 1, characterized in that, In step (4), the ultrafiltration membrane used in the tangential flow ultrafiltration has a molecular weight cutoff of 100~150 kDa; Preferably, the transmembrane pressure of the tangential flow ultrafiltration is 0.1~0.2 MPa; Preferably, the flow rate of the tangential flow ultrafiltration is 10~15 mL / min.

10. The extraction and enrichment method according to claim 1, characterized in that, In step (5), the molecular weight cutoff for the ultracentrifugation concentration is 100~150 kDa; Preferably, the rotation speed of the ultracentrifugation concentration is 3500~5000 g; the ultracentrifugation concentration time is 15~20 min; Preferably, the temperature of the ultracentrifugation concentration is 4~6℃.

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