A solid detection reagent for real-time fluorescence PCR and its preparation method

Abstract

This invention relates to a real-time fluorescent PCR solid detection reagent and its preparation method. The real-time fluorescent PCR solid detection reagent is prepared by freeze-drying a mixture of a protective system, a glycerol-free buffer solution and dNTPs premix, specific primers and probes, and glycerol-free Taq polymerase. The real-time fluorescent PCR solid detection reagent of this invention can maintain its original diagnostic efficacy and sensitivity for at least two years at room temperature, and can detect nucleic acid molecules at a minimum of two copies. This solves the problems of existing real-time PCR detection reagents requiring immediate preparation and strict cold chain technology during long-distance transportation, which significantly increases transportation and storage costs, leading to long transportation times and insufficient reagent inventory in some underdeveloped areas.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically a solid real-time fluorescent PCR solid detection reagent and its preparation method. Background Technology

[0002] Polymerase chain reaction (PCR) provides a universal method for replicating specific DNA sequences for further research. Real-time fluorescence PCR, developed based on PCR technology, monitors the amplification of target DNA sequences or molecules during PCR by detecting the fluorescence signal induced by fluorescent dyes in the reaction system. It achieves extremely high sensitivity, enabling detection limits (LODs) of less than 5 copies of target DNA (in some cases, only 1 copy). This technique has rapidly developed and begun to be widely used in biological research, clinical diagnosis, criminal investigation, and biomedical research. In real-time fluorescence PCR detection, besides the sample extract being tested, the main reagents are polymerase, primers / probes, dNTPs, and aqueous solutions prepared with buffer. These components are highly sensitive to temperature changes and require constant low temperatures (generally around -20 degrees Celsius) during production, transportation, and storage. When stored in liquid form at room temperature (25 degrees Celsius), their shelf life is no more than two days. Furthermore, each component needs to be stored separately at low temperatures and prepared fresh for each use. This significantly increases the transportation and storage costs of the reagents and has led to shortages in many countries and regions, particularly in developing and underdeveloped areas. This makes it difficult for these regions to promptly detect and respond to public health emergencies such as the COVID-19 pandemic. Freeze-drying technology is considered one solution to this problem; however, current freeze-dried reagent products still suffer from short shelf life, low sensitivity, and difficulties in maintaining stable control of both.

[0003] Currently, most real-time PCR test kits are supplied and stored in liquid form to various testing units. To ensure the efficacy of these kits, all components must be kept at a constant low temperature during storage and transportation, and uninterrupted cold chain transportation is required during inter-regional distribution to ensure the product remains at a sufficiently low temperature at the production, transportation, and usage stages. Testing institutions store reagent components, or pre-treated mixtures of several components, along with other necessary components, separately at low temperatures, preparing kits only as needed. A few reports have described freeze-drying techniques for real-time fluorescent PCR test kits to extend shelf life at room temperature; however, this still presents challenges such as difficulty in controlling product quality and detection sensitivity, relatively short shelf life, and the need for strictly controlled dry storage. Furthermore, the requirement for continuous low-temperature storage and uninterrupted constant-temperature cold chain transportation during inter-regional distribution makes these stringent requirements difficult to meet in some less developed regions. At room temperature, most real-time fluorescent PCR test kits containing polymerase can only maintain their original efficacy for approximately 24 hours. Meanwhile, temperature fluctuations and insufficiently low storage conditions can affect the detection performance of polymerases and even other components in the test reagents, leading to misdiagnosis and missed diagnosis. Even in the few existing products that use freeze-drying technology and protective formulations to process real-time fluorescence PCR solid test reagents, problems remain, including difficulty in controlling product quality, relatively low detection sensitivity, relatively short shelf life, and the need for continuous, strictly controlled drying conditions. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a real-time fluorescence PCR solid detection reagent and its preparation method. The solid detection reagent is prepared by freeze-drying a mixture of a protective system, a glycerol-free buffer solution and dNTPs premix, specific primers and probes, and a glycerol-free Taq polymerase. The protective system is an aqueous solution containing the following components at the following mass concentrations: trehalose 0.1–0.5 g / mL, Type A gelatin 5–25 mg / mL, dextrin 0.5–2.5 mg / mL, chitosan 0.1–0.5 mg / mL, sucrose 0.05–0.25 g / mL, and cellulose 0.5–1.0 mg / mL, with ultrapure water as the solvent.

[0005] Furthermore, the glycerol-free buffer solution and dNTPs premix consists of the following components in molar concentrations: 0.2 mol / L KCl, 0.2 mol / L Tris-HCl, 0.2 mol / L (NH4)2SO4, 0.05 mol / L MgCl2, and 0.5 mmol / L dNTPs, with ultrapure water as the solvent.

[0006] Furthermore, the cellulose is one of hydroxyethyl cellulose and methyl cellulose or a mixture of both.

[0007] The preparation method of the real-time fluorescence PCR solid detection reagent includes the following steps:

[0008] 1) Premix the above protection system;

[0009] 2) Mix 5-15 μL of the above-mentioned glycerol-free buffer solution and dNTPs premix, 2-6 μL of 1 μmol / L specific primers and probes, and 10-30 μL of the above-mentioned protection system by shaking until homogeneous;

[0010] 3) Add 1-3 units of glycerol-free Taq polymerase and vortex to mix thoroughly;

[0011] 4) Freeze the prepared material in step 3) at a low temperature of -25 to -30°C for 2 to 6 hours;

[0012] 5) Immediately transfer the material obtained in step 4) to a vacuum dryer and vacuum dry at room temperature for 1-3 hours;

[0013] 6) After drying, the real-time fluorescence PCR solid detection reagent is obtained. The obtained real-time fluorescence PCR solid detection reagent is placed in a PP reagent tube, sealed, and stored at room temperature.

[0014] Furthermore, the room temperature in step 5) is 16-35℃.

[0015] Furthermore, the vacuum drying process in step 5) takes 2 hours.

[0016] Compared with the prior art, the present invention provides a real-time fluorescent PCR solid detection reagent and its preparation method, which has the following beneficial effects:

[0017] 1. Following the formulation and manufacturing process described in this invention, all components necessary for real-time fluorescence PCR detection are mixed and pretreated. After freeze-drying, a dry, multi-well real-time fluorescence PCR solid detection reagent is obtained. It can maintain its original detection effect and sensitivity for at least 2 years at room temperature and can detect nucleic acid molecules with a minimum copy number of 2. The protection system of this invention mainly consists of trehalose, gelatin, dextrin, chitosan, sucrose, hydroxyethyl cellulose, and methylcellulose. Trehalose is a widely used biological cryoprotectant. It forms hydrogen bonds with polymerases and other active ingredients in the reagent to replace water molecules surrounding these components, or isolates water molecules from polymerases and other active ingredients, thereby stabilizing the folded structure of the polymerase and protecting the activity of the active ingredients during the pre-freezing process before drying. It also forms hydrogen bonds with proteins to ensure that functional substances can be stored for a longer time at higher temperatures. The gelatin described in this invention serves as an important carrier substance for the product, rather than the traditional bovine serum albumin as a carrier. When bovine serum albumin (BSA) is used in real-time fluorescent PCR, it denatures irreversibly with increasing amplification temperature, forming a white solid substance that affects nucleic acid amplification and fluorescence signal acquisition. Gelatin, however, exhibits significantly temperature-dependent gel-forming properties, forming a stable gel at low temperatures in a reversible process, effectively avoiding this problem. Experiments showed that using Type A gelatin minimizes its interference with fluorescence signals. Dextrin, an intermediate product generated during the hydrolysis of large starch molecules, makes the system more fluffy during freezing, preventing damage to functional substances caused by large ice crystals. It also facilitates water molecule flow, resulting in faster drying and a more uniform product texture. Chitosan, a polysaccharide with slightly lower water solubility compared to other components, interacts with other protective substances. Adding a small amount without affecting the overall reconstitution rate improves the moisture resistance of the dried product. Sucrose, due to its multi-hydroxyl structure, can interact with functional substances and protect their stability during high-temperature storage. In addition, sucrose can further regulate the viscosity, melting point, and glass transition temperature of the system, allowing the drying process to be carried out in a more readily available environment. Hydroxyethyl cellulose and methyl cellulose can respectively adjust the viscosity of the system at different temperatures, resulting in faster drying and a more uniform, stable, and moisture-resistant porous material. Meanwhile, because glycerol absorbs a large amount of moisture from the air, causing the solid detection reagent to absorb moisture and partially dissolve, the porous structure of the material disappears, solubility decreases, and the absorbed moisture directly leads to the deactivation of functional components in the material, the detection material components of this invention avoid the use of glycerol.

[0018] 2. It solves the problems of existing real-time PCR detection reagents needing to be prepared on demand and requiring strict cold chain technology during long-distance transportation, which greatly increases transportation and storage costs, and thus leads to long transportation times and insufficient reagent inventory in some underdeveloped areas.

[0019] 3. The real-time fluorescent PCR solid detection reagent processed according to the formula and process described in this invention can be used to design primers and probes to match different detection targets, thus producing solid detection reagents for different viruses.

[0020] 4. The solid detection reagent obtained by applying the formula and process described in this invention has a fast reconstitution speed, a longer shelf life at room temperature, higher sensitivity, and a more stable form with better moisture resistance. Attached Figure Description

[0021] Figure 1 The real-time fluorescent PCR solid detection reagent prepared in Example 1;

[0022] Figure 2 Photograph of the internal structure of the real-time fluorescence PCR solid detection reagent prepared in Example 1;

[0023] Figure 3 The graph shows a comparison of the detection performance of the solid detection reagent prepared in Example 1 after 24 months of storage with a solid detection reagent containing glycerol and a freshly prepared detection reagent (without a protective system).

[0024] Figure 4 The graph shows a comparison of the detection results of the solid detection reagent prepared in Example 1 after 24 months of storage with solid detection reagents without trehalose and freshly prepared detection reagents (without the protection system).

[0025] Figure 5 A comparison of the detection results of the solid detection reagent prepared in Example 1 after 24 months of storage with the solid detection reagent with Type A gelatin replaced by BSA and the freshly prepared detection reagent (without the protection system);

[0026] Figure 6 The graph shows a comparison of the detection results of the solid detection reagent prepared in Example 1 after 24 months of storage with solid detection reagents without added gelatin or BSA and freshly prepared detection reagents (without a protective system).

[0027] Figure 7 The graph shows a comparison of the detection results of the solid detection reagent prepared in Example 1 after 24 months of storage with the solid detection reagent without dextrin and the freshly prepared detection reagent (without the protection system).

[0028] Figure 8 The graph shows a comparison of the detection performance of the solid detection reagent prepared in Example 1 after being stored at room temperature for 24 months with the solid detection reagent without chitosan and the freshly prepared detection reagent (without the protection system).

[0029] Figure 9The graph shows a comparison of the detection performance of the solid detection reagent prepared in Example 1 after being stored at room temperature for 24 months with solid detection reagents without added sucrose and freshly prepared detection reagents (without a protection system).

[0030] Figure 10 The graph shows a comparison of the detection results of the solid detection reagent prepared in Example 1 after being stored at room temperature for 24 months with solid detection reagents without added cellulose and freshly prepared detection reagents (without a protective system).

[0031] Figure 11 Comparison of detection results of the solid detection reagent prepared in Example 1 after being stored at room temperature for 24 months;

[0032] Figure 12 Comparison of detection results of the solid detection reagent prepared in Example 1 after being stored at 45°C for 12 months;

[0033] Figure 13 The detection sensitivity test graph of the solid detection reagent prepared in Example 1 after being stored at room temperature for 24 months;

[0034] Figure 14 The detection sensitivity test chart for freshly prepared test reagents (without protection system);

[0035] Figure 15 A comparison of the detection sensitivity Ct values ​​of the solid detection reagent prepared in Example 1 after 24 months of storage at room temperature with those of the freshly prepared detection reagent (without the protection system). Detailed Implementation

[0036] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0037] Example 1: Preparation of a real-time fluorescent PCR solid detection reagent

[0038] Includes the following steps:

[0039] 1) Preparation of the premixed solution of the protection system: The premixed solution of the protection system in this embodiment is prepared by adding 0.05g of trehalose, 2.5mg of Type A gelatin, 0.25mg of dextrin, 0.05mg of chitosan, 50mg of sucrose and 0.25mg of hydroxyethyl cellulose to 0.5mL of ultrapure water;

[0040] 2) Mix 5 μL of the glycerol-free buffer solution and dNTPs premix, 1 μL of 1 μmol / L specific primer probes, and 10 μL of the above protection system uniformly; the glycerol-free buffer solution and dNTPs premix comprises the following components: 0.2 mol / L KCl, 0.2 mol / L Tris-HCl, 0.2 mol / L (NH4)2SO4, 0.05 mol / L MgCl2, and 0.5 mmol / L dNTPs, with ultrapure water as the solvent;

[0041] 3) Add 1 unit of glycerol-free Taq polymerase and vortex to mix thoroughly;

[0042] 4) Freeze the mixture prepared in step 3) at -25°C for 4 hours;

[0043] 5) Immediately transfer the material obtained in step 4) to a vacuum dryer and vacuum dry at room temperature for 1-3 hours;

[0044] 6) After drying, the real-time fluorescent PCR solid detection reagent is obtained. The obtained real-time fluorescent PCR solid detection reagent is divided into PP reagent tubes, sealed and stored at room temperature.

[0045] Furthermore, the room temperature in step 5) is 22.5-25℃.

[0046] Furthermore, the vacuum drying process in step 5) is preferably 2 hours.

[0047] The real-time fluorescent PCR solid detection reagent prepared according to the above method ( Figure 1 It has a porous structure, and after being stored in 50% humidity for a period of time, the porous structure did not change significantly. Figure 2 (ab). This porous structure and framework ensure that this solid detection reagent maintains extremely high water solubility and dissolution rate. In use, simply add ultrapure water and the sample to be tested for rapid testing. Using the formulation and process described in this invention, the resulting solid detection reagent can achieve a detection sensitivity of at least 2 copies. For mass production, the entire production process for a batch of products only requires 4-5 hours, significantly improving production efficiency.

[0048] Example 2: Component Screening Experiment of a Real-Time Fluorescent PCR Solid Detection Reagent

[0049] All the test reagents used in the reference group described in the following experiments were freshly prepared before the experiment and contained only: glycerol-free buffer solution and dNTPs premix, specific primers and probes, glycerol-free Taq polymerase, and no protection system. All components were used in the same proportions as in this invention, and ultrapure water was used instead of the protection system.

[0050] 2.1 About Glycerin

[0051] Existing real-time fluorescence PCR solid detection reagents all contain glycerol. During the drying process, glycerol absorbs a large amount of water, making it impossible for the reagent to dry completely, forming a gel-like clump. This clump is difficult to dissolve in water and its stability decreases at room temperature, causing the solid detection reagent to gradually lose its activity after being stored at room temperature for about 21 days. Therefore, this invention completely avoids the use of glycerol. The screening and comparison experiment is as follows: Components identical to those used in the preparation steps of the real-time fluorescence PCR solid detection reagent were added to the PCR detection tube according to steps 1) and 2). The 5 units / μL glycerol-free Taq polymerase in step 3) was replaced with 0.2–0.6 μL of commercially available Taq polymerase containing 50% glycerol (v / v). The reagent was dried using the same method as described in this invention to obtain a glycerol-containing solid detection reagent. The obtained glycerol-containing solid detection reagent sample was sealed and stored at room temperature for 21 days. Then, the real-time fluorescence PCR solid detection reagent prepared in Example 1 and the glycerol-containing solid detection reagent sample stored for 21 days were tested for the same concentration of viral template. The amplification data were compared using freshly prepared detection reagent (without a protection system) as the reference group. Figure 3 It was found that the real-time fluorescence PCR solid detection reagent of the present invention, after being stored at room temperature for 24 months, still has the same detection ability as the freshly prepared detection reagent (reference group), and can obtain similar amplification curves. The Ct value and the final fluorescence intensity are similar to those of the reference group. However, the solid detection reagent sample containing glycerol gradually loses its activity after being stored for 21 days, the Ct value shows a significant lag, and the amplification efficiency and the final fluorescence intensity are significantly reduced.

[0052] 2.2 About Trehalose

[0053] Trehalose is a biological cryoprotectant that forms hydrogen bonds with Taq polymerase and other active ingredients in the reagent to replace water molecules surrounding these ingredients, or to isolate water molecules from the polymerase and other active ingredients, thereby stabilizing the polymerase's folded structure and protecting the activity of the active ingredients during the pre-freezing process before drying. It also binds to proteins via hydrogen bonds, ensuring that functional substances can be stored for longer periods at higher temperatures. The screening experiment for trehalose is as follows: 0.1–0.5 g / mL of trehalose in step 1) of the solid detection reagent preparation described in Example 1 was replaced with ultrapure water, while the other components remained identical. The preparation was carried out according to the same method as described in the present invention, following steps 1), 2), and 3). The resulting solid detection reagent sample was stored at room temperature. Using the solid detection reagent sample lacking trehalose protection and the solid detection reagent of the present invention to detect the same concentration of viral template, it was found that the detection activity of the solid detection reagent sample lacking trehalose decreased significantly after freezing and drying, with a significant lag in Ct values, and a significant reduction in amplification efficiency and final fluorescence intensity, failing to meet the requirements for subsequent room temperature storage and detection. The solid detection reagent sample lacking trehalose was compared with the solid detection reagent of the present invention stored at room temperature for 24 months to detect the same concentration of viral template. The amplification data were compared using a freshly prepared detection reagent (without protection system) as a reference group. Figure 4 .

[0054] from Figure 4 It is evident that the solid detection reagent described in this invention, after being stored at room temperature for 24 months, still exhibits the same detection capability as the freshly prepared detection reagent (reference group) (excluding the protection system), yielding similar amplification curves, with Ct values ​​and final fluorescence intensities comparable to the reference group. However, the solid detection reagent sample lacking trehalose shows a significant decrease in detection activity after freezing and drying, with a marked lag in Ct values, and a significant reduction in amplification efficiency and final fluorescence intensity, failing to meet the requirements for subsequent room temperature storage and detection.

[0055] 2.3 About Type A Gelatin

[0056] In existing technologies, bovine serum albumin (BSA) is commonly used instead of Type A gelatin. However, comparisons have shown that using BSA significantly reduces the fluorescence signal during detection after reagent drying, failing to meet detection requirements. Conversely, completely replacing BSA with Type A gelatin significantly improves the fluorescence signal released during detection. The screening experiment for Type A gelatin and BSA is as follows: The 5–25 mg / mL Type A gelatin in step 1) of the solid detection reagent preparation process described in Example 1 was replaced with 5–25 mg / mL BSA, with all other components remaining identical. The preparation was carried out using the same drying method as described in steps 1), 2), and 3), and the resulting solid detection reagent samples were stored at room temperature. The solid detection reagent samples with Type A gelatin replaced by BSA and the solid detection reagent of this invention were used to detect the same concentration of viral template. The detection data of the BSA-replaced solid detection reagent samples were compared with those of the solid detection reagent of this invention stored at room temperature for 24 months, using freshly prepared detection reagents (without a protection system) as a reference group. Figure 5 .from Figure 5 The study found that when solid test reagent samples with Type A gelatin replaced by BSA were frozen and dried, the fluorescence signals collected during testing were significantly lower, failing to meet the testing requirements.

[0057] If gelatin or other carriers such as BSA are not used at all, ultrapure water is used instead of the 5-25 mg / mL Type A gelatin in step 1) of the solid detection reagent preparation process described in Example 1. All other components are identical. The solid detection reagent is prepared using the same drying method as described in steps 1), 2), and 3), and the resulting solid detection reagent sample is stored at room temperature. It was found that the solid detection reagent sample prepared in this way cannot form a water-soluble powder cake, which is not conducive to the rapid dissolution and use of the solid detection reagent. The solid detection reagent sample lacking Type A gelatin or BSA, stored at room temperature for 30 days, and the solid detection reagent of the present invention, stored at room temperature for 24 months, were used to detect the same concentration of viral template. The amplification data were compared with the freshly prepared detection reagent (without the protection system) as the reference group. Figure 6 .from Figure 6 The study found that the solid test reagent sample lacking Type A gelatin or BSA lost its activity after being stored at room temperature for about 30 days.

[0058] 2.4 About Dextrin

[0059] Existing solid detection reagents require relatively low temperatures to maintain their activity, and the drying process is slow with slow water evaporation and prolonged drying time. Therefore, precise control of the ambient temperature during drying is crucial to prevent drying failure and potential melting of the reagent before complete drying. This invention addresses this problem by utilizing dextrin, an intermediate substance produced during the hydrolysis of macromolecular starch. Dextrin makes the system more porous during freezing, preventing damage to functional substances caused by large ice crystals. It also facilitates water molecule flow, resulting in faster drying and a more uniform product texture. The dextrin screening experiment is as follows: The 0.5–2.5 mg / mL dextrin in step 1) of the solid detection reagent preparation process described in Example 1 was replaced with ultrapure water, while all other components remained identical. The drying process was performed using the same method as described in this invention, following steps 1), 2), and 3). It was found that the absence of dextrin increased the complete freeze-drying time of the solid detection reagent sample by 2–4 hours, and the dextrin-deficient solid detection reagent sample showed accelerated inactivation at room temperature. After being stored at room temperature for 15 days, the same concentration of viral template was detected using the solid detection reagent sample lacking dextrin and the solid detection reagent of the present invention stored at room temperature for 24 months. The amplification data were compared with the freshly prepared detection reagent as the control group (excluding the protection system). Figure 7 It was found that the drying process was incomplete without the addition of dextrin; therefore, the solid test reagent sample lacking dextrin gradually lost its activity after being stored at room temperature for 15 days.

[0060] 2.5 About Chitosan

[0061] Chitosan, a polysaccharide with slightly lower solubility in water compared to other components, interacts with other protective substances. Adding a small amount can improve the moisture resistance of the dried product without affecting the overall reconstitution rate. Most existing technologies lack protective formulations to enhance reagent moisture resistance, making the dried product highly susceptible to deliquescence and reducing its stability at room temperature. This invention addresses this problem by using chitosan. It reveals that without chitosan in the protective system, the dried test reagent readily absorbs moisture and deliquesces during storage, turning into a gel or droplet-like form. Its stability at room temperature is significantly reduced, and it loses its activity after approximately 30 days of storage at room temperature. The chitosan screening experiment is as follows: In step 1) of the solid detection reagent preparation described in Example 1, 0.1–0.5 mg / mL chitosan was replaced with ultrapure water, while the remaining components remained identical. The preparation was carried out using the same drying method as described in steps 1), 2), and 3). The resulting chitosan-free solid detection reagent sample was stored at room temperature. It was found that even in a capped PCR reaction tube, this solid detection reagent sample began to deliquesce within 15 days, indicating a faster rate of inactivation. After storage at room temperature for 30 days, the chitosan-free solid detection reagent sample and the solid detection reagent of this invention stored at room temperature for 24 months were used to detect the same concentration of viral template. The freshly prepared detection reagent was used as the reference group (without the protection system), and the amplification data were compared to obtain… Figure 8 It was found that without the addition of chitosan, the solid detection reagent would deliquesce and completely lose its activity after being stored at room temperature for 30 days.

[0062] 2.6 About sucrose

[0063] Due to its polyhydroxyl structure, sucrose can interact with functional substances and protect their stability during high-temperature storage. Furthermore, sucrose can further regulate the viscosity, melting point, and glass transition temperature of the system, allowing the drying process to be carried out in more readily available environments. Existing technologies lack similar protective formulations that can optimize the drying process while protecting the activity of the reagent's active ingredients at room temperature. This results in reagents with relatively short shelf lives at room temperature, and precise temperature gradient control is required during drying to prevent drying failure and melting of the reagent before complete drying. The sucrose used solves this problem. This invention reveals that if the protective system lacks sucrose, the reagent preparation process must involve pre-freezing at extremely low temperatures or strict temperature gradient control during drying. Moreover, the dried test reagent cannot be stored at room temperature for extended periods, losing its activity after 60 days at room temperature. The screening experiment for sucrose was as follows: In step 1) of the preparation of the solid detection reagent described in Example 1, 0.05–0.25 g / mL of sucrose was replaced with ultrapure water, while the other components remained identical. The drying process was carried out according to the same procedures as in steps 1), 2), and 3). It was found that without the addition of sucrose, the frozen solid detection reagent sample would quickly melt into a liquid when moved to room temperature for drying. Pre-freezing at approximately -78 degrees Celsius for 3 hours was necessary to ensure proper drying at room temperature. However, even with this method, the inactivation rate of the sample increased rapidly when stored at room temperature. After 60 days of storage at room temperature, the same concentration of viral template was detected using the sucrose-free solid detection reagent sample and the solid detection reagent of this invention stored at room temperature for 24 months. The amplification data were compared using a freshly prepared detection reagent (without a protection system) as a reference group. Figure 9 It was found that without the addition of sucrose, the detection performance of solid test reagent samples decreased significantly after being stored at room temperature for 60 days.

[0064] 2.7 Regarding hydroxyethyl cellulose and methyl cellulose

[0065] Hydroxyethyl cellulose and methyl cellulose can be used to adjust the viscosity of the system at different temperatures, resulting in faster drying and a more uniform, stable, and moisture-resistant porous material. Existing technologies lack similar formulations to alter viscosity and facilitate moisture evaporation during reagent drying, leading to prolonged drying time. Precise control of the ambient temperature during drying is crucial to prevent drying failure and reagent melting before complete drying. This invention reveals that the absence of hydroxyethyl cellulose and methyl cellulose in the protective system results in prolonged or incomplete drying during reagent preparation, reduced reagent stability at room temperature, and gradual loss of activity after approximately 120 days of storage. A cellulose screening experiment was conducted as follows: 0.5–1.0 mg / mL of cellulose in step 1) of the solid detection reagent preparation process described in Example 1 was replaced with ultrapure water, while all other components remained identical. The drying process was performed using the same method as described in this invention, following steps 1), 2), and 3). It was found that without cellulose, the complete drying time of the sample required at least 3 hours; otherwise, local dissolution occurred upon sample removal, forming a gel-like clot that was difficult to dissolve quickly in ultrapure water during use. After extending the drying time, the obtained solid detection reagent sample was stored at room temperature for 120 days. The same concentration of viral template was then detected using both the cellulose-free solid detection reagent sample and the solid detection reagent of this invention stored at room temperature for 24 months. The amplification data were compared with a freshly prepared detection reagent (without a protection system) as a reference group. Figure 10 It was found that without the addition of sucrose, solid test reagent samples gradually lost their activity after being stored at room temperature for 120 days.

[0066] Through the above screening experiments and comparative tests of different concentrations of components, the protection system of the real-time fluorescence PCR solid detection reagent of the present invention was finally determined to be an aqueous solution containing the following components at the following mass concentrations: trehalose 0.1-0.5 g / mL, Type A gelatin 5-25 mg / mL, dextrin 0.5-2.5 mg / mL, chitosan 0.1-0.5 mg / mL, sucrose 0.05-0.25 g / mL, cellulose 0.5-1.0 mg / mL, and ultrapure water as the solvent.

[0067] Example 3 tested the detection performance of the real-time fluorescence PCR solid detection reagent prepared in Example 1 after storage at room temperature and high temperature.

[0068] The real-time fluorescence PCR solid detection reagent prepared in Example 1 was divided into two groups. One group was stored at room temperature (22.5-25℃), and the other group was stored at a higher, artificially controlled temperature of 45℃. After a specific storage period, a portion of the detection material sample was periodically taken, dissolved in 24 μL of ultrapure water, and 1 μL of 10... -4The detection nucleic acid template was prepared at ng / μL (approximately 20,000 copies of nucleic acid molecules), and then directly placed into a real-time fluorescence PCR assay for 45 amplification cycles. In the control group experiment, the detection targets were nucleic acid templates of the same concentration gradient, but the detection reagents were freshly prepared and contained only the basic functional components of the formula: glycerol-free PCR buffer and dNTPs premix, specific primers and probes, and glycerol-free Taq polymerase (in the same proportions as described in the invention, with ultrapure water used instead of the protection system). The detection performance of the solid detection reagent after storage at room temperature and 45°C for a certain period of time was obtained, as shown in the figure. Figure 11 and Figure 12 As shown. From Figure 11 and Figure 12 It was found that the solid detection reagent of Example 1, after being stored at room temperature for 24 months ( Figure 11 After being stored at high temperature for 12 months ( Figure 12 The amplification curve of the template nucleic acid was almost identical to that of the freshly prepared control reagent, and the difference in Ct value was no greater than 0.5 compared to the control reagent. This indicates that the detection performance of the solid detection reagent did not change after being stored at room temperature (22.5-25℃) for 24 months and at 45℃ for 12 months.

[0069] The real-time fluorescence PCR solid detection reagent prepared by the method described in this invention only needs to be kept at room temperature during vacuum drying, without the need for additional temperature control, and the conditions are readily available. The method of this invention can effectively protect the active ingredients in the real-time fluorescence PCR solid detection reagent, improve its stability and moisture resistance, thus enabling the solid detection reagent to maintain extremely high stability at room temperature or even higher temperatures. It can be stored at up to 45 degrees Celsius for more than one year and at around 25 degrees Celsius for at least two years without any change in detection function. Therefore, the use of low-temperature technology is avoided during the storage, transportation, and use of the detection reagent, and the more expensive cold chain transportation technology is avoided when distributing products to certain regions.

[0070] Example 4 tested the sensitivity of the real-time fluorescence PCR solid detection reagent prepared in Example 1 after storage at room temperature for 24 months.

[0071] The detection sensitivity of the real-time fluorescence PCR solid detection reagent prepared in Example 1 was tested after 24 months of storage, and compared with the detection sensitivity and Ct value of the freshly prepared reagent. The solid detection reagent sample from Example 1, stored for 2 years, was dissolved in 24 μL of ultrapure water, and 1 μL of 10... -9 ~10 -2Nucleic acid template of ng / μL (approximately corresponding to 1-2,000,000 copies of nucleic acid molecules). Then, it was directly placed into a real-time fluorescence PCR assay for detection, and 45 amplification cycles were performed. The detection sensitivity test results were obtained after the reagents were stored at room temperature for 2 years. Figure 13 In the control group experiment, the detection targets were nucleic acid templates of the same concentration gradient, but the detection reagents were freshly prepared and only contained the basic functional formulation of the above-described formula: glycerol-free buffer solution and dNTPs premix, specific primers and probes, and glycerol-free Taq polymerase (in the same proportions as described in the invention, with ultrapure water used instead of the protection system). After performing the same detection cycles, the detection sensitivity test chart of the newly prepared reagents was obtained. Figure 14 Read the Ct values ​​corresponding to the detection curves of each template concentration in both graphs, and plot a comparison graph of the Ct values ​​after 24 months of storage of the solid test reagent and the newly prepared reagent. Figure 15 ).

[0072] contrast Figure 13 and Figure 14 And from Figure 15 As can be seen from the comparison images, the sensitivity of the solid detection reagent prepared in Example 1 after being stored at room temperature for 2 years is not much different from that of the newly prepared detection reagent. Both can detect nucleic acid molecules with a minimum of 2 copies, and the corresponding curves and Ct values ​​coincide, indicating that the solid detection reagent prepared in Example 1 can meet the requirement of being stored at room temperature for 2 years without changing detection performance.

[0073] Despite the embodiments shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A real-time fluorescent PCR solid detection reagent, characterized in that, The solid detection reagent is prepared by freeze-drying a mixture of a protective system, a glycerol-free buffer solution and a dNTPs premix, specific primers and probes, and a glycerol-free Taq polymerase. The protective system is an aqueous solution containing the following components at the following concentrations: trehalose 0.1–0.5 g / mL, Type A gelatin 5–25 mg / mL, dextrin 0.5–2.5 mg / mL, chitosan 0.1–0.5 mg / mL, sucrose 0.05–0.25 g / mL, and cellulose 0.5–1.0 mg / mL, with ultrapure water as the solvent. The glycerol-free buffer solution and the dNTPs premix consist of the following components at the following molar concentrations: 0.2 mol / L KCl, 0.2 mol / L Tris-HCl, and 0.2 mol / L (NH₄)₂SO₄. 4、 0.05 mol / L MgCl 2、 0.5 mmol / L dNTPs, with ultrapure water as the solvent; the cellulose is hydroxyethyl cellulose or methyl cellulose.

2. A method for preparing the real-time fluorescence PCR solid detection reagent according to claim 1, characterized in that, Includes the following steps: 1) Premix the protection system; 2) Mix 5-15 µL of the glycerol-free buffer solution and dNTPs premix, 2-6 µL of 1 µmol / L specific primers and probes, and 10-30 µL of the above protection system by shaking until homogeneous; 3) Add 1-3 units of glycerol-free Taq polymerase and vortex to mix thoroughly; 4) Place the mixture prepared in step 3) in a low temperature environment of -25 ~ -30℃ and freeze for 2 ~ 6 hours; 5) Immediately transfer the material obtained in step 4) to a vacuum dryer and vacuum dry at room temperature for 1-3 hours; 6) After drying, the real-time fluorescent PCR solid detection reagent is obtained. The obtained real-time fluorescent PCR solid detection reagent is divided into PP reagent tubes, sealed and stored at room temperature.

3. The method for preparing the real-time fluorescence PCR solid detection reagent according to claim 2, characterized in that, The room temperature in step 5) is 16-35℃.

4. The method for preparing the real-time fluorescence PCR solid detection reagent according to claim 2, characterized in that, The vacuum drying process in step 5) takes 2 hours.

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