Composite freeze-drying protective agent based on CRISPR-Cas13a, microsphere molecular diagnostic reagent and preparation method

Abstract

The embodiment of the invention discloses a composite freeze-drying protective agent based on CRISPR-Cas13a, a microsphere molecular diagnostic reagent and a preparation method. The composite freeze-drying protective agent comprises a vitrification matrix component, a structure support component, a protein aggregation inhibitor and a surfactant, the vitrification matrix component comprises mannitol and trehalose, and the mass ratio of mannitol to trehalose is (3-7): (3-7); the structural support component comprises any one of PEG8000, PEG6000 and PEG4000 and any one of glucan 10000, glucan 8000 and glucan 20000, and the mass ratio of the PEG8000 to the PEG6000 to the glucan 8000 to the glucan 20000 is (1-5): (1-5); the protein aggregation inhibitor is bovine serum albumin, and the surfactant is polyoxyethylene sorbitan monolaurate; the mass ratio of the structure supporting component to the vitrification matrix component is (1-5): (3-7), the mass ratio of the protein inhibitor to the structure supporting component is (0.01-1): (2-10), and the mass ratio of the surfactant to the structure supporting component is (0.001-0.1): (2-10).

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a CRISPR-Cas13a-based composite lyophilization protectant, microsphere molecular diagnostic reagents, and their preparation methods. Background Technology

[0002] Molecular diagnostic technologies, with their advantages of high specificity and high sensitivity, have been widely used in fields such as infectious disease detection and genetic disease screening. The CRISPR / Cas system, as a next-generation gene editing and detection tool, utilizes the Cas13 protein, which possesses RNA-targeting recognition and incidental cleavage activity. This allows it to specifically recognize target RNA and then cleave surrounding reporter probes, amplifying the signal and making it one of the core tools of molecular diagnostics.

[0003] Currently, most Cas13a-based molecular diagnostic reagents are liquid systems, containing functional components such as Cas13a protein, T7 reverse transcriptase, crRNA, signal probes, and rNTPs. However, liquid reagents have significant drawbacks. Lyophilization technology solidifies reagents into microspheres through low-temperature dehydration, which can significantly improve the stability of bioactive components. Patent document CN114574228A discloses a CRISPR / Cas13a nucleic acid detection kit, whose core components are liquid Cas13a protein, T7 polymerase, crRNA, and fluorescent probes. The protective agent is a single trehalose (8% concentration), which requires freezing and storage at -20°C. Patent document WO2022156431A1 discloses a lyophilized CRISPR detection reagent. The protectant consists of trehalose (10%) and BSA (0.5%). The lyophilization procedure is pre-freezing at -50℃ for 3 hours, sublimation at -30℃ for 8 hours, and desorption drying at 25℃ for 6 hours. It is suitable for a 30μL detection system. After reconstitution, the Cas13a activity retention rate is about 75%. The article "Lyophilization of CRISPR-Cas13a Reagents for Point-of-Care Testing" published in the Journal of Pharmaceutical Sciences in 2023 reported a lyophilized protectant containing mannitol (8%) and PEG4000 (2%). After lyophilization, the reagent needs to be refrigerated at 4℃, has a shelf life of about 6 months, and a reconstitution time of more than 5 minutes.

[0004] However, existing lyophilization methods for CRISPR diagnostic reagents have at least the following technical problems:

[0005] First, lyophilization protectants are mostly single-component (such as trehalose or mannitol alone), which cannot simultaneously preserve the activity of nucleic acids (crRNA, rNTPs) and two types of enzymes, resulting in a significant decrease in detection sensitivity after reconstitution. Second, lyophilized systems are mostly in powder form, and the lyophilization process is poorly designed, leading to problems such as collapse and slow reconstitution of lyophilized products. Third, the component ratios are poorly compatible with the lyophilization process, resulting in insufficient system uniformity after reconstitution, which affects the repeatability of detection. Fourth, there are limitations in storage and transportation; lyophilized reagents still require low-temperature refrigeration and cannot be stored and transported at room temperature, thus logistics costs have not been fundamentally reduced. Summary of the Invention

[0006] In view of this, some embodiments disclose a composite lyophilization protectant based on CRISPR-Cas13a, comprising: a vitrification matrix component, a structural support component, a protein aggregation inhibitor, and a surfactant; wherein the vitrification matrix component comprises mannitol and trehalose in a mass ratio of 3–7:3–7; the structural support component comprises any one of PEG8000, PEG6000, and PEG4000 and any one of dextran 10000, dextran 8000, and dextran 20000 in a mass ratio of 1–5:1–5; the protein aggregation inhibitor is bovine serum albumin, and the surfactant is polyoxyethylene sorbitan monolaurate; the mass ratio of the vitrification matrix component to the structural support component is 3–7:1–5, the mass ratio of the protein inhibitor to the structural support component is 0.01–1:2–10, and the mass ratio of the surfactant to the structural support component is 0.001–0.1:2–10.

[0007] Furthermore, some embodiments disclose a CRISPR-Cas13a-based composite freeze-drying protectant, which, by weight, comprises: 5 parts mannitol, 5 parts trehalose, 3 parts PEG8000, 3 parts dextran10000, 0.3 parts bovine serum albumin, and 0.05 parts polyoxyethylene sorbitan monolaurate.

[0008] On the other hand, some embodiments disclose microsphere molecular diagnostic reagents based on CRISPR-Cas13a, which are prepared from the composite lyophilization protectant, buffer solution and molecular diagnostic functional components disclosed in the embodiments of the present invention.

[0009] Furthermore, some embodiments disclose CRISPR-Cas13a-based microsphere molecular diagnostic reagents, with buffers comprising Tris-HCl buffer and MgCl2, wherein the pH of the Tris-HCl buffer is 8.

[0010] Some embodiments disclose CRISPR-Cas13a-based microsphere molecular diagnostic reagents, the molecular diagnostic functional components of which include:

[0011] Mixture of 100–150 nM Cas13a protein, 2–8 U / μL T7 reverse transcriptase, 80–120 nM specific crRNA, 200–300 nM signal probe 7U-FAM-BHQ, and 1–2 mM rNTPs.

[0012] Furthermore, some embodiments disclose methods for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, including the following steps:

[0013] S1, Preparation of premixed solution

[0014] S11. Adjust the pH of the Tris-HCl buffer to 8.0 to serve as the basic buffer system;

[0015] S12. Add rNTPs mixture, bovine serum albumin and polyoxyethylene sorbitan monolaurate to the basic buffer system in sequence. Dissolve by magnetic stirring at room temperature for 10 min with the stirring speed set to 300 rpm.

[0016] S13. Add the vitrified matrix component and structural support component of the composite freeze-drying protectant, place them in a 37°C water bath and stir for 20 minutes until a clear and transparent protectant solution is formed.

[0017] S14. After the protective agent solution cools to 4°C, add Cas13a protein, T7 reverse transcriptase, specific crRNA, and signal probe 7U-FAM-BHQ, mix well, and let stand at 4°C for 30 min to obtain the premixed solution before lyophilization.

[0018] S2. Packaging and freeze-drying

[0019] S21. Dispense the filtered premixed solution before lyophilization into 96-well lyophilization plates or individual lyophilization tubes at a volume of 20 μL / part, ensuring that the liquid level in each well / tube is consistent.

[0020] S22. Place the dispensed freeze-dried plates / tubes into the freeze dryer and perform freeze-drying according to the following procedure:

[0021] Pre-freezing stage: Keep at -40 to -35℃ for 2 to 2.5 hours under normal pressure;

[0022] Sublimation drying first stage: -40~-35℃ for 1~1.5h, pressure 10~20Pa;

[0023] Second stage of sublimation drying: keep at -30 to -25℃ for 4 to 5 hours, with a pressure of 10 to 20 Pa;

[0024] Third stage of sublimation drying: keep at -20 to -15℃ for 5 to 6 hours, with a pressure of 10 to 20 Pa;

[0025] Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa;

[0026] The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa.

[0027] The third stage of drying was analyzed: the temperature was kept at 30℃ for 4 hours, and the pressure was 5-10 Pa.

[0028] S23. Maintain a vacuum state inside the freeze dryer, fill the chamber with nitrogen to atmospheric pressure, and seal the freeze-drying plate / tube.

[0029] Furthermore, some embodiments disclose a method for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, characterized in that lyophilization is performed in step S22 according to the following procedure:

[0030] Pre-freezing stage: -40℃ for 2 hours, at normal pressure;

[0031] First stage of sublimation drying: -40℃ for 1 hour, pressure 10~20Pa;

[0032] Second stage of sublimation drying: keep at -30℃ for 5 hours, with a pressure of 10-20 Pa;

[0033] Third stage of sublimation drying: keep at -20℃ for 5 hours, with a pressure of 10-20 Pa;

[0034] Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa;

[0035] The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa.

[0036] The third stage of drying was carried out at 30℃ for 4 hours under a pressure of 5-10 Pa.

[0037] Some embodiments disclose methods for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, and also include steps for using the microsphere molecular diagnostic reagents, specifically including:

[0038] Take one portion of lyophilized microsphere molecular diagnostic reagent, reconstitute it with 45 μL of water, add 5 μL of the sample to be tested, reconstitute at room temperature for 1–2 min, incubate at 37 °C for 25–30 min, and read the signal value using a fluorescence detector.

[0039] The present invention discloses a CRISPR-Cas13a-based composite lyophilization protectant, microsphere molecular diagnostic reagent, and preparation method. It provides a composite lyophilization protectant that achieves synergistic stability protection of nucleic acids, Cas13a protein, and T7 reverse transcriptase; it provides an optimized lyophilization process to obtain lyophilized microspheres with low residual moisture content, fast reconstitution speed, and uniform morphology; it enables the reagent to be stored and transported at room temperature (2-30℃), extending the shelf life to over 12 months; and it has promising application prospects in the field of molecular diagnostic reagents. Attached Figure Description

[0040] Figure 1 Example 1: Flowchart of the preparation method of microsphere molecular diagnostic reagents;

[0041] Figure 2 Example 2: Experimental curve of activity retention rate of freeze-dried microspheres;

[0042] Figure 3 Example 1: Comparison of the relative activity retention rate of the protective agents in the comparative example. Detailed Implementation

[0043] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in these embodiments of the invention, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in these embodiments is merely for describing particular implementations and is not intended to limit the scope of the disclosure of these embodiments.

[0044] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain; other experimental methods and technical means not specifically noted in the embodiments of this invention refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0045] The terms “basic” and “approximately” as used herein are used to describe small fluctuations. For example, they can mean less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. Numerical data presented or expressed in range format herein are used for convenience and brevity only, and should therefore be interpreted flexibly to include not only the explicitly listed values ​​that define the range, but also all independent values ​​or subranges contained within that range. For example, a numerical range of “1–5%” should be interpreted to include not only the explicitly listed values ​​from 1% to 5%, but also the independent values ​​and subranges within the indicated range. Thus, this numerical range includes independent values ​​such as 2%, 3.5%, and 4%, and subranges such as 1%–3%, 2%–4%, and 3%–5%, etc. This principle also applies to ranges that list only one value. Furthermore, this interpretation applies regardless of the width of the range or the characteristics described.

[0046] In this document, including in the claims, conjunctions such as "comprising," "including," "with," "having," "containing," "involving," and "accommodating" are understood to be open-ended, meaning "including but not limited to." Only the conjunctions "consisting of" and "composed of" are closed conjunctions.

[0047] To better illustrate the content of this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail, in order to highlight the main points of the invention.

[0048] Without conflict, the technical features disclosed in the embodiments of the present invention can be combined arbitrarily, and the resulting technical solutions belong to the content disclosed in the embodiments of the present invention.

[0049] In some embodiments, the CRISPR-Cas13a-based composite lyophilization protectant comprises: a vitrification matrix component, a structural support component, a protein aggregation inhibitor, and a surfactant. The vitrification matrix component comprises mannitol and trehalose in a mass ratio of 3–7:3–7. The structural support component comprises any one of PEG8000, PEG6000, and PEG4000 and any one of dextran 10000, dextran 8000, and dextran 20000, in a mass ratio of 1–5:1–5. The protein aggregation inhibitor is bovine serum albumin, and the surfactant is polyoxyethylene sorbitan monolaurate. The mass ratio of the structural support component to the vitrification matrix component is 1–5:3–7, the mass ratio of the protein inhibitor to the structural support component is 0.01–1:2–10, and the mass ratio of the surfactant to the structural support component is 0.001–0.1:2–10.

[0050] Generally, mannitol, as an excipient in the vitrification matrix component, contributes to the formation of a porous and robust "cake" structure during freeze-drying due to its crystallization properties. This ensures the freeze-dried product has a good appearance, is easy to reconstitute, and protects the structural integrity of active substances during drying and storage. Trehalose, as a molecular-level protectant in the vitrification matrix component, provides protection at the molecular level. Under stresses such as water shortage, high temperature, and freezing, it can replace water molecules to form hydrogen bonds with polar groups of biomolecules. During dehydration, it replaces water molecules, maintaining the native conformation of proteins, forming a high-viscosity glassy state, and preventing protein denaturation and membrane structure rupture. Polyethylene glycol components such as PEG8000, PEG6000, or PEG4000 in the structural support component, as nonionic polymers, compete for water molecules through steric exclusion effects, promoting the aggregation and precipitation of macromolecules. Dextran in the structural support component... Dextran 10000, Dextran 8000, or Dextran 2000 are biocompatible polysaccharides that can increase the effective concentration in solution and affect protein folding. Polyoxyethylene sorbitan monolaurate, namely Tween-20, is a nonionic surfactant that emulsifies, solubilizes, wets, and disperses other substances by reducing the surface tension of the liquid. Bovine serum albumin (BSA), as an inert protein, can adsorb onto the container surface, preventing the non-specific adsorption and inactivation of other important low-concentration biomolecules (such as enzymes and antibodies). Under the synergistic effect of the six components, in the resulting lyophilized microsphere molecular diagnostic reagent, the activity retention rate of Cas13a and T7 reverse transcriptase in the molecular diagnostic functional components is increased by more than 30% compared with a single protective agent, and the degradation rate of crRNA and rNTPs after 12 months of storage at room temperature is ≤10%.

[0051] Some embodiments disclose a CRISPR-Cas13a-based composite freeze-drying protectant, which, by weight, comprises: 5 parts mannitol, 5 parts trehalose, 3 parts PEG8000, 3 parts dextran10000, 0.3 parts bovine serum albumin, and 0.05 parts polyoxyethylene sorbitan monolaurate.

[0052] Some embodiments disclose CRISPR-Cas13a-based microsphere molecular diagnostic reagents, which are prepared from the composite lyophilization protectant, buffer solution and molecular diagnostic functional components disclosed in the embodiments of the present invention.

[0053] In some embodiments, the buffer solution comprises Tris-HCl buffer and MgCl2, wherein the pH of the Tris-HCl buffer solution is 8.

[0054] In some embodiments, the molecular diagnostic functional components include:

[0055] Mixture of 100–150 nM Cas13a protein, 2–8 U / μL T7 reverse transcriptase, 80–120 nM specific crRNA, 200–300 nM signal probe 7U-FAM-BHQ, and 1–2 mM rNTPs.

[0056] Some embodiments disclose methods for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, including the following steps:

[0057] S1, Preparation of premixed solution

[0058] S11. Adjust the pH of the Tris-HCl buffer to 8.0 to serve as the basic buffer system;

[0059] S12. Add the rNTPs mixture, bovine serum albumin, and polyoxyethylene sorbitan monolaurate to the basic buffer system in sequence. Dissolve by magnetic stirring at room temperature for 10 minutes with the stirring speed set to 300 rpm.

[0060] S13. Add the vitrified matrix component and structural support component of the composite freeze-drying protectant, place them in a 37°C water bath and stir for 20 minutes until a clear and transparent protectant solution is formed.

[0061] S14. After the protective agent solution cools to 4°C, add Cas13a protein, T7 reverse transcriptase, specific crRNA, and signal probe 7U-FAM-BHQ, mix well, and let stand at 4°C for 30 min to obtain the premixed solution before lyophilization.

[0062] S2. Packaging and freeze-drying

[0063] S21. Dispense the filtered premixed solution before lyophilization into 96-well lyophilization plates or individual lyophilization tubes at a volume of 20 μL / part, ensuring that the liquid level in each well / tube is consistent.

[0064] S22. Place the dispensed freeze-dried plates / tubes into the freeze dryer and perform freeze-drying according to the following procedure:

[0065] Pre-freezing stage: Keep at -40 to -35℃ for 2 to 2.5 hours under normal pressure;

[0066] Sublimation drying first stage: -40~-35℃ for 1~1.5h, pressure 10~20Pa;

[0067] Second stage of sublimation drying: keep at -30 to -25℃ for 4 to 5 hours, with a pressure of 10 to 20 Pa;

[0068] Third stage of sublimation drying: keep at -20 to -15℃ for 5 to 6 hours, with a pressure of 10 to 20 Pa;

[0069] Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa;

[0070] The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa.

[0071] The third stage of drying was analyzed: the temperature was kept at 30℃ for 4 hours, and the pressure was 5-10 Pa.

[0072] S23. Maintain a vacuum state inside the freeze dryer, fill the chamber with nitrogen to atmospheric pressure, and seal the freeze-drying plate / tube.

[0073] Typically, the prepared CRISPR-Cas13a-based microsphere molecular diagnostic reagents are lyophilized microspheres, appearing as a white, loose powder without collapse or clumping. They dissolve completely within 1 minute after reconstitution, leaving a clear, turbid solution. Moisture residue testing shows that the moisture residue rate, detected by the Karl Fischer method, is ≤1.5%, and generally less than 5% is considered acceptable. Activity testing shows that after reconstitution and addition of the target RNA template, incubation at 37°C for 30 minutes results in a fluorescence signal intensity ≥90% of that of the fresh liquid reagent. Stability testing shows that after storage at room temperature (25°C) for 12 months, the activity retention rate is ≥85%.

[0074] Typically, in the preparation of microsphere molecular diagnostic reagents, mannitol and trehalose in the composite lyophilization protectant form a vitrified matrix, while PEG8000 and dextran 10000 construct the spatial support structure. Bovine serum albumin (BSA), as a protein aggregation inhibitor, can effectively inhibit the aggregation of protein components. Polyoxyethylene sorbitan monolaurate can reduce interfacial tension. Under the synergistic effect of the six components, the activity retention rate of Cas13a and T7 reverse transcriptase in the molecular diagnostic functional components is increased by more than 30% compared with a single protectant, and the degradation rate of crRNA and rNTPs after 12 months of storage at room temperature is ≤10%.

[0075] Microsphere molecular diagnostic reagents have strong volume system adaptability. The reagent dosage can be flexibly adjusted within the system volume range of 15 to 50 μL. After reconstitution, they can be directly used in the detection process without additional dilution, making them suitable for POCT scenarios such as microfluidic chips and portable detection devices.

[0076] The optimized freeze-drying process pre-freezes the system at -40℃ for 2 hours to ensure complete freezing, uses segmented sublimation drying to avoid microsphere collapse, and employs gradient heating during the desorption drying stage to thoroughly remove residual moisture. The final product has a moisture content of ≤1.5% and a reconstitution rate of ≤1 min.

[0077] The stability of microsphere molecular diagnostic reagents is significantly improved. After 12 months of storage at room temperature (2–30°C), the activity retention rate is ≥85%, and cold chain transportation is not required, reducing logistics costs by more than 70%.

[0078] Some embodiments disclose a method for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, characterized in that lyophilization is performed in step S22 according to the following procedure:

[0079] Pre-freezing stage: -40℃ for 2 hours, at normal pressure;

[0080] First stage of sublimation drying: -40℃ for 1 hour, pressure 10~20Pa;

[0081] Second stage of sublimation drying: keep at -30℃ for 5 hours, with a pressure of 10-20 Pa;

[0082] Third stage of sublimation drying: keep at -20℃ for 5 hours, with a pressure of 10-20 Pa;

[0083] Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa;

[0084] The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa.

[0085] The third stage of drying was carried out at 30℃ for 4 hours under a pressure of 5-10 Pa.

[0086] Some embodiments disclose methods for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents, and also include steps for using the microsphere molecular diagnostic reagents, specifically including:

[0087] Take one portion of lyophilized microsphere molecular diagnostic reagent, reconstitute it with 45 μL of water, add 5 μL of the sample to be tested, reconstitute at room temperature for 1–2 min, incubate at 37 °C for 25–30 min, and read the signal value using a fluorescence detector.

[0088] Generally, microsphere molecular diagnostic reagents are easy to use. Freeze-dried microspheres only need to be added to the sample for reconstitution and detection, without the need for additional reagents, thus reducing human error. They have high detection efficiency: results can be obtained after incubation for 25-30 minutes after reconstitution, which is 20% shorter than the detection time of existing liquid reagents. They have a wide range of applications: by designing different specific crRNAs, they can detect a variety of RNA pathogens such as SARS-CoV-2, influenza virus, and hepatitis B virus, and can also be used for screening RNA targets related to genetic diseases.

[0089] The present invention discloses a CRISPR-Cas13a-based composite lyophilization protectant, microsphere molecular diagnostic reagent, and preparation method. It provides a composite lyophilization protectant that achieves synergistic stability protection of nucleic acids, Cas13a protein, and T7 reverse transcriptase; it provides an optimized lyophilization process to obtain lyophilized microspheres with low residual moisture content, rapid reconstitution, and uniform morphology; it enables room temperature storage and transportation of the reagents at 2–30°C, extending the shelf life to over 12 months; and it has promising application prospects in the field of molecular diagnostic reagents.

[0090] The technical details are further illustrated below with reference to the embodiments.

[0091] Example 1

[0092] Preparation of lyophilized microspheres in a 20 μL system

[0093] In Example 1, the basic buffer consisted of 20 mM Tris-HCl (pH 8.0) and 5 mM MgCl2, with a volume of 6.6 μL; the functional components included Cas13a protein 120 nM, T7 reverse transcriptase 6 U / μL, crRNA (targeting the SARS-CoV-2 N gene) 100 nM, signal probe 7 U-FAM-BHQ 250 nM, and rNTPs 0.8 mM, with a total volume of 6.6 μL; the composite lyophilization protectant consisted of mannitol 5%, trehalose 5%, PEG8000 3%, dextran 10000 3%, BSA 0.3%, and Tween-20 0.05%, with a volume of 6.8 μL.

[0094] CRISPR-Cas13a-based microsphere molecular diagnostic reagents were prepared using the following method, such as... Figure 1 As shown, the steps include:

[0095] S11. Adjust the pH of the Tris-HCl buffer to 8.0 and add MgCl2 to form the basic buffer system;

[0096] S12. Add the rNTPs mixture, BSA, and Tween-20 to the basic buffer system in sequence, and stir magnetically for 10 minutes at room temperature to dissolve them. The stirring speed is set to 300 rpm. Ensure complete dissolution.

[0097] S13. Add the vitrified matrix component and structural support component of the composite freeze-drying protectant, place them in a 37°C water bath and stir for 20 minutes until a clear and transparent protectant solution is formed.

[0098] S14. After the protective agent solution has cooled to 4°C, add Cas13a protein, T7 reverse transcriptase, specific crRNA, and signal probe 7U-FAM-BHQ. Gently invert and mix to avoid protein denaturation caused by clustering and stirring. Let stand at 4°C for 30 minutes to obtain the premixed solution before lyophilization.

[0099] S21. Dispense the filtered premixed solution before lyophilization into 96-well lyophilization plates or individual lyophilization tubes at a volume of 20 μL / part, ensuring that the liquid level in each well / tube is consistent.

[0100] S22. Place the dispensed freeze-dried plates / tubes into the freeze dryer and perform freeze drying:

[0101] Pre-freezing stage: -40℃ for 2 hours, at normal pressure;

[0102] First stage of sublimation drying: -40℃ for 1 hour, pressure 10Pa;

[0103] Second stage of sublimation drying: -30℃ for 5 hours, pressure 10Pa;

[0104] Third stage of sublimation drying: -20℃ for 5 hours, pressure 10Pa;

[0105] Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5Pa;

[0106] The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5Pa.

[0107] The third stage of drying was analyzed: the temperature was kept at 30℃ for 4 hours, and the pressure was 5Pa.

[0108] S23. Maintain a vacuum state inside the freeze dryer, fill the chamber with nitrogen to atmospheric pressure, and seal the freeze-drying plate / tube.

[0109] The freeze-dried microspheres obtained in Example 1 had a diameter of 2.86 mm and a moisture content of 1.43%. The system exhibited good homogeneity after reconstitution, with a CV of 3.2%. After storage at room temperature (25°C) for 12 months, the activity retention rate was 95.2%.

[0110] Example 2

[0111] Preparation of lyophilized microspheres in a 30 μL system

[0112] In Example 2, the amounts of each component were 10 μL for the basic buffer, 10 μL for the functional component, and 10 μL for the protective agent.

[0113] The preparation method is the same as in Example 1;

[0114] The microspheres obtained in Example 2 had a diameter of approximately 3.43 mm and a moisture content of 2.20%. After storage at room temperature (25°C) for 12 months, the activity retention rate was 98%. The activity retention rate experimental curve is shown below. Figure 2 As shown.

[0115] Example 3

[0116] Preparation of lyophilized microspheres in a 40 μL system

[0117] In Example 3, the amounts of each component were 13.3 μL for the basic buffer, 13.3 μL for the functional component, and 13.3 μL for the protective agent.

[0118] The preparation method is the same as in Example 1;

[0119] The finished microspheres obtained in Example 3 had a diameter of approximately 4.11 mm, a moisture content of 2.38%, and an activity retention rate of 97% after 12 months of storage at room temperature.

[0120] The performance test results of the freeze-dried microspheres in Examples 1 to 3 are shown in Tables 1 and 2; Table 1 shows the residual moisture rate detected by Karl Fischer method, and Table 2 shows the diameter of the finished microspheres.

[0121] Table 1. Residual moisture content of freeze-dried microspheres in Examples 1-3

[0122]

[0123] Table 2. List of freeze-dried microsphere diameters in Examples 1-3

[0124]

[0125] Example 4

[0126] Detection application verification

[0127] Take the lyophilized microspheres prepared in Example 1, add 15 μL of water to reconstitute the lyophilized microspheres; then add 5 μL of sample containing SARS-CoV-2 N gene RNA, the sample being the RAA product of a sample with a concentration of 10² copies / μL SARS-CoV-2 N gene RNA, reconstitute at room temperature for 1 min, incubate at 37°C for 30 min, and read the signal value using a fluorescence detector.

[0128] The fluorescence detector detected the FAM signal, and the signal value was 92% of that of the fresh liquid reagent. Specificity detection showed no cross-reactivity and no signal response to influenza virus or respiratory syncytial virus.

[0129] Comparative Example

[0130] Following the method of Example 1, lyophilization protectants were prepared using trehalose, mannitol, PEG8000, sucrose, PVP10, and dextran as protectants, respectively.

[0131] The lyophilization protectants obtained in Example 1, trehalose, mannitol, PEG8000, sucrose, PVP10, and dextran obtained in the comparative example were used as protectants to prepare lyophilization protectants, and their enzyme activity retention rates were compared with those of liquid molecular diagnostic reagents (liquid control). The liquid molecular diagnostic reagents included: 100 nM Cas13a protein, 2 U / μL T7 reverse transcriptase, 80 nM specific crRNA, 200 nM signal probe 7U-FAM-BHQ, and 1 mM rNTPs.

[0132] The results are shown in Table 3. Figure 3 As shown, the enzyme activity retention rate of the composite protectant is the highest compared to that of the liquid molecular diagnostic reagent (liquid control).

[0133] Table 3. List of test results for relative activity retention rate of protective agents (%)

[0134]

[0135] The present invention discloses a CRISPR-Cas13a-based composite lyophilization protectant, microsphere molecular diagnostic reagent, and preparation method. It provides a composite lyophilization protectant that achieves synergistic stability protection of nucleic acids, Cas13a protein, and T7 reverse transcriptase; it provides an optimized lyophilization process to obtain lyophilized microspheres with low residual moisture content, fast reconstitution speed, and uniform morphology; it enables the reagent to be stored and transported at room temperature (2-30℃), extending the shelf life to over 12 months; and it has promising application prospects in the field of molecular diagnostic reagents.

[0136] The technical solutions and technical details disclosed in the embodiments of this invention are merely illustrative of the inventive concept of this invention and do not constitute a limitation on the technical solutions of the embodiments of this invention. Any conventional changes, substitutions, or combinations made to the technical details disclosed in the embodiments of this invention have the same inventive concept as this invention and are within the protection scope of the claims of this invention.

Claims

1. A CRISPR-Cas13a-based composite lyophilization protectant, characterized in that, include: Vitrification matrix components, structural support components, protein aggregation inhibitors, and surfactants: among which, The vitrification matrix component includes mannitol and trehalose in a mass ratio of 3-7:3-7; The structural support component includes any one of PEG8000, PEG6000, and PEG4000 and any one of dextran 10000, dextran 8000, and dextran 20000, with a mass ratio of 1-5:1-5. The protein aggregation inhibitor is bovine serum albumin, and the surfactant is polyoxyethylene sorbitan monolaurate. The mass ratio of the vitrified matrix component to the structural support component is 3-7:1-5, the mass ratio of the protein inhibitor to the structural support component is 0.01-1:2-10, and the mass ratio of the surfactant to the structural support component is 0.001-0.1:2-10.

2. The CRISPR-Cas13a-based composite lyophilization protectant according to claim 1, characterized in that, By weight, it includes: 5 parts mannitol, 5 parts trehalose, 3 parts PEG8000, 3 parts dextran10000, 0.3 parts bovine serum albumin, and 0.05 parts polyoxyethylene sorbitan monolaurate.

3. A CRISPR-Cas13a-based microsphere molecular diagnostic reagent, prepared from the composite lyophilization protectant, buffer solution, and molecular diagnostic functional components described in claim 1.

4. The CRISPR-Cas13a-based microsphere molecular diagnostic reagent according to claim 3, characterized in that, The buffer solution comprises Tris-HCl buffer and MgCl2, and the pH of the Tris-HCl buffer solution is 8.

5. The CRISPR-Cas13a-based microsphere molecular diagnostic reagent according to claim 3, characterized in that, The molecular diagnostic functional components include: Mixture of 100–150 nM Cas13a protein, 2–8 U / μL T7 reverse transcriptase, 80–120 nM specific crRNA, 200–300 nM signal probe 7U-FAM-BHQ, and 1–2 mM rNTPs.

6. The method for preparing the CRISPR-Cas13a-based microsphere molecular diagnostic reagent according to any one of claims 3 to 5, characterized in that, Including the following steps: S1. Preparation of premixed solution: S11. Adjust the pH of the Tris-HCl buffer to 8.0 to serve as the basic buffer system; S12. Add rNTPs mixture, bovine serum albumin and polyoxyethylene sorbitan monolaurate to the basic buffer system in sequence. Dissolve by magnetic stirring at room temperature for 10 min with the stirring speed set to 300 rpm. S13. Add the vitrified matrix component and structural support component of the composite freeze-drying protectant, place them in a 37°C water bath and stir for 20 minutes until a clear and transparent protectant solution is formed. S14. After the protective agent solution cools to 4°C, add Cas13a protein, T7 reverse transcriptase, specific crRNA, and signal probe 7U-FAM-BHQ, mix well, and let stand at 4°C for 30 min to obtain the premixed solution before lyophilization. S2. Packaging and freeze-drying: S21. Dispense the filtered premixed solution before lyophilization into 96-well lyophilization plates or individual lyophilization tubes at a volume of 20 μL / part, ensuring that the liquid level in each well / tube is consistent. S22. Place the dispensed freeze-dried plates / tubes into the freeze dryer and perform freeze-drying according to the following procedure: Pre-freezing stage: Keep at -40 to -35℃ for 2 to 2.5 hours under normal pressure; Sublimation drying first stage: -40~-35℃ for 1~1.5h, pressure 10~20Pa; Second stage of sublimation drying: keep at -30 to -25℃ for 4 to 5 hours, with a pressure of 10 to 20 Pa; Third stage of sublimation drying: keep at -20 to -15℃ for 5 to 6 hours, with a pressure of 10 to 20 Pa; Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa; The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa. The third stage of drying was analyzed: the temperature was kept at 30℃ for 4 hours, and the pressure was 5-10 Pa. S23. Maintain a vacuum state inside the freeze dryer, fill the chamber with nitrogen to atmospheric pressure, and seal the freeze-drying plate / tube.

7. The method for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents according to claim 6, characterized in that, In step S22, freeze drying is performed according to the following procedure: Pre-freezing stage: -40℃ for 2 hours, at normal pressure; First stage of sublimation drying: -40℃ for 1 hour, pressure 10~20Pa; Second stage of sublimation drying: keep at -30℃ for 5 hours, with a pressure of 10-20 Pa; Third stage of sublimation drying: keep at -20℃ for 5 hours, with a pressure of 10-20 Pa; Analysis of the first stage of drying: 0℃ for 1 hour, pressure of 5-10 Pa; The second stage of drying was carried out at 20℃ for 1 hour under a pressure of 5-10 Pa. The third stage of drying was carried out at 30℃ for 4 hours under a pressure of 5-10 Pa.

8. The method for preparing CRISPR-Cas13a-based microsphere molecular diagnostic reagents according to claim 6 or 7, characterized in that, It also includes the usage steps of the microsphere molecular diagnostic reagent, specifically including: Take one portion of lyophilized microsphere molecular diagnostic reagent, reconstitute it with 45 μL of water, add 5 μL of the sample to be tested, reconstitute at room temperature for 1–2 min, incubate at 37 °C for 25–30 min, and read the signal value using a fluorescence detector.

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