A sample release agent and its preparation method and application

Abstract

The present invention provides a composite sample release agent and a preparation method and application thereof. The release agent has a fast release speed, avoids complicated extraction processes, and can release different samples in 5-15 minutes through simple release, and can be inactivated at the same time, thereby improving the sample detection efficiency. It also has the characteristics of high detection sensitivity, response to weak positive samples, good preservation effect on samples, long preservation time, good compatibility with multiple samples, etc.

Description

Technical Field

[0001] The invention belongs to the field of biological reagents, and in particular relates to a sample release agent and a preparation method and application thereof. Background Art

[0002] Nucleic acid extraction technology is the basis of molecular biology research and is now widely used in the fields of pathogenic microorganism detection, environmental microorganism detection, food safety detection, etc. Traditional nucleic acid extraction methods include physical methods, chemical methods and biological methods, among which physical methods include boiling method, magnetic bead method, ultrasonic method, etc.; chemical methods include surfactants, alkaline lysis method, etc.; biological methods mainly include enzyme method, etc. At present, the main problem with the release agent used in nucleic acid extraction is that the cell lysis is incomplete, resulting in incomplete nucleic acid release, and the obtained nucleic acid (especially RNA) is unstable and easily degraded by factors such as temperature and enzymes; in addition, due to the high protein content in the cell and a large amount of substances that may interfere with nucleic acid detection, the extraction reagent and the detection system are often not well matched. Therefore, it is also necessary to use traditional methods through multiple steps and use a variety of different reagents and other cumbersome technical means to remove polymerase chain reaction (PCR) inhibitors.

[0003] The nucleic acid extraction methods currently used mainly include the following four methods: (1) Traditional boiling method: directly mix the alkaline lysis solution with the pathogen-containing liquid, lyse at high temperature, and obtain nucleic acid by centrifugation; (2) Concentration boiling method: first use polysaccharide to concentrate and precipitate the pathogen, then add alkaline lysis solution to lyse at high temperature, and obtain nucleic acid by centrifugation; (3) Centrifugal column method: use silica gel membrane or glass fiber membrane to adsorb, purify, and elute to obtain nucleic acid; (4) Magnetic bead method: use silica powder or polyethylene glycol-coated magnetic beads for adsorption, and then purify and elute to obtain nucleic acid.

[0004] The extraction using the above methods (1) and (2) requires multiple tube changes and centrifugation steps, which takes a long time to process the sample and results in a low yield. Although the centrifugal column method is better than methods (1) and (2), it still has many steps and low extraction efficiency. The magnetic bead method has simple extraction steps, high recovery rate, and is easy to automate, but the product is very expensive and is not widely used at present.

[0005] Sample releasers can release various substances to be tested in cells, such as nucleic acids, by destroying cell membranes and organelle membranes, which facilitates nucleic acid extraction. However, existing sample releasers have shortcomings such as easy missed detection, low detection sensitivity, low accuracy, high detection cost, and slow speed. Therefore, the development of new sample releasers is of great significance. Summary of the invention

[0006] In order to improve the above technical problems, the present invention provides a sample releaser, which, based on the total volume of the sample releaser, includes: a surfactant with a concentration of 0.01-10% (w / v), an auxiliary salt with a concentration of 0-5M, a pH regulator with a concentration of 0.01-500mM, a promoter with a concentration of 0.01-20% (w / v), a stabilizer with a concentration of 0.01-5M and an antibacterial agent with a concentration of 0.01-3% (w / v).

[0007] According to an embodiment of the present invention, the surfactant is selected from at least one of Savantine, tetrabutylammonium fluoride, tetrabutylammonium bromide, tetrabutylammonium chloride, saponin powder, Morwet EFW (alkyl naphthalene sulfonate), sodium lauryl sarcosinate (sodium N-lauroyl sarcosinate), sodium lauryl sulfate, sodium lauryl sulfonate, potassium lauryl alcohol ether phosphate, cocamidopropyl betaine, lauroyl propyl betaine, cetyl trimethylammonium bromide, Tween-20, Tween-40, Tween-60, Tween-80, Brij-58, and Triton X-100; preferably at least one of sodium lauryl sarcosinate, Savantine, and lauroyl propyl betaine.

[0008] According to an embodiment of the present invention, the concentration of the surfactant in the sample release agent is 0.01-10% (w / v), for example, 0.01-8% (w / v), 0.05-5% (w / v), 0.1-2.5% (w / v); exemplarily, the concentration is 1% (w / v).

[0009] According to an embodiment of the present invention, the auxiliary salt can be selected from sodium salt or potassium salt, for example, selected from at least one of sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium fluoride, potassium bifluoride, cesium fluoride, sodium chloride, potassium chloride and sodium bromide; preferably, the auxiliary salt is selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium fluoride, sodium chloride or sodium bromide; more preferably selected from at least one of sodium chloride, sodium bicarbonate and potassium chloride.

[0010] According to an embodiment of the present invention, the concentration of the auxiliary salt in the sample release agent is 0-5M, for example, 30-200mM, 2-4M; illustratively, the concentration is 150mM.

[0011] According to an embodiment of the present invention, the pH regulator can be selected from at least one of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), sodium morpholine acetate, sodium acetate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, sodium hydroxide, etc.; preferably at least one of tris(hydroxymethyl)aminomethane hydrochloride, sodium acetate, sodium citrate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0012] According to an embodiment of the present invention, the concentration of the pH regulator in the sample release agent is 0.01-500 mM, for example, 10-200 mM; illustratively, the concentration is 50 mM.

[0013] According to an embodiment of the present invention, the accelerator is selected from at least one of dimethyl sulfoxide, formamide, N,N-dimethylformamide, crown ether (such as: 18-crown-6), mannitol, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, tetradecanol, hexadecanol, octadecyl alcohol, polyvinyl pyrrolidone (K=10-20), glycogen, linear polyacrylamide, N-n-alkylphenylisothiazolone, etc.; preferably at least one of dimethyl sulfoxide, polyethylene glycol 8000, formamide, and hexadecanol.

[0014] According to an embodiment of the present invention, the concentration of the enhancer in the sample release agent is 0.01-20% (w / v), for example, 1-10% (w / v); illustratively, the concentration is 10% (w / v).

[0015] According to an embodiment of the present invention, the stabilizer is selected from at least one of guanidine thiocyanate, guanidine hydrochloride, urea, gelatin, sucrose fatty acid ester, and light white oil; preferably at least one of guanidine thiocyanate and light white oil.

[0016] According to an embodiment of the present invention, the concentration of the stabilizer in the sample release agent is 0.01-5M, for example, 10-20mM, 0.5-4M; illustratively, the concentration is 0.5M.

[0017] According to an embodiment of the present invention, the antibacterial agent is selected from at least one of sodium azide, sorbitol, potassium sorbate, phenoxyethanol, carbendazim, thiophanate-methyl, etc.; preferably at least one of sodium azide, potassium sorbate, phenoxyethanol, carbendazim.

[0018] According to an embodiment of the present invention, the concentration of the antibacterial agent in the sample release agent is 0.01-3% (w / v), for example, 0.1-1% (w / v); illustratively, the concentration is 0.3% (w / v).

[0019] According to an exemplary embodiment of the present invention, the sample release agent is composed of 50 mM Tris-HCl with a concentration of pH = 8, 150 mM NaCl, 0.5 M guanidine thiocyanate (GuSCN), 1% (w / v) sodium N-lauroyl sarcosinate (SLS), 1% (w / v) Savantine, 10% (v / v) DMSO, 0.5 M light white oil and 0.3% (w / v) sodium azide and the remainder sterile water, based on the total volume of the sample release agent.

[0020] According to an exemplary embodiment of the present invention, the sample release agent is composed of 50 mM sodium acetate, 150 mM sodium bicarbonate, 0.5 M urea, 1% (w / v) Tween-20, 1% (w / v) lauramidopropyl betaine, 5% (w / v) polyethylene glycol 8000, 2% (v / v) light white oil and 0.2% (w / v) potassium sorbate and the balance sterile water, based on the total volume of the sample release agent.

[0021] According to an exemplary embodiment of the present invention, the sample releaser is composed of 50 mM disodium hydrogen phosphate, 50 mM sodium dihydrogen phosphate, 150 mM sodium chloride, 0.5 M guanidine hydrochloride, 1% (w / v) Triton X-100, 1% (w / v) lauramidopropyl betaine, 10% (v / v) formamide, 0.5 M guanidine isothiocyanate and 0.2% (w / v) phenoxyethanol and the balance sterile water, based on the total volume of the sample releaser.

[0022] According to an exemplary embodiment of the present invention, the sample releaser is composed of 50 mM sodium citrate, 150 mM potassium chloride, 0.5 M urea, 1% (w / v) hexadecyltrimethylammonium bromide, 1% (w / v) lauramide propyl betaine, 10% (w / v) hexadecanol, 2% (v / v) light white oil and 0.2% (w / v) carbendazim and the balance sterile water, based on the total volume of the sample releaser.

[0023] The present invention also provides a method for preparing the sample release agent, comprising the step of mixing a surfactant, an auxiliary salt, a pH regulator, a promoter, a stabilizer and an antibacterial agent.

[0024] The present invention also provides application of the sample releasing agent in nucleic acid release.

[0025] The present invention also provides a nucleic acid release method, comprising the following steps: adding a sample into the sample release agent, then oscillating and mixing, placing at room temperature, and absorbing the supernatant to obtain the released nucleic acid.

[0026] According to an embodiment of the present invention, the sample may be bacteria, epithelial cells, whole blood (blood cells) or viruses.

[0027] Beneficial Effects

[0028] The present invention provides a compound sample release agent and its preparation method and application. The release agent has a fast release speed, avoids complicated extraction processes, and can release different samples in 5-15 minutes through simple release, and can be inactivated at the same time, thereby improving the sample detection efficiency. It also has the characteristics of high detection sensitivity, response to weak positive samples, good preservation effect on samples, long preservation time, good compatibility with multiple samples, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison chart of the sample release of the release agent and the control group in Example 1. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0031] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0032] The control release agent described below is Shengxiang biological release agent; the extraction reagent is magnetic bead nucleic acid extraction reagent purchased from Daan Biotechnology Co., Ltd.

[0033] Example 1 Release speed test

[0034] Prepare the sample release agent according to the following table, formula 1:

[0035] Components concentration Tris-HCl (pH = 8) 50mM NaCl (Sodium Chloride) 150mM GuSCN (Guanidine Thiocyanate) 0.5M SLS (Sodium Lauroyl Sarcosinate) 1%(w / v) Savantine 1%(w / v) DMSO (Dimethyl Sulfoxide) 10%(v / v) Light white oil 0.5M Sodium Azide 0.3%(w / v) Sterile water margin

[0036] The oral swab sample was directly immersed in the release agent, the tail was removed, and it was mixed upside down for 30 seconds, and then left to stand at room temperature. Samples were taken at 0, 1, 2, 5, 10, and 30 minutes, and the state of epithelial cells was observed under a microscope (magnification: 16X 40). At the same time, physiological saline, control release agent, and inactivated sample preservation solution were used as controls to complete the above test.

[0037] The results show:

[0038] 1) The state of saline epithelial cells does not change over time;

[0039] 2) The release agent reported in the present invention can release about 90% of the epithelial cell sample within 2 minutes, and at the same time, destroy the cell membrane and other membrane tissues to inactivate the bacteria, pathogenic microorganisms, viruses, etc. in the sample, while the control release agent needs 30 minutes to complete 90% release;

[0040] 3) The cell state of the inactivated sample preservation solution has changed, but the cell membrane is still intact, see Figure 1 .

[0041] Example 2: COVID-19 Fake Virus Sample Release

[0042] The nasopharyngeal swab sample was directly immersed in the release agent prepared in Example 1, the tail was removed, and the COVID-19 pseudovirus was added thereto, with the final concentration after addition being 200-10 4 copies / mL, mix by inversion for 30 seconds, let stand at room temperature for 10 minutes, mix by inversion again for 30 seconds, centrifuge instantaneously, take the supernatant and add it to the fluorescent quantitative PCR reagent for quantitative amplification. At the same time, use the control release agent to complete the above test, and compare the results with the results of the nucleic acid extraction and purification reagent.

[0043]

[0044] The results show:

[0045] 1) The test results of the release agent according to the present invention are comparable to those of the nucleic acid extraction reagent, and it still responds to weak positive samples;

[0046] 2) Although the control release agent has better detection data for conventional samples, it still misses weak positive samples.

[0047] Example 3: COVID-19 pseudovirus sample storage time test

[0048] The nasopharyngeal swab head sample was directly immersed in the release agent prepared in Example 1, the tail was removed, and the COVID-19 pseudovirus was added thereto, with a final concentration of 10 3 copies / mL, mix by inverting for 30 seconds, let stand at room temperature for 10 minutes, mix by inverting again for 30 seconds, centrifuge instantly, and add the supernatant to the fluorescent quantitative PCR reagent on days 0, 1, 2, 4, 7, and 14 for quantitative amplification. At the same time, use the control release agent to complete the above test for comparison.

[0049]

[0050] The results show that the release agent test results according to the present invention have a better preservation effect on the sample

[0051] Example 4 African swine fever release (whole blood sample, cotton swab sample, environmental sample)

[0052] The samples of the quality control products containing African swine fever virus were mixed with 200 μL of the release agent prepared in Example 1, wherein 200 μL of the pig whole blood sample (A series samples in the table), 200 μL of the cotton swab (B series samples in the table), and the environmental sample (C series samples in the table) were centrifuged at 10000 r / min, the supernatant was removed, 200 μL of the release agent was added, and the mixture was mixed upside down for 30 seconds, and then stood at room temperature for 10 minutes, and then mixed upside down again for 30 seconds. After instantaneous centrifugation, the supernatant was added to the fluorescent quantitative PCR reagent for quantitative amplification. At the same time, the control release agent was used to complete the above test, and the results were compared with the results of the nucleic acid extraction and purification reagent.

[0053]

[0054] The results show:

[0055] 1) The test results of the release agent according to the present invention are comparable to those of the nucleic acid extraction reagent and are compatible with all samples;

[0056] 2) In the detection data of the control release agent for routine samples, there are omissions in the detection of whole blood samples and environmental samples.

[0057] Example 5 Release agent with the same functional formula

[0058] After a lot of experiments and explorations, the present invention finally obtained a variety of sample release agent formulas with consistent release effect and similar preservation function. Similar to Example 1, the following sample release agent formulas 2 to 4 according to the present invention were obtained by replacing the corresponding components according to the following table:

[0059] Sample release agent formulation 2:

[0060] Components concentration Sodium acetate 50mM Sodium bicarbonate 150mM Urea 0.5M Tween-20 1%(w / v) Lauramidopropyl Betaine 1%(w / v) Polyethylene glycol 8000 5%(w / v) Light white oil 2%(v / v) Potassium Sorbate 0.2%(w / v) Sterile water margin

[0061] Sample release agent formulation 3:

[0062]

[0063]

[0064] Sample release agent formulation 4

[0065] Components concentration Sodium Citrate 50mM Potassium chloride 150mM Urea 0.5M Hexadecyltrimethylammonium bromide 1%(w / v) Lauramidopropyl Betaine 1%(w / v) Cetyl Alcohol 10%(w / v) Light white oil 2%(v / v) Carbocillin 0.2%(w / v) Sterile water margin

[0066] The sampling operation was performed according to Example 2, and the sample release agent Formula 1 (Example 1), Formula 2, Formula 3, and Formula 4 of the present invention were used to release the COVID-19 pseudovirus sample, complete the fluorescent quantitative PCR test, and compare the results with the nucleic acid extraction and purification results.

[0067]

[0068]

[0069] The results showed that there was no significant difference among the four formulations of sample release agents, no missed detection occurred, and all were able to meet the needs of actual testing.

[0070] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sample release agent, which, based on the total volume of the sample release agent, consists of 1% (w / v) Savantine, 1% (w / v) sodium N-lauroyl sarcosine, 150 mM sodium chloride, 50 mM tris(hydroxymethyl)aminomethane hydrochloride, 10% by volume dimethyl sulfoxide, 0.5 M guanidine isothiocyanate, 0.5 M light white oil, 0.3% (w / v) sodium azide and the balance sterile water.

2. The method for preparing the sample release agent according to claim 1, comprising the step of mixing Savantine, sodium N-lauroyl sarcosinate, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride), dimethyl sulfoxide, guanidine isothiocyanate, light white oil, sodium azide and sterile water.

3. Use of the sample release agent according to claim 1 in nucleic acid release.

4. A method for releasing nucleic acid, comprising the following steps: The sample is added to the sample release agent according to claim 1, then shaken and mixed, left at room temperature, and the supernatant is aspirated to obtain the released nucleic acid.

5. The method according to claim 4, characterized in that The sample is bacteria, epithelial cells, blood cells or viruses.

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