Drying protective agent, drying method and nucleic acid quality control product

Abstract

The invention relates to the technical field of molecular biology, in particular to a drying protective agent, a drying method and a nucleic acid quality control product. The invention provides a drying protective agent combination comprising monosaccharide, polysaccharide and gelatin and a preparation process thereof. The dry quality control product is stable in performance, low-temperature storage or dry ice transportation is not needed, and the storage and transportation cost is reduced. The dry quality control product is packaged by a single person, so that performance reduction caused by repeated freezing and thawing is avoided. The redissolved quality control product can be used for nucleic acid extraction and direct amplification of splitting decomposition of a releasing agent, and the operation is simple and convenient.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a drying protective agent combination and a process method for nucleic acid fragments or inactivated viruses containing nucleic acid fragments and armored virus quality control products. Background Art

[0002] In the field of molecular biology, laboratories must set up quality control products when conducting tests. Common quality control products are divided into negative quality control products and positive quality control products. Negative quality control products can eliminate false positive results caused by reagents or environmental pollution, and positive quality control products can monitor whether the performance of reagents or personnel operations are normal, and are used to eliminate false negative results. Quality control products are important methods and quality indicators for measuring and evaluating the performance of a test kit, such as accuracy and stability, and provide reference standards for laboratory test results to ensure the accuracy of test results.

[0003] There are two main forms of existing nucleic acid quality control products: 1. Liquid quality control products, which generally need to be stored below -20°C. After opening the bottle, the shelf life is short, and repeated freezing and thawing will affect the stability of the nucleic acid sample; 2. Freeze-dried quality control products. After the quality control products are prepared, they are packaged in vials for freeze-drying. Freeze-drying is a drying technology that sublimates the frozen solid ice in the mixture into a gas without melting into liquid water, and finally removes moisture and retains other effective components. After freeze-drying, the plug is pressed. Compared with liquid, freeze-dried powder has better stability and longer shelf life, but the freeze-drying process is complicated. During the freeze-drying process, the refrigeration system and heating system consume a lot of energy, and the equipment cost and operating cost are high. It is necessary to develop suitable freeze-drying protectants and freeze-drying processes, and the development cycle is long. Generally, freeze-dried quality control products are large in volume and need to be immediately packaged and placed below -20°C after reconstitution. The storage time is generally not more than 1 month.

[0004] In order to solve the problems of convenience and poor stability of quality control products during use and reduce the cost of the test kit, adding protective agents to the quality control products can solve the above problems. Summary of the invention

[0005] In view of this, the present invention provides a drying protective agent combination and drying method for quality control products, wherein monosaccharides, polysaccharides and gelatin are used to prepare drying protective agents. The quality control products with added protective agents are dried, and the performance is stable after the treatment, and there is no need for low-temperature storage and transportation, which reduces costs. Performance degradation caused by repeated freezing and thawing is avoided. The reconstituted quality control products can be used for nucleic acid extraction and direct amplification by release agent cleavage, which is easy to operate.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a drying protective agent, which comprises monosaccharide, polysaccharide and gelatin.

[0008] In some specific embodiments of the present invention, the above-mentioned drying protective agent:

[0009] The monosaccharide includes: one of erythrose, arabinose, and glucose;

[0010] The polysaccharide comprises: one of hydroxyethyl starch, dextran and chitosan.

[0011] In some specific embodiments of the present invention, the above-mentioned drying protective agent, in parts by weight: the monosaccharide is 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts;

[0012] The polysaccharide is 1 part, 2 parts, 3 parts, 4 parts or 5 parts;

[0013] The gelatin is 0.2 parts, 0.35 parts, 0.5 parts, 0.75 parts, 1 parts, 2 parts or 3 parts.

[0014] The present invention also provides a nucleic acid protection solution, which comprises the above-mentioned drying protection agent.

[0015] In some specific embodiments of the present invention, the mass volume percentage of the monosaccharide in the nucleic acid protection solution is 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%; and / or

[0016] The mass volume percentage of the polysaccharide is 1%, 2%, 3%, 4% or 5%; and / or

[0017] The mass volume percentage of the gelatin is 0.2%, 0.35%, 0.5%, 0.75%, 1%, 2% or 3%.

[0018] In some specific embodiments of the present invention, the nucleic acid protection solution further comprises at least one of a salt buffer, a preservative, a defoaming agent, and a stabilizer.

[0019] In some specific embodiments of the present invention, the nucleic acid protection solution is:

[0020] The salt buffer comprises at least one of Tris-HCl, HEPES and Tricine; and / or

[0021] The preservative includes NaN 3 ; and / or

[0022] The defoamer comprises SE-15; and / or

[0023] The stabilizer includes BSA.

[0024] In some specific embodiments of the present invention, the nucleic acid protection solution is:

[0025] The salt buffer has a content of 0 mM, 25 mM, 50 mM, 75 mM, 80 mM, 90 mM or 100 mM;

[0026] The mass volume percentage of the preservative is 0.5%, 0.75%, 1%, 2%, 3%, 4% or 5%; and / or

[0027] The mass volume percentage of the defoaming agent is 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2% or 3%;

[0028] The mass volume percentage of the stabilizer is 0.5%, 0.75%, 1%, 2%, 3%, 4% or 5%.

[0029] The present invention also provides a nucleic acid quality control product, comprising the above-mentioned drying protective agent or the above-mentioned nucleic acid protective solution.

[0030] In some specific embodiments of the present invention, the final concentration of the monosaccharide in the above-mentioned nucleic acid quality control product is 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0031] In some specific embodiments of the present invention, the final concentration of the polysaccharide in the above-mentioned nucleic acid quality control product is 1%, 2%, 3%, 4% or 5%.

[0032] In some specific embodiments of the present invention, the final concentration of the gelatin in the above-mentioned nucleic acid quality control product is 0.2%, 0.35%, 0.5%, 0.75%, 1%, 2% or 3%.

[0033] In some specific embodiments of the present invention, the final concentration of the preservative in the nucleic acid quality control product is 0.5%, 0.75%, 1%, 2%, 3%, 4% or 5%; and / or

[0034] The final concentration of the defoaming agent is 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2% or 3%; and / or

[0035] The final concentration of the stabilizer is 1%, 2%, 3%, 4% or 5%.

[0036] In some specific embodiments of the present invention, the final concentration of the above-mentioned drying protective agent, the above-mentioned nucleic acid protection solution, and the above-mentioned buffer of the quality control product is 10 mM, 25 mM, 50 mM, 75 mM, 80 mM, 90 mM or 100 mM.

[0037] The present invention also provides a method for drying a quality control product, which is based on drying with the above-mentioned drying protective agent or the above-mentioned nucleic acid protective solution.

[0038] In some specific embodiments of the present invention, the drying in the above drying method is achieved by electric hot air drying.

[0039] In some specific embodiments of the present invention, the drying method comprises: mixing the nucleic acid and the nucleic acid protection solution, placing the mixture in an electric heated air drying oven, and drying the mixture to obtain a quality control product.

[0040] In some specific embodiments of the present invention, the drying method comprises: placing the nucleic acid quality control product in an electric heated air drying oven, drying, and obtaining the quality control product.

[0041] In some specific embodiments of the present invention, the drying conditions of the above drying method include a drying temperature of 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C, and a drying time of 0.5 h, 1 h, 2.5 h, 3 h, 3.5 h or 5 h.

[0042] In some specific embodiments of the present invention, the nucleic acid quality control product is prepared by the above drying method.

[0043] The drying process of the present invention has the following effects:

[0044] For quality control products, the added drying protective agents include: monosaccharides (3%~10%), polysaccharides (1%~5%), and gelatin (0.2%~3%). Among them, monosaccharides protect the stability of nucleic acid fragments during the drying process, polysaccharides can withstand higher drying temperatures, and gelatin is a good dispersant. Gelatin molecules and water molecules interact with each other to form a stable colloidal reaction system. These protective agents have a certain viscosity and can play a role in fixing nucleic acid fragments. During the drying process, they can maintain a high water holding rate and a low coefficient of variation in water loss, which can better maintain the performance of nucleic acid fragments after drying, and the product has high uniformity. It can also withstand higher drying temperatures. With the addition of this drying protective agent, the quality control product can be stored for 1 year at 2~30℃. DETAILED DESCRIPTION

[0045] The present invention discloses a quality control product drying protective agent combination and a drying method. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] The present invention provides a drying process for nucleic acid quality control products. The drying process mainly includes a drying protective agent combination and a drying curve. At present, the drying process is mainly used in the air drying of reagents. For example, in two patents, CN116200469 A "An air-dried qPCR reagent and its preparation method" of Guangzhou Daan Biotechnology Co., Ltd. and CN116004780 A "PCR reagent drying method and its application" of Zhuhai Lizhu Reagent Co., Ltd., the drying process is used to dry the PCR reagent. The protective agents mainly include trehalose, pullulan, BSA, etc. These protective agents are mainly used to protect the activity of DNA Taq enzyme and reverse transcriptase. The maximum drying temperature is 55°C. The present invention is aimed at the drying of quality control products. In some embodiments, monosaccharides, polysaccharides and gelatin as protective agents are mainly used to maintain the stability of nucleic acids during the drying process, and can tolerate 60~70°C.

[0047] Specific embodiments of the present invention include:

[0048] (1) Quality control products are obtained through a drying process. Quality control products are divided into negative quality control products and positive quality control products. Quality control products are stored in a preservation solution that can provide a stable environment.

[0049] The storage solution mainly includes: 10~100 mM salt buffer (at least one of Tris-HCl, HEPES, Tricine), 0.5%~5% preservative NaN 3 , 0.1%~3% defoamer SE-15, 1%~5% stabilizer.

[0050] (2) The dry quality control product also includes a drying protective agent of 4% to 18%, and the protective agent includes at least one or more combinations of monosaccharides (3% to 10%), polysaccharides (1% to 5%), and gelatin (0.2% to 3%).

[0051] (3) The drying process includes: preparation of quality control products - packaging of quality control products - drying - packaging.

[0052] The percentages (%) involved in the present invention, unless otherwise specified, are percentages by mass volume, i.e. g / 100 mL. For example, 5% means that there are 5 g of solute per 100 mL of liquid.

[0053] The erythrose involved in the present invention is also known as D-(-)-erythrose, and its CAS number is 583-50-6.

[0054] The arabinose involved in the present invention is also known as L-(+)-arabinose, and its CAS number is 5328-37-0.

[0055] The glucose molecular formula of the present invention is C 6 H 12 O 6, CAS number is 50-99-7.

[0056] The CAS number of the hydroxyethyl starch involved in the present invention is 9005-27-0 (Shanghai Yuanye Biotechnology Co., Ltd., V35074).

[0057] The dextran molecular formula of the present invention is (C 6 H 10 O 5 ) n , CAS number is 9004-54-0 (Shanghai Yuanye Biotechnology Co., Ltd., S14099).

[0058] The chitosan molecular formula (C 8 H 13 O 5 ) n , CAS number is 9012-76-4 (Shanghai Yuanye Biotechnology Co., Ltd., S22208).

[0059] The gelatin involved in the present invention has a CAS number of 9000-70-8 (Shanghai Yuanye Biotechnology Co., Ltd., S30952). In some embodiments, the gelatin is derived from the skin of cold-water fish.

[0060] The electric heating blast drying oven involved in the present invention is: an electric heating blast drying oven, manufacturer: Tianjin Shengtongda Experimental Equipment Co., Ltd., model: DL-101-4.

[0061] Unless otherwise specified, the remaining raw materials, reagents, consumables and instruments involved in the present invention are common commercial products and can be purchased from the market.

[0062] The present invention will be further described below in conjunction with the embodiments.

[0063] Example 1: Application of Drying Process for Flu-A / Flu-B / RSV Positive Controls

[0064] (1) Preparation of positive quality control products

[0065] Influenza A virus / influenza B virus / human respiratory syncytial virus (Flu-A / Flu-B / RSV) inactivated virus strains (purchased from Beina Biotechnology, BNCC372983, BNCC373174, BNCC375795) Other instruments, materials, and reagents used, unless otherwise specified, can be obtained from commercial channels.

[0066] Table 1: Dry quality control stock solution without added protective agent

[0067]

[0068] Table 2: Dry quality control stock solution with added protective agent

[0069]

[0070] (2) Drying of quality control products

[0071] Prepare the quality control solution according to the formula in Table 1 and Table 2. After preparation, dispense 30 μL of the quality control solution into the sample tube and place it in an electric heated air drying oven for drying. Table 3 shows the drying curve.

[0072] Table 3: Drying curve

[0073]

[0074] (3) Experimental results

[0075] ①Water loss rate detection

[0076] The water loss rates of the samples without protective agents and with protective agents were tested. The results in Table 4 show that the average water loss rates of the dried quality control products without protective agents and with other polysaccharides added were above 98%, and the water holding rate (1-water loss rate) and water loss coefficient of variation were poor. The water loss rates of the dried quality control products with protective agents were all between 93% and 95%, and the water loss coefficient of variation was small. Among them, the water loss coefficient of variation CV = (standard deviation SD / mean value) × 100%.

[0077] Table 4: Water loss rate test results

[0078]

[0079]

[0080] ②Performance verification

[0081] The dried quality control product was reconstituted with 800 μL, and the reconstituted quality control product was purified by nucleic acid extraction to obtain nucleic acid, and PCR amplification was performed using the Influenza A Virus / Influenza B Virus / Human Respiratory Syncytial Virus Nucleic Acid Detection Kit (National Medical Device Registration No.: 20223401421) of Zhengzhou Antu Biotechnology Co., Ltd. The results are shown in Table 4.

[0082] Table 4: Performance verification results

[0083]

[0084]

[0085] The results showed that the quality control products without added protective agent and the quality control products with added melezitose and raffinose alone were 2 to 4 CT delayed after drying compared with before drying, and the quality control products with added only one protective agent were 1 to 2.5 CT delayed after drying compared with before drying. In the public perception of PCR amplification, for every 1 Ct lower in Ct value, the theoretical concentration of the detection target should be 2 times higher, indicating that the performance of the quality control products without protective agent or with a single protective agent decreased during the drying process. The difference in CT values ​​before and after drying of the quality control products with added three-component protective agent was ≤1, and the performance did not decrease, indicating that the protective agent played a protective role during the drying process.

[0086] ③ Thermal acceleration simulation test of real-time stability of dry quality control products at room temperature

[0087] The dry quality control product with protective agent 4 was placed at 55°C for accelerated stability test to simulate the real-time stability of the dry quality control product at room temperature. According to the Arrhenius formula, 60 days of thermal acceleration at 55°C is approximately equivalent to 12 months of real-time placement at room temperature. The results are shown in Table 5.

[0088] Table 5: Accelerated stability test of influenza A virus / influenza B virus / human respiratory syncytial virus (Flu-A / Flu-B / RSV) dried quality control products at 55°C

[0089]

[0090] The results showed that the performance of the dry quality control product without a protective agent began to decline after being accelerated at 55°C for 15 days; the CT value of the dry quality control product with a protective agent did not delay after being accelerated at 55°C for 60 days, indicating that after adding a protective agent and using this drying process, the quality control product can be stored at room temperature for 12 months.

[0091] Example 2: Application of Drying Process for Mycoplasma Pneumoniae (MP) Positive Control

[0092] (1) Preparation of positive quality control products

[0093] The positive samples of Mycoplasma pneumoniae (MP) were from Hanheng Biotech's armored virus (product name: LV-MP pseudovirus). Other instruments, materials and reagents used were available from commercial channels unless otherwise specified.

[0094] Table 6: Dry quality control products with added protective agent

[0095]

[0096] (2) Drying of quality control products

[0097] Prepare the quality control solution according to the formula in Table 6. After the preparation is completed, use a pipette or automatic injector to dispense 30 μL of the quality control solution into the sample tube, and use an electric heated air drying oven to dry it according to the different drying conditions in Table 7.

[0098] Table 7: Drying curve

[0099]

[0100] (3) Experimental results:

[0101] ①Performance verification

[0102] The dried quality control products were reconstituted with 800 μL, and three reconstituted quality control products and the liquid quality control products before drying were randomly selected for nucleic acid extraction using the nucleic acid extraction or purification reagent (registration number: Yu Zheng Xie Bei 20180037) of Zhengzhou Antu Biotechnology Co., Ltd. The nucleic acid amplification was performed using the Mycoplasma pneumoniae, Chlamydia pneumoniae, and Adenovirus Nucleic Acid Detection Kit (registration number: Guo Xie Zhu Zhu 20223401535) marketed by Zhengzhou Antu Biotechnology Co., Ltd.

[0103] Table 8: Performance verification of dried Mycoplasma pneumoniae quality control products

[0104]

[0105]

[0106] The results are shown in Table 8. Under different drying conditions and different drying protective agent concentrations, the difference in CT value before and after drying is ≤1, indicating that the performance of this drying protective agent is stable under the drying process of temperature 30~65℃ and drying time 0.5~6 h.

[0107] Example 3: Application of Drying Process for Group B Streptococcus (GBS) Positive Control

[0108] (1) Preparation of positive quality control products

[0109] Group B Streptococcus (GBS) DNA standards (nucleic acid fragments) were purchased from Beina Biotechnology Group B Streptococcus DNA Standards (BNCC363486). Other instruments, materials, and reagents used were available from commercial sources unless otherwise specified.

[0110] Table 9: Dry quality control products without adding protective agent

[0111]

[0112] (2) Drying of quality control products

[0113] Prepare the quality control solution according to the formula in Table 9. After the preparation is completed, use a pipette or automatic injector to dispense 80 μL of the quality control solution into the sample tube and place it in an electric air drying oven for drying according to Table 10.

[0114] Table 10: Drying curve of Group B Streptococcus (GBS) positive control

[0115]

[0116] (3) Experimental results

[0117] ①Performance verification

[0118] The dried quality control product was reconstituted with 200 μL sample release agent (purchased from Zhuocheng Huisheng Jingchang Medical Equipment No. 20230013), and 30 μL of the liquid quality control product before drying was mixed with 170 μL of sample release agent (purchased from Zhuocheng Huisheng Jingchang Medical Equipment No. 20230013), and the sample nucleic acid was released. 20 μL nucleic acid was added to the (Group B Streptococcus Nucleic Acid Detection Kit (National Medical Equipment No. 20233401646) for amplification. The results are shown in Table 11.

[0119] Table 11: Performance verification of Group B Streptococcus (GBS) positive dry quality control

[0120]

[0121] The results showed that the difference in CT value before and after drying was ≤1, indicating that the drying process had no effect on the performance.

[0122] In summary, the drying protective agent combination of nucleic acid fragments or inactivated viruses containing nucleic acid fragments and armored virus quality control products provided by the present invention can stabilize the sample performance during the drying process, accelerate the performance to a qualified level, and the single-person drying is simple and convenient to use.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drying protective agent, characterized in that: Including monosaccharides, polysaccharides and gelatin; The monosaccharide includes: one of erythrose, arabinose, and glucose; The polysaccharide comprises: one of hydroxyethyl starch, dextran and chitosan.

2. The drying protective agent according to claim 1, characterized in that: By weight: The monosaccharide is 3 to 10 parts; The polysaccharide is 1 to 5 parts; The gelatin is 0.2 to 3 parts.

3. A nucleic acid protection solution, characterized in that: The desiccant protective agent according to claim 1 or 2; The mass volume percentage of the monosaccharide is 3-10%; The mass volume percentage of the polysaccharide is 1-5%; The mass volume percentage of the gelatin is 0.2-3%.

4. The nucleic acid protection solution according to claim 3, characterized in that: It also includes at least one of a salt buffer, a preservative, a defoaming agent, and a stabilizer.

5. The nucleic acid protection solution according to claim 4, characterized in that: The salt buffer comprises at least one of Tris-HCl, HEPES and Tricine; and / or The preservative and defoamer are NaN3 and SE-15 respectively; and / or The stabilizer is BSA.

6. The nucleic acid protection solution according to claim 5, characterized in that: The content of the salt buffer is 10-100 mM; and / or The mass volume percentage of the preservative is 0.5-5%; and / or The mass volume percentage of the defoamer is 0.1-3%; and / or The mass volume percentage of the stabilizer is 1-5%.

7. A method for drying nucleic acid quality control products, characterized in that: Drying based on the drying protective agent according to claim 1 or 2 or the nucleic acid protection solution according to any one of claims 3 to 6.

8. The drying method according to claim 7, characterized in that The drying is achieved by electric heating air blowing drying.

9. The drying method according to claim 8, characterized in that include: Mixing the quality control product with the nucleic acid protection solution according to any one of claims 3 to 6, and drying the mixture in an electric heated blast drying oven to obtain a quality control product; The drying method comprises: placing the quality control product in a fixed container and placing it in an electric blast drying oven, with the drying temperature being 30-65° C. and the drying time being 0.5-5 h.

10. Nucleic acid quality control product, characterized by: (i) comprising the drying protective agent according to claim 1 or 2 or the nucleic acid protective solution according to any one of claims 3 to 6; or (ii) A nucleic acid quality control product obtained by the drying method according to any one of claims 7 to 9.

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