Search

CN-121991901-A - Extraction and enrichment method of coral symbiota virus

CN121991901ACN 121991901 ACN121991901 ACN 121991901ACN-121991901-A

Abstract

The invention discloses an extraction and enrichment method of coral symbiont viruses, which comprises the following steps of obtaining coral tissues, grinding the coral tissues in sterile seawater, carrying out vortex treatment on coral tissue suspension after grinding, carrying out low-temperature centrifugation on the coral tissue suspension after vortex treatment, carrying out graded filtration and purification on supernatant after centrifugation, wherein the graded filtration and purification comprise filter membrane primary screening filtration and tangential flow ultrafiltration, and carrying out ultracentrifugation concentration on coral tissue filtrate after graded filtration and purification. The extraction and enrichment method provided by the invention can be used for efficiently capturing various known and unknown viruses in the coral symbiotic system, is suitable for low-abundance viruses and covering known and unknown virus groups, and is simple and convenient to operate, free from complex professional equipment and controllable in cost.

Inventors

  • ZHOU JIN
  • WU MENGJIE
  • CAI ZHONGHUA

Assignees

  • 清华大学深圳国际研究生院

Dates

Publication Date
20260508
Application Date
20251218

Claims (10)

  1. 1. The extraction and enrichment method of the coral symbiont virus is characterized by comprising the following steps of: (1) Obtaining coral tissue, and grinding the coral tissue in sterile seawater; (2) Carrying out vortex treatment on the coral tissue suspension after grinding; (3) Centrifuging the coral tissue suspension subjected to vortex treatment at a low temperature; (4) Subjecting the centrifuged supernatant to fractional filtration and purification, wherein the fractional filtration and purification comprises primary filtration by a filter membrane and tangential flow ultrafiltration; (5) And (5) performing ultracentrifugation concentration on the coral tissue filtrate after the classification, filtration and purification.
  2. 2. The extraction and enrichment method of claim 1, wherein in step (1), the salinity of the sterile seawater is consistent with the native environment of the coral; preferably, the sterile seawater is subjected to pre-cooling treatment at a temperature of 3-4 ℃; Preferably, the sterile seawater is seawater that has been filter sterilized through a filter membrane of less than 0.22 μm.
  3. 3. The extraction and enrichment method according to claim 1, wherein in step (2), the grinding is performed to a slurry state.
  4. 4. The method of claim 1, wherein in step (2), the container containing coral tissue is placed on ice during the grinding process; preferably, the container for containing coral tissue is a sterile container.
  5. 5. The extraction and enrichment method according to claim 1, wherein the coral tissue is obtained from a branch tissue of a healthy coral without significant disease, and has a diameter of 2-3 cm.
  6. 6. The extraction and enrichment method according to claim 1, wherein in step (2), the vortex is added with sterilized glass beads; preferably, the diameter of the sterilized glass beads is 1-3 mm; preferably, the vortex is repeated 3-5 times at a frequency of 2500-3500 rpm for 30-60 seconds each time.
  7. 7. The extraction and enrichment method according to claim 1, wherein in the step (3), the temperature of the low-temperature centrifugation is 4-6 ℃, the rotating speed of the low-temperature centrifugation is 3500-5000 g, and the time of the low-temperature centrifugation is 15-20 min.
  8. 8. The method according to claim 1, wherein in the step (4), the primary filtration with the filter membrane is performed by sequentially using filter membranes of 8-10 μm, 3-5 μm and 0.45-1 μm.
  9. 9. The extraction and enrichment method according to claim 1, wherein in the step (4), the cutoff molecular weight of an ultrafiltration membrane used for tangential flow ultrafiltration is 100-150 kDa; Preferably, the transmembrane pressure of the tangential flow ultrafiltration is 0.1-0.2 MPa; preferably, the flow speed of the tangential flow ultrafiltration is 10-15 mL/min.
  10. 10. The extraction and enrichment method according to claim 1, wherein in the step (5), the molecular weight cut-off of the ultracentrifugation concentration is 100-150 kDa; Preferably, the rotating speed of the ultracentrifugation concentration is 3500-5000 g, and the ultracentrifugation concentration time is 15-20 min; preferably, the temperature of the ultracentrifugation concentration is 4-6 ℃.

Description

Extraction and enrichment method of coral symbiota virus Technical Field The invention belongs to the technical field of marine organisms, and particularly relates to an extraction and enrichment method of coral symbiota viruses. Background The coral reef ecosystem is one of the most diverse ecosystems in the ocean, and has an irreplaceable effect on maintaining the balance of the ocean ecology. In recent years, global coral reefs face serious threats such as albinism and diseases, wherein viruses serve as important microorganism groups in a coral symbiotic system, and the association of infection mechanisms and coral health has become a research hotspot. However, enrichment of viruses in coral symbiota faces three core problems, namely extremely low virus content, host cells (such as thorn cells and interstitial cells) in coral tissues, symbiotic algae (such as yellow worm algae) and biomass of symbiotic microorganisms (bacteria and archaea) account for more than 99%, virus particles are wrapped by a large amount of impurities, the existing enrichment method is strong in pertinence and poor in broad spectrum, such as an immunoaffinity method relies on specific antibodies, only known viruses can be enriched, unknown viruses cannot be covered, an ultracentrifugation method can separate viruses, professional equipment (such as an ultracentrifuge) is needed, operation is time-consuming (4 hours for single centrifugation) and virus particles are easily broken due to overlarge centrifugal force, and polysaccharide, protein and free nucleic acid (host DNA/RNA) released by coral tissues can adsorb the virus particles or generate false positive signals in subsequent detection (such as virus sequencing and electron microscopy). Therefore, the development of the extraction and enrichment method of the coral symbiont viruses, which is simple and convenient to operate, has broad spectrum, high efficiency and low cost, is a key for breaking through the technical bottleneck of coral virus research. Disclosure of Invention Aiming at the technical problems, the invention provides an extraction and enrichment method of coral symbiont viruses, which is suitable for broad-spectrum enrichment of various known and unknown viruses in a coral symbiont system and provides key technical support for coral virus diversity analysis, virus-host interaction research and coral disease diagnosis. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: The invention provides a method for extracting and enriching coral symbiont viruses, which comprises the following steps: (1) Obtaining coral tissue, and grinding the coral tissue in sterile seawater; (2) Carrying out vortex treatment on the coral tissue suspension after grinding; (3) Centrifuging the coral tissue suspension subjected to vortex treatment at a low temperature; (4) Subjecting the centrifuged supernatant to fractional filtration and purification, wherein the fractional filtration and purification comprises primary filtration by a filter membrane and tangential flow ultrafiltration; (5) And (5) performing ultracentrifugation concentration on the coral tissue filtrate after the classification, filtration and purification. As 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 is subjected to pre-cooling treatment at a temperature of 3-4 ℃; in certain specific embodiments, the sterile seawater is seawater that is filter sterilized through a filter membrane of less than 0.22 μm; Preferably, the grinding is grinding to a slurry; Preferably, in the grinding process, the container for containing coral tissue is placed on ice to prevent virus inactivation due to high temperature; Preferably, the container for containing coral tissue is a sterile container; In some specific embodiments, the coral tissue is obtained from a healthy and disease-free branched tissue of coral, having a diameter of 2-3 cm. In a preferred embodiment, in step (2), the vortex is added to sterilized glass beads; preferably, the diameter of the sterilized glass beads is 1-3 mm; preferably, the vortex is repeated 3-5 times at a frequency of 2500-3500 rpm for 30-60 seconds each time. In the step (3), the temperature of the low-temperature centrifugation is 4-6 ℃, the rotating speed of the low-temperature centrifugation is 3500-5000 g, and the time of the low-temperature centrifugation is 15-20 min. In the step (4), as a preferable implementation manner, the primary screening and filtering of the filter membrane are sequentially carried out by adopting filter membranes of 8-10 mu m, 3-5 mu m and 0.45-1 mu m; preferably, the cutoff molecular weight of an ultrafiltration membrane used for tangential flow ultrafiltration is 100-150 kDa; Preferably, the transmembrane pressure of the tangential flow ultrafiltration is 0.1-0.2 MPa; preferably, the flow speed of the tange