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CN-122012492-A - Lysate composition and application thereof in preparation of reagent for extracting genome DNA

CN122012492ACN 122012492 ACN122012492 ACN 122012492ACN-122012492-A

Abstract

The application belongs to the technical field of molecular biology and in-vitro diagnosis, and particularly discloses a lysate composition and application thereof in preparation of a reagent for extracting genomic DNA. The lysate composition comprises a pH regulator, 0.05-1.0% (v/v) nonionic surfactant, 0.1-2 mg/mL bovine serum albumin, 0.5-5 mM reducing agent and 50-500 mug/mL proteinase K, wherein the pH is 7.0-8.0. The application provides a genome DNA quick release lysate which is reasonable in composition and simple and convenient to operate, and can realize efficient release and stable preservation of DNA in complex biological samples under the condition of no need of traditional purification steps, so that the obtained DNA can be directly used for nucleic acid amplification detection such as PCR, LAMP and the like, thereby improving detection efficiency, repeatability and application convenience, and meeting the actual requirements of quick molecular diagnosis and field detection.

Inventors

  • ZHANG ZHANG
  • LI LIAN
  • YAO JUAN

Assignees

  • 西南医科大学附属中医医院

Dates

Publication Date
20260512
Application Date
20260320

Claims (10)

  1. 1. The lysate composition is characterized by comprising a pH regulator, a nonionic surfactant, bovine serum albumin, a reducing agent and proteinase K, wherein the volume concentration of the nonionic surfactant is 0.05-1.0%, the concentration of the bovine serum albumin is 0.1-2 mg/mL, the concentration of the reducing agent is 0.5-5 mM, and the concentration of the proteinase K is 50-500 mug/mL; the reducing agent is used for preventing proteinase K from being oxidized and deactivated in the high-temperature cracking process and maintaining the proteolytic capability of proteinase K; The pH value of the lysate composition is 7.0-8.0.
  2. 2. The composition of claim 1, wherein the composition of lysate is a water-soluble complex lysate system.
  3. 3. The lysate composition according to claim 2, wherein the pH regulator is selected from buffers having a pH of 7.0-8.0.
  4. 4. The lysate composition of claim 3, wherein the buffer is selected from Tris-HCl buffer.
  5. 5. The lysate composition according to claim 1, wherein the nonionic surfactant is selected from Tween-series nonionic surfactants; And/or the reducing agent is selected from dithiothreitol.
  6. 6. The lysate composition according to claim 5, wherein the nonionic surfactant is at least one selected from the group consisting of Tween-20, tween-40 and Tween-60.
  7. 7. The lysate composition according to any one of claims 1-6, wherein: the lysate composition comprises the following components: a) Tris-HCl buffer solution with the concentration of 10-100 mM and the pH of 7.0-8.0; b) Tween-20 with a volume concentration of 0.05-1.0%; c) Bovine serum albumin at a concentration of 0.1-2 mg/mL; d) Dithiothreitol with the concentration of 0.5-5 mM; e) Proteinase K at a concentration of 50-500. Mu.g/mL.
  8. 8. The lysate composition according to claim 7, wherein the lysate composition comprises the following components: a) Tris-HCl buffer solution with the concentration of 50 mM and the pH of 7.0-8.0; b) Tween-20, volume concentration of 1.0%; c) Bovine serum albumin at a concentration of 1 mg/mL; d) Dithiothreitol at a concentration of 5 mM; e) Proteinase K at a concentration of 200. Mu.g/mL.
  9. 9. Use of the lysate composition according to any one of claims 1-8 for the preparation of a reagent for extracting genomic DNA.
  10. 10. The use according to claim 9, characterized in that: Releasing and preserving the biological sample genomic DNA from the biological sample with the agent; and/or extracting the obtained genome DNA by the reagent for nucleic acid amplification detection.

Description

Lysate composition and application thereof in preparation of reagent for extracting genome DNA Technical Field The application relates to the technical fields of molecular biology and in-vitro diagnosis, in particular to a lysate composition and application thereof in preparation of a reagent for extracting genomic DNA. Background Nucleic acid detection technology is a core means in molecular diagnostics, biomedical research, and genetic analysis. Wherein, the efficient release and stable acquisition of genomic DNA (gDNA) are the preconditions for the accurate implementation of subsequent nucleic acid amplification, genotyping and molecular detection. Currently, for complex biological samples such as blood, swab and the like, a silica gel membrane column method, a magnetic bead method or an organic solvent extraction method is commonly adopted in the prior art to extract DNA. The methods generally depend on multi-step operation flow, including steps of sample cracking, protein removal, nucleic acid adsorption, washing, elution and the like, are complex in operation, consume long time, have high requirements on experimental conditions and technical levels of operators, and are not beneficial to popularization and application of rapid detection and point-of-CARE TESTING (POCT) on site. Furthermore, frequent centrifugation or magnetic separation steps also limit the use of such methods in primary medical institutions and resource-constrained environments. To simplify the DNA release procedure, there have been studies attempting to release DNA directly from whole blood or cell samples using chemical lysis systems. For example, some of the prior art uses Tris-HCl buffer system to maintain pH stability of the solution to reduce DNA hydrolysis, protease K (Proteinase K) is added to lyse cell structure and degrade nucleic acid binding protein to promote DNA release, and nonionic surfactant (such as Tween-20) is used to destroy cell membrane and nuclear membrane structure to improve the lysis efficiency. However, the simplified cleavage system still has certain disadvantages in practical application. On the one hand, the blood sample is rich in hemoglobin, immunoglobulin and other inhibitory components, which are easy to inhibit downstream nucleic acid amplification reaction, and on the other hand, during the process of cracking and preserving, the protease activity is easy to be influenced by oxidizing environment to reduce, thereby influencing DNA release efficiency and repeatability. In addition, the existing partial lysis system is still difficult to consider in terms of DNA stability, inhibitor tolerance and downstream amplification compatibility, and is difficult to meet the requirements of rapid and direct amplification detection. Therefore, there is a need to provide a nucleic acid release system which has reasonable composition, is easy to operate, can release and stably preserve DNA in complex biological samples with high efficiency, so as to overcome the problems of complex operation, obvious amplification inhibition, insufficient detection consistency and the like in the prior art. Disclosure of Invention In view of the above-mentioned drawbacks of the prior art, the present application aims to provide a lysate composition and application thereof in preparing a reagent for extracting genomic DNA, which are used for solving the problems of complicated DNA extraction process, long time consumption, easy introduction of amplification inhibition, etc. in complex biological samples such as whole blood in the prior art. In order to achieve the above and other related objects, the application provides a lysate composition, which comprises a pH regulator, a nonionic surfactant, bovine serum albumin (Bovine Serum Albumin, BSA), a reducing agent and protease K (Proteinase K), wherein the volume concentration of the nonionic surfactant is 0.05-1.0% (v/v), the concentration of the bovine serum albumin is 0.1-2 mg/mL, the concentration of the reducing agent is 0.5-5 mM, the concentration of the proteinase K is 50-500 mug/mL, the reducing agent is used for preventing oxidation deactivation of the proteinase K in the high-temperature cracking process and maintaining the proteolytic capability of the proteinase K, and the pH of the lysate composition is 7.0-8.0. Further, the lysate composition is a water-soluble complex lysate system. Further, the pH regulator is selected from buffers with pH of 7.0-8.0. Further, the buffer is selected from Tris-HCl buffer. Further, the nonionic surfactant is selected from the group consisting of Tween-series nonionic surfactants including, but not limited to, combinations of one or more of Tween-20, tween-40, tween-60, and the like. Further, the reducing agent is selected from dithiothreitol (Dithiothreitol, DTT). Further, the lysate composition comprises the following components: a) Tris-HCl buffer solution with the concentration of 10-100 mM and the pH of 7.0-8.0; b) Tween-20 with a