Drying protectant, drying method, and nucleic acid quality control

Abstract

The present application relates to the field of molecular biology technology, and particularly relates to a dry protective agent, a drying method and nucleic acid quality control products. The present application provides a dry protective agent combination comprising monosaccharide, polysaccharide and gelatin and a preparation process thereof. The dry quality control product of the present application is stable in performance, does not need low-temperature storage or dry ice transportation, and reduces storage and transportation costs. The dry quality control product is packaged in a single serving, avoiding performance degradation caused by repeated freeze-thawing. The reconstituted quality control product can be used for nucleic acid extraction and release agent lysis direct amplification, and the operation is simple.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a combination of desiccant and process for quality control products of nucleic acid fragments or inactivated viruses or armored viruses containing nucleic acid fragments. Background Technology

[0002] In the field of molecular biology, quality control materials are essential for laboratory testing. Commonly used quality control materials are divided into negative and positive controls. Negative controls eliminate false positive results caused by reagent or environmental contamination, while positive controls monitor reagent performance and operator technique to eliminate false negative results. Quality control materials are crucial methods and quality indicators for measuring and evaluating the accuracy and stability of a test kit, providing a reference standard for laboratory test results and ensuring their accuracy.

[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℃. After opening, they have a short shelf life, and repeated freeze-thaw cycles can affect the stability of nucleic acid samples; 2. Lyophilized quality control products, which are prepared, dispensed into vials, and then freeze-dried. Lyophilization is a drying technique that directly sublimates the frozen solid ice in the mixture into a gaseous state without melting it into liquid water, ultimately removing moisture and retaining other effective components. After lyophilization, the vials are sealed with rubber stoppers. Lyophilized powder has better stability and a longer shelf life than liquid, but the lyophilization process is complex. During lyophilization, the refrigeration and heating systems consume a lot of energy, resulting in high equipment and operating costs. It is necessary to develop suitable lyophilization protectants and lyophilization processes, which has a long development cycle. Generally, lyophilized quality control products are large in volume and need to be immediately dispensed and stored below -20℃ after reconstitution. The general storage time is no more than one month.

[0004] To address the issues of convenience, poor stability, and reduced reagent costs associated with quality control products, adding a protective agent to the quality control products can resolve these problems. Summary of the Invention

[0005] In view of this, the present invention provides a desiccant combination and drying method for quality control products, using monosaccharides, polysaccharides, and gelatin in the preparation of the desiccant. The quality control products with added desiccant undergo drying treatment, resulting in stable performance and eliminating the need for low-temperature storage and transportation, thus reducing costs. This avoids performance degradation caused by repeated freeze-thaw cycles. The reconstituted quality control products can be used for nucleic acid extraction and direct amplification via lysis of release agents, simplifying the operation.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides desiccant agents, including monosaccharides, polysaccharides, and gelatin.

[0008] In some specific embodiments of the present invention, the above-mentioned desiccant is:

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

[0010] The polysaccharide includes one of the following: hydroxyethyl starch, dextran, and chitosan.

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

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

[0013] The gelatin is in the form of 0.2 parts, 0.35 parts, 0.5 parts, 0.75 parts, 1 part, 2 parts, or 3 parts.

[0014] The present invention also provides a nucleic acid protection solution comprising the above-mentioned desiccant.

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

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

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

[0018] In some specific embodiments of the present invention, the above-mentioned nucleic acid protection solution further includes at least one of saline buffer, preservative, defoamer, and stabilizer.

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

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

[0021] The preservative includes NaN3; and / or

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

[0023] The stabilizer includes BSA.

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

[0025] The concentration of the saline buffer is 0 mM, 25 mM, 50 mM, 75 mM, 80 mM, 90 mM or 100 mM;

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

[0027] The defoamer has a mass-volume percentage of 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, or 3%.

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

[0029] The present invention also provides nucleic acid quality control products, including the above-mentioned desiccant or the above-mentioned nucleic acid protection 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 above-mentioned nucleic acid quality control product is 0.5%, 0.75%, 1%, 2%, 3%, 4%, or 5%; and / or

[0034] The final concentration of the defoamer 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 buffer solution of the above-mentioned desiccant, the above-mentioned nucleic acid protection solution, and the above-mentioned 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 quality control products, based on the above-mentioned desiccant or the above-mentioned nucleic acid protection solution.

[0038] In some specific embodiments of the present invention, the drying in the above-described drying method is achieved by electric heating and blowing.

[0039] In some specific embodiments of the present invention, the above-mentioned drying method includes: mixing nucleic acid and the above-mentioned nucleic acid protection solution, placing them in an electric heating drying oven, drying them, and obtaining a quality control product.

[0040] In some specific embodiments of the present invention, the above-mentioned drying method includes: placing the nucleic acid quality control sample in an electrically heated drying oven and drying it to obtain the quality control sample.

[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 above-mentioned nucleic acid quality control product is prepared by the above-mentioned drying method.

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

[0044] For quality control products, the added desiccant includes: monosaccharides (3%~10%), polysaccharides (1%~5%), and gelatin (0.2%~3%). 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 to form a stable colloidal reaction system. These desiccant agents have a certain viscosity, which helps to fix the nucleic acid fragments, maintaining a high water retention rate and a low coefficient of variation during drying, thus better preserving the performance of the dried nucleic acid fragments and resulting in high product uniformity. They can also withstand higher drying temperatures. With the addition of this desiccant, the quality control products can be stored at 2~30℃ for one year. Detailed Implementation

[0045] This invention discloses a combination of desiccant and drying method for quality control products. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0046] This invention provides a drying process for nucleic acid quality control products. The drying process mainly includes a combination of drying protectants and a drying curve. Currently, drying processes are mainly used for reagent air drying. For example, two patents, CN116200469 A ("An Air-Dried qPCR Reagent and Its Preparation Method") by Guangzhou Da'an Biotechnology Co., Ltd. and CN116004780 A ("Drying Method and Application of PCR Reagents") by Zhuhai Lizhu Reagent Co., Ltd., utilize drying processes to dry PCR reagents. The protectants in these patents mainly include trehalose, pullulanose, BSA, etc. These protectants are mainly used to protect the activity of DNA Taq enzyme and reverse transcriptase, and the maximum tolerable drying temperature is 55℃. However, this invention is for drying quality control products. In some embodiments, the monosaccharides, polysaccharides, and gelatin used as protectants are mainly used to maintain the stability of nucleic acids during the drying process, and can tolerate 60~70℃.

[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 storage solution, which can provide a stable environment.

[0049] The preservation solution mainly includes: 10~100 mM salt buffer (at least one of Tris-HCl, HEPES, and Tricine), 0.5%~5% preservative NaN3, 0.1%~3% defoamer SE-15, and 1%~5% stabilizer.

[0050] (2) The drying quality control product also includes 4% to 18% of desiccant, which 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 - dispensing of quality control products - drying - packaging.

[0052] Unless otherwise specified, all percentages (%) in this invention are mass-volume percentages, 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 this invention is also known as D-(-)-erythrose, and its CAS number is 583-50-6.

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

[0055] The glucose involved in this invention has the molecular formula C6H. 12 O6, CAS number 50-99-7.

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

[0057] The dextran involved in this invention has the molecular formula (C6H). 10 O5) n The CAS number is 9004-54-0 (Shanghai Yuanye Biotechnology Co., Ltd., S14099).

[0058] The chitosan involved in this invention has the molecular formula (C8H). 13 O5) n The CAS number is 9012-76-4 (Shanghai Yuanye Biotechnology Co., Ltd., S22208).

[0059] The gelatin involved in this invention has CAS number 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 forced-air drying oven involved in this invention is: electric heating forced-air drying oven, manufacturer: Tianjin Shengtongda Experimental Equipment Co., Ltd., model: DL-101-4.

[0061] Unless otherwise specified, all other raw materials, reagents, consumables and instruments involved in this invention are commercially available products and can be purchased from the market.

[0062] The present invention will be further illustrated below with reference to the embodiments.

[0063] Example 1: Application of drying process for positive quality control samples of influenza A virus / influenza B virus / human respiratory syncytial virus (Flu-A / Flu-B / RSV)

[0064] (1) Preparation of positive control samples

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

[0066] Table 1: Stock solution of dried quality control material without added protective agent

[0067]

[0068] Table 2: Stock solutions of dried quality control products with added protective agents

[0069]

[0070] (2) Drying of quality control products

[0071] Prepare the quality control solutions according to the formulas in Tables 1 and 2. After preparation, dispense 30 μL of the quality control solution into sample tubes and place them in an electric heating drying oven for drying. Table 3 shows the drying curves.

[0072] Table 3: Drying Curve

[0073]

[0074] (3) Experimental results

[0075] ①Water loss rate test

[0076] The water loss rate was tested for both the untreated and treated samples. Table 4 shows that the average water loss rate of the dried quality control samples without protective agents and those with added polysaccharides was above 98%, with poor water retention (1 - water loss rate) and coefficient of variation for water loss. The water loss rate of the dried quality control samples with protective agents was between 93% and 95%, with a smaller coefficient of variation for water loss. The coefficient of variation for water loss (CV) is calculated as: CV = (standard deviation SD / average) × 100%.

[0077] Table 4: Results of Water Loss Rate Test

[0078]

[0079]

[0080] ②Performance Verification

[0081] The dried quality control sample was reconstituted with 800 μL of solution. The reconstituted quality control sample was then purified by nucleic acid extraction, 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 Certificate: 20223401421) from 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 samples without protective agents and those with only menotriose or raffinose showed a 2-4 CT delay after drying compared to before drying. The samples with only one protective agent showed a 1-2.5 CT delay after drying compared to before drying. In the common understanding of PCR amplification, for every 1 CT decrease in the Ct value, the theoretical concentration of the target should be twice as high. This indicates that the performance of the sample without a protective agent or with a single protective agent decreased during the drying process. The quality control samples with three protective agents showed a CT value difference of ≤1 before and after drying, and their performance did not decrease, indicating that the protective agents played a protective role during the drying process.

[0086] ③ Accelerated thermal simulation test of the real-time stability of dried quality control products at room temperature during the effective period

[0087] The desiccated quality control sample containing protectant 4 was subjected to accelerated stability testing at 55°C to simulate the real-time stability of the desiccated quality control sample under normal temperature conditions. Based on the Arrhenius equation, 60 days of accelerated thermal stress at 55°C is approximately equivalent to 12 months of normal temperature conditions. The results are shown in Table 5.

[0088] Table 5: Accelerated Stability Test of Dry Control Samples of Influenza A Virus / Influenza B Virus / Human Respiratory Syncytial Virus (Flu-A / Flu-B / RSV) at 55℃

[0089]

[0090] The results showed that the performance of the dried quality control products without a preservative began to decline after 15 days of accelerated drying at 55°C; the CT value of the dried quality control products with a preservative did not lag after 60 days of accelerated drying at 55°C, indicating that with the addition of a preservative, this drying process can achieve 12 months of room temperature storage for the quality control products.

[0091] Example 2: Application of drying process for Mycoplasma pneumoniae (MP) positive quality control samples

[0092] (1) Preparation of positive control samples

[0093] The positive samples of Mycoplasma pneumoniae (MP) were obtained from Hanheng Bio-armored virus (product name: LV-MP pseudovirus). Other instruments, materials and reagents used can be obtained from commercial sources unless otherwise specified.

[0094] Table 6: Drying control products with added protective agents

[0095]

[0096] (2) Drying of quality control products

[0097] Prepare the quality control solution according to the formula in Table 6. After preparation, use a pipette or automatic dispensing device to dispense 30 μL of the quality control solution into sample tubes and dry them in an electric heating drying oven 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 samples were reconstituted with 800 μL of solution. Three reconstituted quality control samples and their undried liquid quality control samples were randomly selected and retained for nucleic acid extraction using nucleic acid extraction or purification reagents from Zhengzhou Antu Biotechnology Co., Ltd. (Registration No.: Yu Zheng Xie Bei 20180037). Nucleic acid amplification was performed using the Mycoplasma pneumoniae, Chlamydia pneumoniae, and Adenovirus Nucleic Acid Detection Kits (Registration No.: Guo Xie Zhu Zhun 20223401535) marketed by Zhengzhou Antu Biotechnology Co., Ltd.

[0103] Table 8: Performance Verification of Mycoplasma pneumoniae Dry Quality Control Products

[0104]

[0105]

[0106] The results are shown in Table 8. Under different drying conditions and with different concentrations of desiccant, the difference in CT values ​​before and after drying is ≤1, indicating that this desiccant has stable performance under drying processes with temperatures of 30~65℃ and drying times of 0.5~6 h.

[0107] Example 3: Application of drying process for Group B Streptococcus (GBS) positive control samples

[0108] (1) Preparation of positive control samples

[0109] Group B Streptococcus (GBS) DNA standard (nucleic acid fragment) was purchased from Beina Biotechnology Group B Streptococcus DNA Standard (BNCC363486). Other instruments, materials and reagents used can be obtained commercially unless otherwise specified.

[0110] Table 9: Drying control samples with added protective agents

[0111]

[0112] (2) Drying of quality control products

[0113] Prepare the quality control solution according to the formula in Table 9. After preparation, use a pipette or automatic dispensing device to dispense 80 μL of the quality control solution into sample tubes and place them in an electric heating drying oven to dry according to Table 10.

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

[0115]

[0116] (3) Experimental results

[0117] ①Performance Verification

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

[0119] Table 11: Performance Validation of Group B Streptococcus (GBS) Positive Dry Control Samples

[0120]

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

[0122] In summary, the desiccant combination of nucleic acid fragments or inactivated viruses and armored viruses containing nucleic acid fragments provided by this invention can stabilize sample performance and accelerate performance to meet standards during the drying process, and is simple and convenient to use for single-person drying.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for drying nucleic acid quality control articles, characterized in that, Drying based on a dry protection agent or a nucleic acid protection solution; The dry protection agent comprises monosaccharide, polysaccharide and gelatin; The monosaccharide comprises one of erythrose, arabinose and glucose; The polysaccharide comprises one of hydroxyethyl starch, dextran and chitosan; The nucleic acid protection solution comprises the dry protection agent, wherein the mass-volume percentage of the monosaccharide is 3-10%, the mass-volume percentage of the polysaccharide is 1-5%, and the mass-volume percentage of the gelatin is 0.2-3%. The drying condition of the drying method comprises that the drying temperature is 30-65℃, and the drying time is 0.5-5 h.

2. The drying method according to claim 1, characterized in that, In the dry protection agent, by weight: The monosaccharide is 3-10 parts; The polysaccharide is 1-5 parts; The gelatin is 0.2-3 parts.

3. The drying method according to claim 1, wherein The nucleic acid protection solution further comprises at least one of a salt buffer, a preservative, an antifoaming agent and a stabilizer.

4. The drying method according to claim 3, characterized in that, Comprises: The salt buffer comprises at least one of Tris-HCl, HEPES and Tricine; and / or The preservative and the antifoaming agent are respectively NaN3 and SE-15; and / or The stabilizer is BSA.

5. The drying method according to claim 4, wherein Comprises: 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 antifoaming agent is 0.1-3%; and / or The mass-volume percentage of the stabilizer is 1-5%.

6. The drying method according to claim 1, wherein The drying is achieved by electrothermal air drying.

7. The drying method according to claim 6, characterized in that, Comprises: Mixing the quality control product and the nucleic acid protection solution, and placing them in an electrothermal air drying oven to dry, thereby obtaining a quality control product; The drying method comprises: placing the quality control product in a fixed container into an electrothermal air drying oven, and the drying temperature is 30-65℃, and the drying time is 0.5-5 h.

8. A nucleic acid quality control characterized in that, The nucleic acid quality control product prepared by the drying method of any one of claims 1-7; The dry protection agent comprises monosaccharide, polysaccharide and gelatin; The monosaccharide comprises one of erythrose, arabinose and glucose; The polysaccharide comprises one of hydroxyethyl starch, dextran and chitosan; The nucleic acid protection solution comprises the dry protection agent, wherein the mass-volume percentage of the monosaccharide is 3-10%, the mass-volume percentage of the polysaccharide is 1-5%, and the mass-volume percentageof the gelatin is 0.2-3%.

9. The nucleic acid quality control of claim 8, wherein In the dry protection agent, by weight: The monosaccharide is 3- 10 parts; The polysaccharide is 1-5 parts; The gelatin is0.2-3 parts.

10. The nucleic acid quality control of claim 8, wherein The nucleic acid protection solution further comprises at least one of the salt buffer, the preservative, the antifoaming agent and the stabilizer.

11. The nucleic acid quality control of claim 10, wherein Comprises: The salt buffer comprises at least one of Tris-HCl,HEPES and Tricine; and / or The preservative and the antifoaming agent are respectivelyNaN3 and SE-15; and / or The stabilizer is BSA.

12. The nucleic acid quality control of claim 11, wherein Comprises: the content of the salt buffer is 10-100 mM; and / or The mass-volume percentageof the preservative is 0.5-5%; and / or The mass-volume percentage ofthe antifoaming agent is 0.1-3%; and / or The mass-volume percentageof the stabilizer is 1-5%.

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