Search

CN-121975914-A - Virus preservation solution for non-guanidine salt type compatible nucleic acid extraction and preparation method and application thereof

CN121975914ACN 121975914 ACN121975914 ACN 121975914ACN-121975914-A

Abstract

The invention relates to a virus preservation solution for extracting non-guanidine salt compatible nucleic acid, a preparation method and application thereof, wherein the virus preservation solution comprises P (HEMA-BAMPD hydrochloride), a buffering agent, a chelating agent, a saccharide stabilizer and an osmotic pressure regulator. The invention uses P (HEMA-BAMPD hydrochloride) to replace guanidine salt and micromolecular quaternary ammonium salt, has good effect on inactivating envelope RNA/DNA viruses such as SARS-CoV-2 virus, influenza virus and the like, has no irritation, is friendly to operators and environment, realizes multipoint adsorption by a high molecular structure, has strong capability of resisting interference of organic matters, and does not influence the inactivating effect of a small amount of blood, sputum and other components in a sample. In addition, the synergistic effect of P (HEMA-BAMPD hydrochloride), chelating agent and saccharide stabilizer can effectively inhibit RNase and DNase activities.

Inventors

  • CHEN BEIBEI
  • ZHANG YUJI
  • WANG PENG
  • WU YUNPING
  • LI MENGNAN

Assignees

  • 江苏宁普医疗科技有限公司

Dates

Publication Date
20260505
Application Date
20260409

Claims (10)

  1. 1. A non-guanidine salt compatible nucleic acid extracted virus preservation solution is characterized by comprising P (HEMA-BAMPD hydrochloride), a buffering agent, a chelating agent, a sugar stabilizer and an osmotic pressure regulator.
  2. 2. The virus preservation solution for non-guanidinium compatible nucleic acid extraction according to claim 1, wherein the P (HEMA-BAMPD hydrochloride) is prepared by random copolymerization of free radicals, the number average molecular weight Mn is 5000-8000, PDI=1.0-1.2, and the content thereof is 0.05% -0.5% w/v.
  3. 3. The virus preservation solution for non-guanidinium compatible nucleic acid extraction according to claim 1, wherein the buffer is selected from 4-hydroxyethylpiperazine ethanesulfonic acid or phosphate buffer, and the content thereof is 10-50 mM/L.
  4. 4. The virus preservation solution extracted from non-guanidine salt compatible nucleic acid according to claim 1, wherein the chelating agent is selected from ethylene glycol bis (2-aminoethylether) tetraacetic acid or sodium citrate, and the content thereof is 1-10 mM/L.
  5. 5. The virus preservation solution extracted from non-guanidine salt compatible nucleic acid according to claim 1, wherein the sugar stabilizer is one or both of trehalose and sucrose, and the content of the sugar stabilizer is 5% -20% w/v.
  6. 6. The virus preservation solution extracted from non-guanidine salt compatible nucleic acid according to claim 1, wherein the osmotic pressure regulator is sodium chloride, and the content of the osmotic pressure regulator is 0.1% -5% w/v.
  7. 7. The virus preservation solution extracted from non-guanidine salt compatible nucleic acid according to claim 1, wherein the pH of the virus preservation solution is 7.0 to 7.5.
  8. 8. The method for preparing a virus preservation solution for non-guanidinium compatible nucleic acid extraction according to any one of claims 1 to 7, wherein a chelating agent, a sugar stabilizer and an osmotic pressure regulator are sequentially added into a buffer, stirred until the mixture is completely dissolved, and then added with P (HEMA-BAMPD hydrochloride) and fully and uniformly dispersed to prepare the virus preservation solution.
  9. 9. The method for preparing a virus preservation solution extracted from non-guanidine salt compatible nucleic acid according to claim 8, wherein the preparation method of P (HEMA-BAMPD hydrochloride) is as follows: Dissolving 1, 3-diamino-2-propanol and (Boc) 2 O in DCM, adding Et 3 N for room temperature reaction to obtain 1, 3-di (tert-butoxycarbonylamino) -2-propanol, namely a product 1, dissolving the product 1 and 10-bromo-1-decanol in DMF, adding K 2 CO 3 and 90 ℃ for reaction to obtain a product 2, dissolving the product 2 in DCM, placing a reaction bottle in an ice bath, adding triethylamine, slowly adding methyl methacrylate, and reacting to obtain a product 3, dissolving the product 3 in DCM, adding an equal volume of TFA, stirring at room temperature, and removing Boc to obtain a product 4, namely BAMPD; Weighing monomer according to a molar ratio HEMA of BAMPD =3:1, adding AIBN, dissolving in anhydrous toluene, introducing high-purity N 2 , sealing, transferring into an oil bath, heating to 70 ℃, stirring for reaction, cooling to room temperature after the reaction is finished to obtain copolymer solution, rotationally evaporating concentrated reaction solution, dissolving crude product by THF, slowly dripping into ice-N-hexane for precipitation, centrifuging/filtering to collect solid, repeating precipitation and washing for 3 times, and vacuum drying at 50 ℃ to obtain copolymer P (HEMA-BAMPD); Dissolving the copolymer P (HEMA-BAMPD) in an absolute ethanol/THF mixed solvent, dropwise adding concentrated hydrochloric acid/ethanol hydrochloride solution until the pH value of the system is 3-4, stirring at room temperature, removing most of the solvent by rotary evaporation, precipitating the residue with absolute ethyl ether, filtering, and drying in vacuum until the weight is constant to obtain the target product P (HEMA-BAMPD hydrochloride).
  10. 10. Use of a virus preservation solution extracted from a non-guanidinium-compatible nucleic acid according to any one of claims 1-7 in preservation of a virus sample.

Description

Virus preservation solution for non-guanidine salt type compatible nucleic acid extraction and preparation method and application thereof Technical Field The invention relates to the technical field of virus preservation solutions, in particular to a non-guanidine salt type compatible nucleic acid extraction virus preservation solution, a preparation method and application thereof. Background The virus nucleic acid detection is a core technical means of infectious disease clinical diagnosis and molecular biology research, and the virus preservation solution is used as a key medium for the links of sample collection and transportation to nucleic acid extraction, and the performance of the virus preservation solution directly determines the accuracy and reliability of a nucleic acid detection result. The ideal virus preservation solution needs to realize three core functions simultaneously, namely, high-efficiency virus inactivation to ensure the biological safety of operators, stable protection of nucleic acid (particularly easily degradable RNA) to avoid detection omission, and high compatibility with the subsequent nucleic acid extraction technology (such as a magnetic bead method and a column extraction method) to ensure the recovery rate of the nucleic acid. Currently, the main stream of virus preservation solution on the market mainly comprises guanidine hydrochloride, guanidine isothiocyanate and other guanidine salts as strong protein denaturants and cracking components, a Tris-HCl buffer system, an EDTA chelating agent, isopropanol, ethanol and other alcohol stabilizers. Although the guanidine salt preservation solution can inactivate by destroying the structure of viral proteins and inhibit nuclease activity to a certain extent, a plurality of technical defects which are difficult to overcome exist: (1) The safety risk is prominent, the guanidine salt has stronger irritation, and can easily generate highly toxic cyanide when being contacted with chlorine-containing disinfectant, thereby causing potential harm to the health of operators and the environment; (2) The nucleic acid extraction compatibility is poor, guanidine salt is easy to combine with the functional groups on the surface of the magnetic beads, the adsorption efficiency of the magnetic beads on the nucleic acid is interfered, the recovery rate of the nucleic acid is reduced, and the effect is poorer when the method is especially suitable for a magnetic bead method (clinical mainstream extraction technology); (3) The stability of the nucleic acid is insufficient, the alcohol stabilizer is easy to volatilize, the protection effect on the nucleic acid can be lost in the long-term storage process at room temperature, the protection effect of guanidine salt on RNA is limited, the RNA is easy to degrade, and the detection of a low-concentration sample is missed; (4) The anti-interference capability is weak, and organic matters such as blood, sputum and the like contained in the sample are easy to combine with guanidine salt or matched small molecular additives, so that the virus inactivation efficiency and the nucleic acid protection effect are reduced. To solve the above problems, CN115948501a discloses an inactivated virus nucleic acid preservation solution containing no guanidine salt, which comprises a protein denaturing agent, a disulfide bond reducing agent, a saccharide compound and water. The protein denaturant is a small molecular compound such as amide, ammonium salt and the like, and the preservation solution can quickly inactivate viruses, protect viral nucleic acid and prevent RNase from decomposing and does not react with common nucleic acid extraction reagents. However, the small molecule inactivating agent still has obvious short plates, for example, has certain irritation to mucous membrane, is not suitable for processing related samples of special people, is easy to be neutralized by organic matters in the samples, has insufficient inactivation stability, has the problems of imperfect nucleic acid protection system, short room temperature preservation period and the like, and cannot meet the requirements of long-distance transportation and long-term preservation of clinical samples. Based on the method, a virus preservation solution extracted from non-guanidine salt compatible nucleic acid is developed, and the virus preservation solution has important clinical application value and market prospect. Disclosure of Invention Aiming at the problems that the existing virus preservation solution depends on guanidine salt, has poor compatibility with nucleic acid extraction by a magnetic bead method, insufficient RNA preservation stability, weak capability of resisting organic matter interference, and the operation safety to be improved, the invention provides a virus preservation solution for extracting non-guanidine salt compatible nucleic acid, a preparation method and application thereof. The long-chain alkane quaternary ammon