Extraction method of latent virus genome

Abstract

The invention belongs to the technical field of biology, and particularly relates to an extraction method of a latent virus genome. The isoelectric point difference of plant virus capsid protein and plant cell protein, the characteristics of pathogenic genetic materials and the requirements of different plant viruses on PEG concentration are utilized. The latent virus genome is successfully separated from mulberry leaves infected with four pathogens by adopting PEG-coated magnetic nuclei, and semi-quantitative and quantitative PCR proves that the obtained genome has higher purity and higher concentration and can be used for PCR experiments and high-throughput sequencing. The problems of low content of latent virus pathogens in woody plants, few indication plants and difficulty in purification are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting a latent virus genome. Background technique [0002] Viruses often cause great harm to humans, and there are countless examples of agricultural and forestry plants suffering heavy losses due to viruses, such as rice Donggelu virus, potato Y virus, wheat yellow dwarf virus, etc. [0003] Viruses are non-cellular organisms with small shapes and simple structures that cannot be observed under an optical microscope. Therefore, the classification and purification of viruses is particularly important for virus research. Viruses cannot be cultivated through selective media like other microorganisms, nor can they be propagated, expanded, and purified on media like bacteria, so viruses must be extracted from host tissues. Because the virus stimulates plants to attract insects and other media, the virus-infected samples collected in the field are often mixe...

AI Technical Summary

Problems solved by technology

[0005] Centrifugation is the earliest method used by people. It mainly uses the difference in density between virus particles and cell debris. However, the purified virus has a lot of impurities. After the advent of high-speed and ultra-high-speed centrifuges, people have obtained a relatively satisfactory purification effect. And developed differential centrifugation and density gradient centrifugation on this basis, but these large-scale equipment are very expensive, and the concentration requirement of virus is higher, 5-10mg / kg concentration is the minimum limit of this method purification, and often needs Multiple experiments to obtain purified virus

[0006] The precipitation method mainly uses PEG to precipitate the virus at a certain particle concentration, but the PEG concentration required by different virus types varies greatly. For example, 4% PEG can precipitate tobacco mosaic virus, and 11% PEG can precipitate the virus. Precipitate Potato S virus, and PEG can also precipitate a part of non-viral substances, resulting in more impurities in the purified virus, and the PEG precipitation method must be combined with low-speed centrifugation, causing virus particles to easily aggregate and fail to precipitate and be lost by centrifugation

At the same time, this method needs to use organic clarifier, which is easy to cause harm to operators.

[0007] Charge method, virus particles can be directly adsorbed to the surface of polyethylene or polystyrene, and polyethylene centrifuge tubes can be used to capture virus particles directly, but the non-specificity of this method is too poor, and the type and amount of genome obtained are too small

[0008] Immunization method, which utilizes the specific combination of antigen and antibody, uses antibody-coated magnetic beads or other easily separated media as a carrier, and forms an antigen-antibody-medium complex through the reaction of antibody and antigen. Under the external force of a magnetic field or other media that can be separated, directional movement occurs to achieve the purpose of separation, and the obtained virus is of high purity. However, this method must know the capsid protein of the virus and prepare polyclonal antibodies, which requires a long period of time.

[0009] The filtration method uses the molecular weight of the virus coat protein to centrifuge and precipitate the virus particles, and then dialyzes them with a dialysis bag with a cutoff of 3KDa. However, the main purpose of this method is to obtain protein, and the operation period is long. 3d, the genome of the virus is severely degraded

[0010] In summary, the problems existing in the existing technology are that the extracted latent virus genome has many impurities, the extraction amount is not enough, or the extraction cycle is long

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