A fully premixed nucleic acid detection freeze-dried reagent and a preparation method thereof

Abstract

The present application relates to a kind of full premix nucleic acid detection freeze-dried reagent and its preparation method, the freeze-dried reagent includes: (1) freeze-dried protection system: trehalose, final concentration is 0.1 M~0.5 M;Mannitol, final concentration is 2.0%~6.0%;Polyethylene glycol 20000, final concentration is 1.0%~5.0%;Defoaming agent SE-15, final concentration is 0.02%~0.15%;(2) effervescent disintegrating system: citric acid, final concentration is 1 mM~5 mM;Sodium bicarbonate, final concentration is 2 mM~8 mM;(3) detection reaction system: specific primer and probe, final concentration is independently 0.1 μM~1.0 μM respectively;qPCR reaction enzyme premix liquid, including DNA polymerase, reverse transcriptase, dNTPs, Mg²⁺ And buffer component.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a fully premixed lyophilized nucleic acid detection reagent that can be stored at room temperature. In particular, it relates to a method for preparing a lyophilized nucleic acid detection reagent that achieves rapid reconstitution based on the principle of effervescent disintegration and its multidimensional standardized quality evaluation system. Background Technology

[0002] Nucleic acid testing, as a core technology in the field of in vitro molecular diagnostics, has been widely applied in various scenarios such as infectious disease control, early cancer screening, and gene testing. Traditional nucleic acid testing reagents are mostly liquid reagents, which contain bioactive components such as DNA polymerase and reverse transcriptase, exhibiting extremely poor stability at room temperature. They require low-temperature (typically -20°C) cold chain transportation and storage. This not only significantly increases the application cost of the reagents but also severely limits their widespread use in remote areas without cold chain facilities, failing to meet the needs of rapid on-site testing.

[0003] To address the preservation challenges of liquid reagents, related research has gradually developed lyophilized nucleic acid detection reagents. These reagents remove moisture through freeze-drying, allowing for storage at room temperature and significantly reducing the requirements for storage and transportation conditions. For example, patents have disclosed lyophilized qPCR reagents for triple detection of influenza A, influenza B, and the novel coronavirus, which are prepared by freeze-drying a liquid premix containing enzymes, primers, probes, dNTPs, Mg²⁺, lyophilization protectants, and additives. Other studies have disclosed lyophilization protectants and methods for qPCR premixes, using cellobiose and BSA as lyophilization protectants.

[0004] However, existing lyophilized nucleic acid reagents still have many shortcomings: First, slow reconstitution speed. Most existing lyophilized reagents are prepared using conventional lyophilization processes, resulting in dense lyophilized products that are prone to clumping and slow dissolution during reconstitution, often requiring prolonged vortexing for complete dissolution. Some lyophilized formulations have reconstitution times as long as 56 seconds or even several minutes, leading to long detection wait times and failing to meet the timeliness requirements of point-of-care testing (POCT). Second, insufficient premixing and cumbersome operation. Some lyophilized reagents require the lyophilized matrix to be dissolved separately before use, followed by mixing of each component according to the specified ratio, a cumbersome process that easily introduces contamination. Even premixed lyophilized reagents often suffer from enzyme activity loss and decreased amplification efficiency after reconstitution due to unreasonable lyophilization protectant formulations or improper lyophilization processes. Third, a lack of standardized quality evaluation systems. Currently, quality evaluation of lyophilized nucleic acid reagents mostly focuses on the detection performance after reconstitution (such as sensitivity and specificity), lacking a standardized evaluation system that covers multiple dimensions such as lyophilization process, reconstitution characteristics, storage stability, and detection sensitivity. This makes it difficult to comprehensively measure the product quality of lyophilized nucleic acid reagents, which is detrimental to the industrial production and quality control of such reagents. Fourth, the stability at room temperature needs to be improved. Although some lyophilized reagents can be stored at room temperature, the shelf life is usually short, or the enzyme activity decreases significantly during room temperature storage, failing to meet the requirements for long-term stability in practical applications.

[0005] Therefore, there is an urgent need in this field for a fully premixed lyophilized reagent for nucleic acid detection that can achieve long-term preservation at room temperature, rapid reconstitution in seconds, simple operation, and a standardized quality evaluation system. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a fully premixed lyophilized nucleic acid detection reagent that can be stored at room temperature, reconstitutes quickly, and has stable performance, along with its preparation method. It also establishes a multi-dimensional standardized quality evaluation system to solve common industry problems associated with existing nucleic acid detection reagents, such as cold chain dependence, cumbersome operation, slow reconstitution, and poor stability.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] One of the objectives of this invention is to provide a fully premixed nucleic acid detection lyophilized reagent, which consists of three parts: a lyophilization protection system, an effervescent disintegration system, and a detection reaction system.

[0009] (1) Freeze-drying protection system

[0010] The freeze-drying protection system consists of the following components:

[0011] Trehalose, with a final concentration of 0.1 M to 0.5 M, preferably 0.3 M, is used as a vitrification protectant to maintain the natural structure of biological macromolecules.

[0012] Mannitol, with a final concentration of 2.0% to 6.0%, preferably 4.0%, is used as a skeletal support agent to prevent the product from collapsing during freeze-drying.

[0013] PEG20000 (polyethylene glycol 20000), with a final concentration of 1.0% to 5.0%, preferably 3.0%; is used as a protein protectant to reduce the activity loss of bioactive components such as enzymes during the freeze-drying process;

[0014] Defoamer SE-15, with a final concentration of 0.02% to 0.15%, preferably 0.08%, is used to reduce the surface tension of the system and inhibit the generation and rupture of bubbles during ice crystal sublimation.

[0015] Defoamer SE-15: A silicone resin product used to prevent or reduce foaming in reaction systems, minimizing damage. Product model: Sigma-Aldrich A8582.

[0016] (2) Effervescent disintegration system

[0017] Effervescent disintegration systems consist of the following components:

[0018] Citric acid, with a final concentration of 1 mM to 5 mM, preferably 2 mM, is used as the acidic component.

[0019] Sodium bicarbonate, with a final concentration of 2 mM to 8 mM, preferably 4 mM, is used as an alkaline component.

[0020] The molar ratio of citric acid to sodium bicarbonate is 1:1 to 1:3, preferably 1:2. When the lyophilized reagent is reconstituted, the citric acid and sodium bicarbonate react with water to produce carbon dioxide gas, achieving rapid disintegration and dissolution of the reagent.

[0021] (3) Detection of reaction system

[0022] The detection reaction system includes:

[0023] Specific primers and probes, each with a final concentration of 0.1 μM to 1.0 μM, are used for the specific recognition and amplification of target nucleic acids.

[0024] The qPCR reaction enzyme premix contains DNA polymerase, reverse transcriptase, dNTPs, Mg²⁺, and buffer components.

[0025] Specifically, the fully premixed nucleic acid detection lyophilized reagent system 1 consists of the following components:

[0026] Freeze-drying protection system Trehalose 0.1 M~0.5 M Freeze-drying protection system Mannitol 2.0%～6.0% Freeze-drying protection system PEG20000 1.0%～5.0% Freeze-drying protection system Defoamer SE-15 0.02%～0.15% Effervescent disintegration system Citric acid 1 mM~5 mM Detection reaction system Primers / Probes 0.1 - 1.0 μM Detection reaction system qPCR reaction enzyme premix Appropriate amount

[0027] Freeze-drying system 2 consists of the following components:

[0028] Effervescent disintegration system Sodium bicarbonate 2 mM~10 mM

[0029] Preferably, the fully premixed nucleic acid detection lyophilized reagent system 1 consists of the following components:

[0030] Freeze-drying protection system Trehalose 0.3M Freeze-drying protection system Mannitol 4.0% Freeze-drying protection system PEG20000 3.0% Freeze-drying protection system Defoamer SE-15 0.08% Effervescent disintegration system Citric acid 2mM Detection reaction system Primers / Probes 0.1 - 1.0 μM Detection reaction system qPCR reaction enzyme premix Appropriate amount

[0031] Preferably, the freeze-drying system 2 consists of the following components:

[0032] Effervescent disintegration system Sodium bicarbonate 4mM

[0033] Another object of the present invention is to provide a method for preparing a fully premixed nucleic acid detection lyophilized reagent, comprising the following steps:

[0034] (1) Preparation of premix: Mix the components of the freeze-drying protection system (trehalose, mannitol, PEG20000, SE-15), the components of the effervescent disintegration system (citric acid), and the components of the detection reaction system (specific primers, probes, qPCR reaction enzyme premix) in sequence to prepare a homogeneous freeze-drying stock solution 1. Sodium bicarbonate, a component of the effervescent disintegration system, is prepared separately as freeze-drying stock solution 2.

[0035] (2) Liquid nitrogen pre-freezing: The freeze-dried stock solution is added dropwise at a distance of 1 mm to 10 mm from the liquid nitrogen surface, so that the droplets are rapidly frozen into spherical microspheres;

[0036] (3) Vacuum freeze drying: The pre-frozen microspheres are transferred to a freeze dryer for three-stage drying;

[0037] (4) Dispensing and storage: Dispense and seal the freeze-dried microspheres in a dry environment and store them at room temperature.

[0038] The specific preparation method includes the following steps:

[0039] Step 1: Preparation of premixed solution

[0040] According to the above formula, the components of the freeze-drying protection system (trehalose, mannitol, PEG20000, SE-15), the components of the effervescent disintegration system (citric acid), and the components of the detection reaction system (specific primers, probes, qPCR reaction enzyme premix) are thoroughly mixed in sequence to prepare a homogeneous freeze-drying stock solution 1. The effervescent disintegration system component sodium bicarbonate is prepared separately as freeze-drying stock solution 2.

[0041] Step 2: Pre-freezing with liquid nitrogen

[0042] The freeze-dried product is shaped using a pipette or an automated microdroplet generation device. When using a pipette manually, the freeze-drying stock solution 1 is added dropwise from a distance of 1 mm to 10 mm (preferably 5 mm) above the liquid nitrogen surface, allowing the droplet to rapidly freeze into spherical microspheres approximately 1 mm to 3 mm (preferably 2 mm) in diameter. Pre-freezing is completed once the microspheres sink to the bottom of the liquid nitrogen. The freeze-drying stock solution 2 is then directly added to an eight-tube pre-freezing process.

[0043] Step 3: Vacuum freeze drying

[0044] The pre-frozen microspheres and the eight-tube pack containing the lyophilized stock solution 2 were transferred to a freeze dryer and dried using a three-stage drying process:

[0045] First stage (deep freezing): Temperature -30℃~-10℃, preferably -20℃, maintained for 1~4 hours, preferably 2 hours, to complete the deep freezing of the sample;

[0046] Second stage (main sublimation): Temperature -50℃~-30℃, preferably -40℃, maintained for 8~16 hours, preferably 12 hours, to complete the main sublimation drying and remove more than 90% of free water;

[0047] The third stage (desorption and drying): The temperature is 20℃~30℃, preferably 25℃, and maintained for 2~5 hours, preferably 3 hours, to complete the desorption and drying, reducing the moisture content to below 2%, preferably to about 1.5%.

[0048] Step 4: Dispense and store

[0049] The freeze-dried microspheres were transferred to an eight-tube array containing freeze-dried sodium bicarbonate under a dry environment, sealed, and then packaged in an aluminum foil bag and stored at room temperature.

[0050] Another objective of this invention is to provide a multi-dimensional standardized quality evaluation method for fully premixed nucleic acid detection lyophilized reagents, including three dimensions: physical stability evaluation, biological activity evaluation, and thermal stability evaluation.

[0051] Physical stability Appearance It is spherical, does not collapse, and is smooth and full. Smooth sphere, without collapse Physical stability Transfer performance It does not stick to consumables and can be picked up and transferred with tweezers. Easy to transfer, no adhesion Physical stability Reconstitution time <30 seconds, the shorter the better ≤15s Physical stability Moisture content ＜3% 1.5% (mean) Bioactivity Detection sensitivity 500 copies / mL 500 copies / mL Bioactivity Storage time at 25℃ ΔCt≤1 RNA (6 months) thermal stability Storage time at 37℃ ΔCt≤1 RNA (60 days) thermal stability Storage time at 56℃ ΔCt≤1 RNA-based products take 3 days.

[0052] Another objective of this invention is to provide the application of fully premixed nucleic acid detection lyophilized reagents in the preparation of pathogen detection reagents, public health monitoring reagents, or clinical diagnostic reagents.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] 1. Breaking free from cold chain dependence and significantly reducing costs. The lyophilized reagent of this invention can be stably stored at room temperature (25°C) for more than 6 months, requiring no cold chain transportation or storage. The overall cost of use is more than 20% lower than that of traditional liquid reagents, making it particularly suitable for nucleic acid testing in remote areas without cold chain conditions.

[0055] 2. Instant reconstitution for true point-of-care testing. This invention innovatively introduces an effervescent disintegration system into the lyophilized reagent for nucleic acid detection. Utilizing the disintegration principle of carbon dioxide gas generated by the reaction of citric acid and sodium bicarbonate with water, the lyophilized microspheres rapidly disintegrate and disperse upon reconstitution, with a reconstitution time of ≤15 seconds, which is 2 to 4 times faster than existing lyophilized reagents. This truly meets the "add and test" point-of-care testing (POCT) requirement.

[0056] 3. Fully premixed system, extremely simple operation. The reagents of this invention are a fully premixed system. All detection components (including enzymes, primers, probes, dNTPs, lyophilization protectants, and effervescent disintegrants) are premixed and lyophilized into microspheres. No pre-treatment preparation is required. The amplification reaction can be carried out directly after adding the sample. The operation time is reduced by more than 80%, the professional requirements of the operators are greatly reduced, and the risk of contamination introduced during the operation is effectively avoided.

[0057] 4. Stable and reliable performance. This invention maximizes the protection of the activity of enzymes and other bioactive components during freeze-drying and storage by optimizing the combination of freeze-drying protectants (trehalose + mannitol + PEG20000 + SE-15) and a three-stage vacuum freeze-drying process. Verification has shown that the freeze-dried reagent of this invention is completely consistent with the liquid fresh reagent in terms of detection sensitivity and specificity. After 6 months of storage at room temperature, the change in Ct value ΔCt ≤ 1, with no statistically significant difference (P > 0.05).

[0058] 5. Pioneering a multi-dimensional standardized evaluation system. This invention establishes for the first time a standardized quality evaluation system for lyophilized nucleic acid detection reagents, encompassing three dimensions and multiple indicators: physical stability (appearance, transfer performance, reconstitution time, moisture content), biological activity (detection sensitivity, room temperature storage stability), and thermal stability (storage stability at 37℃ and 56℃). This fills a gap in domestic quality evaluation standards for lyophilized detection reagents and can be directly used to guide product development and quality control in the industry.

[0059] 6. High versatility and broad application prospects. The core formula and process of this invention have been successfully verified in nucleic acid detection reagents for four types of pathogens: influenza A virus (IAV), novel coronavirus (COV), Pseudomonas aeruginosa (PA), and Streptococcus pneumoniae (SP). It can be easily extended to other nucleic acid detection reagent categories simply by adjusting the primer and probe sequences. This invention can be widely applied in multiple fields such as pathogen detection, public health monitoring, clinical diagnosis, food safety testing, and environmental monitoring.

[0060] The following explanations are provided for the English words or terms appearing in the instruction manual:

[0061] qPCR (Quantitative Real-time PCR)

[0062] Point-of-Care Testing (POCT)

[0063] Ct value cycle threshold

[0064] Difference in the value of ΔCt

[0065] IAV (Influenza A virus)

[0066] COVID-19 (Coronavirus, usually referring to SARS-CoV-2)

[0067] PA Pseudomonas aeruginosa

[0068] SP Streptococcus pneumoniae

[0069] dNTPs are deoxyribonucleoside triphosphates.

[0070] PEG20000 (Polyethylene glycol 20000)

[0071] Defoamer SE-15 (SE-15 / Sigma-Aldrich)

[0072] One Step U + Mix one-step qPCR premix (containing dNTPs, buffer, etc.) / Novizan QL227

[0073] One Step U + Enzyme Mix (Glycerol-free) One-step qPCR enzyme mixture (glycerol-free) / Novizan QL227

[0074] RNA ribonucleic acid

[0075] DNA deoxyribonucleic acid

[0076] Karl Fischer method. Attached Figure Description

[0077] Figure 1 IAV and COV fully premixed lyophilized reagents

[0078] Figure 2 PA, SP fully premixed lyophilized reagents

[0079] Figure 3 Optimization of freeze-drying protectant concentration Detailed Implementation

[0080] The present invention will be further illustrated by the following examples.

[0081] The product and its preparation method according to the present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials can be obtained from publicly available commercial sources.

[0082] Example 1: Preparation and performance verification of fully premixed lyophilized reagent for influenza A virus (IAV).

[0083] 1. Preparation method of lyophilized stock solution 1 (single serving, total volume 37.7 μL)

[0084]

[0085] Preparation method of lyophilized stock solution 2 (single serving, total volume 3.2μL)

[0086]

[0087] In the above formulation, the concentration of mannitol and PEG20000 in "%" is the mass-volume ratio (w / v), and the concentration of defoamer SE-15 in "%" is the volume ratio (v / v).

[0088] Add the above components in sequence to a centrifuge tube without nuclease, mix thoroughly to obtain a homogeneous lyophilized stock solution.

[0089] 2. Liquid nitrogen pre-freezing

[0090] Using a pipette, the above-mentioned lyophilized stock solution 1 was added dropwise from a height of approximately 5 mm above the liquid nitrogen surface, with each drop containing approximately 25–50 μL (preferably 38 μL). The droplets were rapidly frozen into spherical microspheres with a diameter of approximately 2 mm. After the microspheres had completely sunk to the bottom of the liquid nitrogen, they were retrieved using sterile forceps or a sieve and transferred to a pre-cooled lyophilization container. The lyophilized stock solution 2 was then directly added to the eight-tube pre-freezing process.

[0091] 3. Vacuum freeze drying

[0092] The freeze-drying container containing the pre-frozen microspheres and the eight-tube container containing freeze-drying stock solution 2 were transferred to the freeze dryer, and a three-stage drying process was adopted:

[0093] Phase 1: Temperature -20℃, maintained for 2 hours;

[0094] Second stage: Temperature -40℃, maintained for 12 hours, vacuum degree ≤10 Pa;

[0095] Third stage: Temperature 25℃, maintained for 3 hours, vacuum degree ≤5 Pa.

[0096] After freeze-drying, the freeze-dried microspheres were transferred to an eight-tube array containing freeze-dried sodium bicarbonate under a dry environment, sealed, and then packaged in an aluminum foil bag and stored at room temperature.

[0097] 4. Performance Verification

[0098] The performance of the prepared IAV fully premixed lyophilized reagent was verified according to the multi-dimensional standardized quality evaluation method of the present invention:

[0099] Appearance: It is a smooth sphere, without any collapse, and has a full shape;

[0100] Transfer performance: It does not stick to consumables and can be easily picked up and transferred with tweezers;

[0101] Reconstitution time: ≤12 seconds;

[0102] Moisture content: 1.24% (determined by Karl Fischer method);

[0103] Detection sensitivity: 500 copies / mL;

[0104] Stability at 25℃: After storage at 25℃ for 6 months, the Ct value of 10³ copies / μL of IAV positive plasmid was found to be 0.68 different from that of freshly prepared control reagent (ΔCt≤1), which was not statistically significant (P>0.05).

[0105] Stability at 37℃: After 60 days of storage at 37℃, ΔCt≤1.

[0106] Stability at 56℃: After storage at 56℃ for 3 days, ΔCt≤1.

[0107] Primer / Probe Name Base Composition

[0108] IAV-F 5'-GACCRATCCTGTCACCTCTGA C-3'

[0109] IAV-R 5'-GGGCATTYTGGACAAAKCGTCTACG-3'

[0110] IAV-P 5'-FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1-3'

[0111] The primer and probe sequences are derived from the "National Influenza Surveillance Technical Guidelines (2017 Edition)".

[0112] Example 2: Preparation and performance verification of a fully premixed lyophilized reagent for novel coronavirus (COV)

[0113] 1. Preparation method of lyophilized stock solution 1 (single serving, total volume 37.22 μL)

[0114]

[0115] Preparation method of lyophilized stock solution 2 (single serving, total volume 3.2μL)

[0116]

[0117] In the above formulation, the concentration of mannitol and PEG20000 in "%" is the mass-volume ratio (w / v), and the concentration of defoamer SE-15 in "%" is the volume ratio (v / v).

[0118] Add the above components in sequence to a centrifuge tube without nuclease, mix thoroughly to obtain a homogeneous lyophilized stock solution.

[0119] 2-3. Liquid nitrogen pre-freezing and vacuum freeze-drying

[0120] Same as steps 2 and 3 in Example 1.

[0121] 4. Performance Verification

[0122] The performance of the prepared COV fully premixed lyophilized reagent was verified according to the multi-dimensional standardized quality evaluation method of the present invention:

[0123] Appearance: It is a smooth sphere, without any collapse, and has a full shape;

[0124] Transfer performance: It does not stick to consumables and can be easily picked up and transferred with tweezers;

[0125] Reconstitution time: ≤14 seconds;

[0126] Moisture content: 1.41% (determined by Karl Fischer method);

[0127] Detection sensitivity: 500 copies / mL;

[0128] Stability at 25℃: After 6 months of storage at 25℃, ΔCt≤1;

[0129] Stability at 37℃: After storage at 37℃ for 60 days, the Ct value of 10³ copies / μL of COV positive plasmid was found to be 0.72 different from that of the freshly prepared control reagent (ΔCt≤1), which was not statistically significant (P>0.05).

[0130] Stability at 56℃: After storage at 56℃ for 3 days, ΔCt≤1.

[0131] Detection target: ORF1ab:

[0132] COV-F: 5'-CCCTGTGGGTTTTACACTTAA-3'

[0133] COV-R: 5'-ACGATTGTGCATCAGCTGA-3'

[0134] COV-P: 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3'

[0136] Example 3: Preparation of a fully premixed lyophilized reagent for Pseudomonas aeruginosa

[0137] 1. Preparation of premixed solution

[0138] Following the formulation of Example 1, the IAV-specific primers and probes were replaced with Pseudomonas aeruginosa-specific primers and probes (final concentration 0.1–1.0 μM), while the remaining components and amounts remained unchanged, and a lyophilized stock solution was prepared.

[0139] 2-3. Liquid nitrogen pre-freezing and vacuum freeze-drying

[0140] Same as steps 2 and 3 in Example 1.

[0141] 4. Performance Verification

[0142] Performance verification was performed using the multi-dimensional standardized quality evaluation method of the present invention, and the results were consistent with those of Examples 1 and 2, indicating that the core formula and process of the present invention have good versatility.

[0143] Detection target: Pseudomonas aeruginosa outer membrane lipoprotein gene (oprI)

[0144] PA-F: 5'-CTCCAAAGAAACCGAAGCTCGTC-3'

[0145] PA-R: 5'-CACGCTCGTAGCCTCGTC-3'

[0146] PA-P: 5'-FAM-CCAGAGCTTCGTCAGCCTTGCGAT-BHQ1-3'

[0147] Primer and probe sequences were obtained from GenBank Accession NC_002516.2 and designed using Oligo7 software.

[0148] Example 4: Preparation of a fully premixed lyophilized reagent for Streptococcus pneumoniae

[0149] 1. Preparation of premixed solution

[0150] Following the formulation of Example 1, the IAV-specific primers and probes were replaced with Streptococcus pneumoniae-specific primers and probes (final concentration 0.1–1.0 μM), while the remaining components and amounts remained unchanged, and a lyophilized stock solution was prepared.

[0151] 2-3. Liquid nitrogen pre-freezing and vacuum freeze-drying

[0152] Same as steps 2 and 3 in Example 1.

[0153] 4. Performance Verification

[0154] Performance verification was performed using the multi-dimensional standardized quality evaluation method of the present invention, and the results were consistent with those of Examples 1 and 2, further verifying the universality of the core formula and process of the present invention.

[0155] Detection target: N-acetylmuramoyl-L-alanine amidase (LytA) gene

[0156] SP-F: 5'-ACGCAATCTAGCAGATGAAGC-3'

[0157] SP-R: 5'-TGTTTGGTTGGTTATTCGTGC-3'

[0158] SP-P: 5'-FAM-TTTGCCGAAAACGCTTGATACAGGG-BHQ1-3'

[0159] Primer and probe sequences were obtained from GenBank Accession CP137110.1 and designed using Oligo7 software.

[0160] Example 5: Screening and optimization experiment of lyophilization protectant formulation

[0161] 1. Screening of lyophilization protectant concentration:

[0162] Trehalose final concentration settings: 0.1M, 0.2M, 0.3M, 0.5M.

[0163] Final mannitol concentration settings: 2%, 4%, 6%, 8%.

[0164] Final PEG20000 concentration settings: 1%, 3%, 5%, 7%.

[0165] SE-15 final concentration settings: 0.02%, 0.05%, 0.08%, 0.1%.

[0166] Preliminary screening results of lyophilization protectants showed that when the final concentration of trehalose reached 0.3M, the fluorescence signal intensity of the qPCR amplification curve reached its highest level, while the Ct value was the lowest, indicating optimal amplification efficiency under this condition. When the final concentration of mannitol was 4%, the fluorescence intensity of the qPCR amplification curve was the highest, and the Ct value was the lowest. The optimal final concentration of PEG20000 was 3%, under which condition the fluorescence intensity was the highest and the Ct value was the lowest. When the concentration of each additive exceeded the above optimal values, the fluorescence intensity of the qPCR amplification curve decreased, while the Ct value increased, reflecting the inhibitory effect of excessive addition on the amplification reaction. When the final concentration of SE-15 was 0.08%, the fluorescence intensity of the qPCR amplification curve reached its peak, and the Ct value was the lowest, indicating the best amplification effect. Figure 3 .

[0167] 2. Optimization of lyophilization protectant formulation

[0168] To verify the superiority of the freeze-drying protectant combination of the present invention (0.3M trehalose + 4% mannitol + 3% PEG20000 + 0.08% SE-15), the following comparative group was set up:

[0169] Control group 1: Trehalose concentration was 0.1M, and all other components were the same;

[0170] Control group 2: Mannitol concentration was 2%, and all other components were the same;

[0171] Control group 3: PEG20000 concentration was 1.5%, and the other components were the same;

[0172] Control group 4: Contains no SE-15, all other components are the same;

[0173] The composition of this invention is: 0.3M trehalose + 4% mannitol + 3% PEG20000 + 0.08% SE-15.

[0174] Experimental results:

[0175]

[0176] The above description is merely a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the described embodiments. Various changes, modifications, substitutions, combinations, and simplifications can be made without departing from the principle of the present invention, and all such substitutions should be considered equivalent replacements and should also be considered within the scope of protection of the present invention.

Claims

1. A fully premixed lyophilized reagent for nucleic acid detection, characterized in that, The lyophilized reagent consists of a lyophilization protection system, an effervescent disintegration system, and a detection reaction system. The freeze-drying protection system comprises: trehalose 0.1 M to 0.5 M, mannitol 2.0% to 6.0%, PEG20000 1.0% to 5.0%, and defoamer SE-15 0.02% to 0.15%; The effervescent disintegration system comprises: 1 mM to 5 mM citric acid and 2 mM to 8 mM sodium bicarbonate, wherein the molar ratio of citric acid to sodium bicarbonate is 1:1 to 1:3; The detection reaction system includes: specific primers and probes at 0.1 μM to 1.0 μM, and qPCR reaction enzyme premix.

2. The fully premixed nucleic acid detection lyophilized reagent according to claim 1, characterized in that, The final concentration of the trehalose is 0.3 M, the final concentration of the mannitol is 4.0%, the final concentration of the PEG20000 is 3.0%, and the final concentration of the defoamer SE-15 is 0.08%.

3. The fully premixed nucleic acid detection lyophilized reagent according to claim 1, characterized in that, The final concentration of citric acid is 2 mM, the final concentration of sodium bicarbonate is 4 mM, and the molar ratio of citric acid to sodium bicarbonate is 1:

2.

4. The fully premixed nucleic acid detection lyophilized reagent according to claim 1, characterized in that, Composed of the following components composition: 。 5. The fully premixed nucleic acid detection lyophilized reagent according to claim 1, characterized in that, The lyophilized reagent is a spherical microsphere with a diameter of 1 mm to 3 mm.

6. A method for preparing a fully premixed nucleic acid detection lyophilized reagent as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Preparation of premix: Mix the components of the freeze-drying protection system (trehalose, mannitol, PEG20000, SE-15), the components of the effervescent disintegration system (citric acid), and the components of the detection reaction system (specific primers, probes, qPCR reaction enzyme premix) in sequence to prepare a homogeneous freeze-drying stock solution 1. Sodium bicarbonate, a component of the effervescent disintegration system, is prepared separately as freeze-drying stock solution 2. (2) Liquid nitrogen pre-freezing: The freeze-dried stock solution is added dropwise at a distance of 1 mm to 10 mm from the liquid nitrogen surface, so that the droplets are rapidly frozen into spherical microspheres; (3) Vacuum freeze drying: The pre-frozen microspheres are transferred to a freeze dryer for three-stage drying; (4) Dispensing and storage: Dispense and seal the freeze-dried microspheres in a dry environment and store them at room temperature.

7. The preparation method according to claim 6, characterized in that, The three-stage drying process described in step (3) includes: Phase 1: Temperature -30℃ to -10℃, maintained for 1 to 4 hours; Second stage: Temperature -50℃ to -30℃, maintained for 8 to 16 hours; Third stage: Temperature 20℃~30℃, maintained for 2~5 hours.

8. The preparation method according to claim 7, characterized in that, The temperature in the first stage is -20℃ and maintained for 2 hours; the temperature in the second stage is -40℃ and maintained for 12 hours; the temperature in the third stage is 25℃ and maintained for 3 hours.

9. A multi-dimensional standardized quality evaluation method for fully premixed nucleic acid detection lyophilized reagents, characterized in that, Includes the following three dimensions: (1) Physical stability evaluation: including appearance evaluation, transfer performance evaluation, reconstitution time determination and moisture content determination; (2) Evaluation of bioactivity: including determination of detection sensitivity and determination of stability during storage at room temperature; (3) Evaluation of thermal stability: including the determination of storage stability at 37℃ and storage stability at 56℃.

10. The use of the fully premixed nucleic acid detection lyophilized reagent according to any one of claims 1 to 4 in the preparation of pathogen detection reagents, public health monitoring reagents or clinical diagnostic reagents.

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