Freeze-drying protective agent and freeze-drying reagent of PCR (Polymerase Chain Reaction) full premix as well as preparation method and application of freeze-drying protective agent

Abstract

The invention relates to a freeze-drying protective agent and a freeze-drying reagent of a PCR (Polymerase Chain Reaction) full premixed solution as well as a preparation method and an application of the freeze-drying protective agent. The freeze-drying protective agent is prepared from the following components in working concentration: 0.01% v / v to 0.05% v / v of lipid nanoparticles, 4% w / v to 10% w / v of trehalose, 2% w / v to 5.36% w / v of glucan, 0.10% w / v to 0.30% w / v of BSA (Bovine Serum Albumin), 0.10% w / v to 0.30% w / v of PEG8000 (Polyethylene Glycol 8000), 0.01% v / v to 0.05% v / v of a surfactant and 0.01% v / v to 0.03% v / v of a de-foaming agent. And the stability of the spatial structure of some heat-sensitive substances in the PCR full premix reagent in the dehydration process can be protected, so that the heat-sensitive substances still have the original biological activity after being redissolved. The freeze-drying reagent prepared by using the freeze-drying protective agent can be stored at normal temperature for a long time.

Description

Technical Field

[0001] The present application relates to the field of molecular biology detection, and specifically to a lyophilization protectant, a lyophilization reagent, a preparation method and an application of a PCR complete premix. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It is widely used in diagnostic reagents for the detection of viruses, bacteria, and gene mutations. PCR detection reagents usually contain thermosensitive substances such as Taq enzyme, UDG or UNG enzyme, dNTP Mix, primer probes, etc. These substances are sensitive to temperature, and excessive or repeated freezing and thawing will affect the performance and stability of the detection reagents. Therefore, the storage and transportation of PCR detection reagents require special conditions. Usually, the reagents need to be stored separately in 2-3 mixed components to maintain their stability.

[0003] Traditional liquid cryopreservation technology cannot achieve fully premixed cryopreservation of PCR test reagents. Usually, the enzyme solution and other components are separated into 2 or more mixed components for cryopreservation. The stability of cryopreserved reagents is affected by repeated freezing and thawing. Cryopreservation can never get rid of its dependence on temperature, and special equipment is required. Transportation also needs to go through the cold chain. Air-drying technology started late, and its applicability has yet to be verified. At the same time, air-drying technology has the disadvantages of long existence time, slow re-dissolution after air-drying, and the scope of application of air-drying is temporarily limited.

[0004] Freeze drying technology is a method of dehydrating materials at low temperatures and maintaining their original spatial structure by freezing them at low temperatures and then drying them in a vacuum. This technology is often used for long-term storage of food, medicines, biological products, etc., and can effectively prevent the deterioration and corruption of materials.

[0005] The freeze-drying of PCR detection reagents began in the late 20th century. The key to freeze-drying PCR detection reagents is to match them with suitable lyoprotectants, which not only protect the PCR detection reagents, but also act as excipients during the freeze-drying process. However, there are thousands of substances that can be used as lyoprotectants and excipients. Therefore, in order to achieve full premix freeze-drying of PCR detection reagents, it is necessary to select suitable protectants and excipients and use appropriate concentrations to achieve the desired effect. Summary of the invention

[0006] Based on this, one embodiment of the present application provides a lyophilization protectant for a PCR premix, which can protect the stability of the spatial structure of some heat-sensitive substances in the PCR premix during the dehydration process, so that the substances still have their original biological activity after reconstitution. The lyophilized reagent prepared using the lyophilization protectant can be stored at room temperature for a long time.

[0007] The technical solutions include the following:

[0008] A lyophilized protective agent for a complete PCR premix, the lyophilized protective agent comprising components at the following working concentrations: 0.01% v / v ~ 0.05% v / v lipid nanoparticles, 4% w / v ~ 10% w / v trehalose, 2% w / v ~ 5.36% w / v dextran, 0.10% w / v ~ 0.30% w / v BSA, 0.10% w / v ~ 0.30% w / v PEG8000, 0.01% v / v ~ 0.05% v / v surfactant and 0.01% v / v ~ 0.03% v / v defoaming agent.

[0009] In one embodiment, the lyoprotectant satisfies one or more of the following conditions:

[0010] (1) The surfactant includes Tween-20;

[0011] (2) The dextran includes dextran-40;

[0012] (3) The defoaming agent includes a non-ionic TF composite fermentation defoaming agent.

[0013] In one embodiment, the lipid nanoparticles are 10nm~50nm.

[0014] The freeze-dried reagent of the PCR complete premix comprises the freeze-dried protective agent and the PCR reaction system.

[0015] In one embodiment, the PCR reaction system includes PCR Buffer, enzyme, primers, probe, dATP, dCTP, dGTP, dUTP, Mg 2+ and one or more of an RNase inhibitor.

[0016] In one embodiment, the enzyme comprises a DNA polymerase and / or a reverse transcriptase.

[0017] In one embodiment, the enzyme further comprises UNG enzyme and / or UDG enzyme.

[0018] In one embodiment, the freeze-dried reagent of the PCR premix includes one or more of the freeze-dried reagent of the common PCR premix, the freeze-dried reagent of the qPCR premix, the freeze-dried reagent of the RT-qPCR premix, the freeze-dried reagent of the enzyme cutting method PCR premix, and the freeze-dried reagent of the single-tube super multiplex PCR melting curve method premix.

[0019] The preparation method of the freeze-dried reagent comprises the following steps:

[0020] Mixing the lyophilized protective agent with the PCR reaction system to prepare a PCR premix solution; and

[0021] The PCR complete premix is ​​freeze-dried to prepare a freeze-dried reagent.

[0022] In one embodiment, freeze drying includes a pre-freezing process and a sublimation process in sequence;

[0023] In one embodiment, the parameters of the pre-freezing process are:

[0024] Set the temperature to 10℃~-10℃, the temperature rise and fall time is 30min~90min, and the holding time is 0.5h~1h;

[0025] Set the temperature to -20℃~-50℃, the heating and cooling time to 30min~90min, and the holding time to 0.5h~1h;

[0026] Set the temperature to 0℃~-20℃, the heating and cooling time to 30min~90min, and the holding time to 0.5h~1h;

[0027] Set the temperature to -20℃~-50℃, the temperature rise and fall time is 30min~90min, and the holding time is 1h~3h;

[0028] The parameters of the sublimation process are:

[0029] Set the temperature to -20℃~-50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 3h~10h;

[0030] Set the temperature to -35℃~-50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 0.5h~2h;

[0031] Set the temperature to -5℃~-20℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 0.5h~1h;

[0032] Set the temperature to -10℃~-5℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 1Pa~20Pa, and the holding time to 0.5h~1h;

[0033] Set the temperature to 10℃~25℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 0Pa~1Pa, and the holding time to 0.5h~1h;

[0034] Set the temperature to 25℃~50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 0Pa~1Pa, and the holding time to 1h~3h;

[0035] Set the temperature to 25℃~30℃, the heating and cooling time to 0min~30min, the vacuum degree to 0Pa~1Pa, and the holding time to 0.5h~1h.

[0036] The application of the freeze-dried reagent in PCR detection.

[0037] Compared with the traditional technology, this application has the following beneficial effects:

[0038] The present application provides a lyophilized protective agent for a nanoparticle-based PCR detection reagent full premix, which can protect the stability of the spatial structure of some heat-sensitive substances in the PCR full premix during the dehydration process, so that the substances still have the original biological activity after reconstitution; at the same time, the lyophilized protective agent of the present application also contains a certain concentration of excipients, so that the full premix can present a good form after freeze-drying.

[0039] The present application uses a combination of lipid nanoparticles and excipients, which can not only act as crystal nuclei when the liquid is frozen, shortening the freezing time, but also improve the anti-interference ability and amplification efficiency of the freeze-dried reagent.

[0040] The lyophilized protective agent of the present application is used in the PCR full premix detection reagent, and can be stably stored at room temperature for more than 12 months after lyophilization. The lyophilized protective agent of the present application is used in the PCR amplification system to make the amplification efficiency higher, the Ct value relatively smaller, and the sensitivity higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0042] Figure 1 A lyophilized form of a lyophilized reagent that does not contain a lyoprotectant;

[0043] Figure 2 A lyophilized form of a lyophilized reagent that does not contain a nanoparticle lyoprotectant;

[0044] Figure 3 A lyophilized form of a lyophilized reagent containing a nanoparticle lyoprotectant;

[0045] Figure 4 It is the lyophilized form of the fully premixed lyophilized reagent for the HBV PCR detection system;

[0046] Figure 5 It is the lyophilized form of the fully premixed lyophilized reagents for the HCV PCR detection system;

[0047] Figure 6 It is a lyophilized form of the APOE genotyping lyophilized reagent;

[0048] Figure 7 It is the lyophilized form of HPV 31 genotyping lyophilized reagent;

[0049] Figure 8 This is the dissolution peak diagram of the liquid reagent for HPV 31 genotyping types;

[0050] Fig. 9 This is the dissolution peak diagram of the HPV 31 genotyping freeze-dried reagent;

[0051] Fig.10 This is the accelerated stability monitoring chart of freeze-dried reagents containing different protective agents;

[0052] Fig.11 This is a real-time stability monitoring chart of a lyophilized reagent containing the lyophilized protective agent of the present application;

[0053] Fig.12 To test the precision of freeze-dried reagents containing freeze-dried protective agents of the present application;

[0054] Fig.13 The state of the detection reagent without the lyophilized protective agent of the present application after shaking and mixing;

[0055] Fig.14 It is the state of the detection reagent containing the lyophilized protective agent after shaking and mixing. DETAILED DESCRIPTION

[0056] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0058] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0059] In this article, PCR: Polymerase Chain Reaction.

[0060] Herein, lyoprotectants are mainly used to protect heat-sensitive substances or combined substances during the lyophilization process.

[0061] In this article, excipients: act as a supporting framework to maintain a certain shape after freeze-drying.

[0062] One embodiment of the present application provides a lyophilized protective agent for a PCR complete premix, the lyophilized protective agent comprising the following components at working concentrations: 0.01% v / v ~ 0.05% v / v lipid nanoparticles, 4% w / v ~ 10% w / v trehalose, 2% w / v ~ 5.36% w / v dextran, 0.10% w / v ~ 0.30% w / v BSA, 0.10% w / v ~ 0.30% w / v PEG8000, 0.01% v / v ~ 0.05% v / v surfactant and 0.01% v / v ~ 0.03% v / v defoaming agent.

[0063] This application is based on a freeze-drying technology platform. By selecting suitable protective agents and excipients for combined use, and creatively adding nanoparticles to the protective agent, the fully premixed PCR detection reagent after freeze-drying not only gets rid of the low temperature restrictions on transportation and storage, but also improves the amplification efficiency.

[0064] In a specific example, in the lyoprotectant, the working concentration of the lipid nanoparticles can be but is not limited to 0.01% v / v, 0.02% v / v, 0.03% v / v, 0.04% v / v, 0.05% v / v, or a range between any two of the above values.

[0065] In a specific example, the lipid nanoparticles are 10nm to 50nm. In a specific example, the lipid nanoparticles are 10nm, 20nm, 30nm, 40nm, 50nm, or a range between any two of the above values. In a specific example, the lipid nanoparticles are 10nm to 20nm, and the lyophilized protective agent of lipid nanoparticles in this range is used in the PCR amplification system to make the amplification efficiency higher, the Ct value relatively smaller, and the sensitivity higher.

[0066] Lipid nanoparticles are spherical vesicles composed of one (unilayer) or more (multilayer) phospholipid bilayers.

[0067] Those skilled in the art can prepare the lipid nanoparticles according to conventional technical means. As long as the particle size of the lipid nanoparticles meets the requirement of 10nm~50nm, the formed lyophilization protectant or lyophilization reagent is within the protection scope of this application.

[0068] In a specific example, the lipid nanoparticles include phase A and phase B, phase A includes one or more of ionizable lipids, phospholipids and cholesterol, and phase B includes stearic acid. In a specific example, the volume ratio of phase A to phase B is 1: (9-11), and the volume ratio of phase A to phase B is optionally 1: 10. In a specific example, phase A includes ionizable lipids, phospholipids and cholesterol. In a specific example, the mass ratio of ionizable lipids, phospholipids and cholesterol is 1: (2~3): (1~2).

[0069] In a specific example, the ionizable lipid includes an ionizable cationic lipid, and the ionizable cationic lipid includes one or more of DLinDMA, DLin-KC2-DMA, and D-Lin-MC3-DMA.

[0070] In one specific example, the phospholipid includes lecithin.

[0071] In a specific example, the method for preparing lipid nanoparticles comprises the following steps:

[0072] Prepare phase A;

[0073] Prepare phase B;

[0074] Phase A and phase B are mixed to prepare lipid nanoparticles, and the lipid nanoparticles have a size of 10 nm to 50 nm.

[0075] In a specific example, preparing phase A includes mixing ionizable lipids, phospholipids and cholesterol to obtain a mixture, and dissolving the mixture in ethanol at a ratio of W / V=1:(3-5) to form phase A.

[0076] In one specific example, preparing the B phase includes heating and dissolving stearic acid to form the B phase.

[0077] In a specific example, after mixing phase A and phase B, distilled water is added to the mixture to form a slightly turbid emulsion, and the slightly turbid emulsion is added to cold water and mixed, and then crushed for 10 minutes to 30 minutes.

[0078] In a specific example, the volume ratio of the slightly turbid emulsion to the cold water is 1:20-1:50.

[0079] In a specific example, in the lyoprotectant, the working concentration of trehalose can be, but is not limited to, 4% w / v, 5% w / v, 6% w / v, 6.25% w / v, 7% w / v, 7.3% w / v, 8% w / v, 9% w / v, 10% w / v, or a range between any two of the above values.

[0080] In a specific example, in the lyoprotectant, the working concentration of dextran can be, but is not limited to, 2% w / v, 2.5% w / v, 2.92% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v, 5% w / v, 5.36% w / v, or a range between any two of the above values.

[0081] In a specific example, the dextran includes dextran-40. Dextran-40 is used in the freeze-drying protective agent of the present application in combination with other components, which can greatly increase the eutectic point of the full premix solution, thereby increasing the sublimation temperature, shortening the freeze-drying time and reducing energy consumption.

[0082] In a specific example, in the lyoprotectant, the working concentration of BSA can be, but is not limited to, 0.1% w / v, 0.13% w / v, 0.15% w / v, 0.2% w / v, 0.3% w / v, or a range between any two of the above values.

[0083] In a specific example, in the lyoprotectant, the working concentration of PEG8000 can be but is not limited to 0.10% w / v, 0.12% w / v, 0.15% w / v, 0.17% w / v, 0.19% w / v, 0.22% w / v, 0.25% w / v, 0.30% w / v, 0.30% w / v, or a range between any two of the above values.

[0084] In a specific example, in the lyoprotectant, the working concentration of the surfactant can be but is not limited to 0.01% v / v, 0.02% v / v, 0.03% v / v, 0.04% v / v, 0.05% v / v, or a range between any two of the above values.

[0085] In a specific example, the surfactant includes one or more of Ween-20, Tween-80, and polyethylene glycol octylphenyl ether.

[0086] In a specific example, in the lyoprotectant, the working concentration of the antifoaming agent can be, but is not limited to, 0.01% v / v, 0.02% v / v, 0.03% v / v, or a range between any two of the above values.

[0087] In a specific example, the defoaming agent includes a non-ionic TF compound fermentation defoaming agent. The defoaming agent is used in the protective agent of the present application to eliminate bubbles generated when the reagents are mixed and packaged.

[0088] An embodiment of the present application further provides a freeze-dried reagent for a complete PCR premix, comprising the freeze-dried protective agent and a PCR reaction system.

[0089] In a specific example, the lyophilized reagents of the PCR complete premix include, but are not limited to, one or more of the lyophilized reagents of the common PCR complete premix, the lyophilized reagents of the qPCR complete premix, the lyophilized reagents of the RT-qPCR complete premix, the lyophilized reagents of the enzyme cutting method PCR complete premix, and the lyophilized reagents of the single-tube super multiplex PCR melting curve method complete premix (MPA).

[0090] It should be noted that, in other specific examples, those skilled in the art can combine the lyophilized protectant of the present application with any existing PCR reaction system to form a lyophilized reagent for a PCR premix, all of which are within the protection scope of the present application.

[0091] In a specific example, the PCR reaction system includes but is not limited to PCR Buffer, enzyme, primer, probe, dATP, dCTP, dGTP, dUTP, Mg 2+ and one or more of an RNase inhibitor.

[0092] In one specific example, the enzyme includes a DNA polymerase and / or a reverse transcriptase.

[0093] In a specific example, the enzyme further includes UNG enzyme and / or UDG enzyme.

[0094] It should be noted that those skilled in the art can select the corresponding PCR reaction system and the lyophilized protective agent of the present application to form a lyophilized reagent through the required PCR technology. The concentration of each substance in the PCR reaction system can be selected and adjusted according to conventional techniques, or according to the instructions of the existing commercially available kits.

[0095] One embodiment of the present application provides a method for preparing the lyophilized reagent, the preparation method comprising the following steps:

[0096] Mixing the lyophilized protective agent with the PCR reaction system to prepare a PCR premix solution; and

[0097] The PCR complete premix is ​​freeze-dried to prepare a freeze-dried reagent.

[0098] In a specific example, freeze-drying includes a pre-freezing process and a sublimation process in sequence.

[0099] In a specific example, the parameters of the pre-freezing process are:

[0100] Set the temperature to 10℃~-10℃, the heating and cooling time is 30min~90min, and the holding time is 0.5h~1h; set the temperature to -20℃~-50℃, the heating and cooling time is 30min~90min, and the holding time is 0.5h~1h; set the temperature to 0℃~-20℃, the heating and cooling time is 30min~90min, and the holding time is 0.5h~1h; set the temperature to -20℃~-50℃, the heating and cooling time is 30min~90min, and the holding time is 1h ~3h.

[0101] In a specific example, the parameters of the sublimation process are:

[0102] Set the temperature from -20℃~-50℃, the temperature rise and fall time is 30min~90min, the vacuum degree is 10Pa~50Pa, and the holding time is 3h~10h; set the temperature from -35℃~-50℃, the temperature rise and fall time is 30min~90min, the vacuum degree is 10Pa~50Pa, and the holding time is 0.5h~2h; set the temperature from -5℃~-20℃, the temperature rise and fall time is 30min~90min, the vacuum degree is 10Pa~50Pa, and the holding time is 0.5h~1h; set the temperature from -10℃~-5℃, the temperature rise and fall time is 30 min~90min, vacuum degree 1Pa~20Pa, holding time 0.5h~1h; set temperature 10℃~25℃, heating and cooling time 30min~90min, vacuum degree 0Pa~1Pa, holding time 0.5h~1h; set temperature 25℃~50℃, heating and cooling time 30min~90min, vacuum degree 0Pa~1Pa, holding time 1h~3h; set temperature 25℃~30℃, heating and cooling time 0min~30min, vacuum degree 0Pa~1Pa, holding time 0.5h~1h.

[0103] One embodiment of the present application provides the use of the lyophilized reagent in PCR detection.

[0104] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0105] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0106] The main reagents involved in the following examples include the following:

[0107] Stearic acid CAS No.: 57-11-4, phospholipid (lecithin) CAS No.: 8002-43-5; ionizable lipid (ionizable cationic phospholipid) CAS No.: 871258-12-7; cholesterol CAS No.: 57-88-5; trehalose, Aladdin; dextran-40, PEG8000, Tween 20, defoaming agent, Sangon Biotech (Shanghai) Co., Ltd.; BSA, Roche; the PCR products involved in the embodiments are commercially available human APOE genotyping detection kit (fluorescent probe method), hepatitis C virus (HCV) nucleic acid detection kit (fluorescent PCR method), hepatitis B virus (HBV) nucleic acid detection kit (fluorescent PCR method), human papillomavirus (HPV) 31 genotyping detection kit (fluorescent PCR-melting curve method).

[0108] The following preparation methods of lipid nanoparticles with different particle sizes of 10 nm, 20 nm, 50 nm, 100 nm, etc. include:

[0109] Ionizable lipids, phospholipids and cholesterol are selected and mixed in a mass ratio of 1:3:1, and the mixture is dissolved in ethanol in a ratio of W / V=1:4 to form an aqueous phase, stearic acid is heated at 72°C to dissolve, and added to the aqueous phase in a volume ratio of 1:10 to mix, and then distilled water is gradually added until it is slightly turbid, and the slightly turbid emulsion is added to 2°C cold water at a rate of 1 drop / 5s, and the volume of the emulsion and the volume of the cold water are mixed at a ratio of 1:10, 1:20, 1:30 and 1:50, respectively, and then placed in an ultrasonic crusher for crushing for 10 min, 10 min, 20 min and 30 min, respectively, and crushed ice is used to keep the crushing process at a low temperature, thereby forming lipid nanoparticles (which may be referred to as nanoparticles) with average particle sizes of about 100 nm, 50 nm, 20 nm and 10 nm, respectively.

[0110] The freeze-drying parameter settings recommended in the following examples are shown in Table 1:

[0111] Table 1

[0112]

[0113] It is understandable that those skilled in the art can routinely select other freeze-drying parameters for freeze-drying process as needed.

[0114] Example 1

[0115] A freeze-dried protective agent for a PCR complete premix: lipid nanoparticles with a particle size of 20 nm, trehalose, dextran-40, BSA, PEG8000, Tween-20 and a non-ionic TF composite fermentation defoaming agent.

[0116] The above lyophilized protective agent is added to the PCR amplification system to prepare a lyophilized reagent. The final concentration (working concentration) of each component of the lyophilized protective agent in the lyophilized reagent is shown in Table 2.

[0117] Table 2

[0118]

[0119] Example 2

[0120] A freeze-dried protective agent for a PCR complete premix: lipid nanoparticles with a particle size of 20 nm, trehalose, dextran-40, BSA, PEG8000, Tween-20 and a non-ionic TF composite fermentation defoaming agent.

[0121] The lyophilized protective agent is added to the PCR amplification system to prepare a lyophilized reagent. The final concentration (working concentration) of each component of the lyophilized protective agent in the lyophilized reagent is shown in Table 3.

[0122] Table 3

[0123]

[0124] Example 3

[0125] A freeze-dried protective agent for a PCR complete premix: lipid nanoparticles with a particle size of 20 nm, trehalose, dextran-40, BSA, PEG8000, Tween-20 and a non-ionic TF composite fermentation defoaming agent.

[0126] The lyophilized protective agent is added to the PCR amplification system to prepare a lyophilized reagent. The final concentration (working concentration) of each component of the lyophilized protective agent in the lyophilized reagent is shown in Table 4.

[0127] Table 4

[0128]

[0129] Example 4

[0130] A freeze-dried protective agent for a PCR complete premix: lipid nanoparticles with a particle size of 20 nm, trehalose, dextran-40, BSA, PEG8000, Tween-20 and a non-ionic TF composite fermentation defoaming agent.

[0131] The lyophilized protective agent is added to the PCR amplification system to prepare a lyophilized reagent. The final concentration (working concentration) of each component of the lyophilized protective agent in the lyophilized reagent is shown in Table 5.

[0132] Table 5

[0133]

[0134] Example 5

[0135] A freeze-dried protective agent for a PCR complete premix: lipid nanoparticles with a particle size of 20 nm, trehalose, dextran-40, BSA, PEG8000, Tween-20 and a non-ionic TF composite fermentation defoaming agent.

[0136] The lyophilized protective agent is added to the PCR amplification system to prepare a lyophilized reagent. The final concentration (working concentration) of each component of the lyophilized protective agent in the lyophilized reagent is shown in Table 6.

[0137] Table 6

[0138]

[0139] Example 6

[0140] The contents are substantially the same as those of Example 5, except that the lipid nanoparticles are 20 nm in size.

[0141] Example 7

[0142] The contents are substantially the same as those of Example 5, except that the lipid nanoparticles are 50 nm in size.

[0143] Example 8

[0144] The contents are substantially the same as those of Example 5, except that the lipid nanoparticles are 100 nm.

[0145] Example 9

[0146] The lyophilized protective agents containing lipid nanoparticles with particle sizes of 10nm (Example 5), 20nm (Example 6), 50nm (Example 7), and 100nm (Example 8) were added to the amplification system for comparative detection. Each concentration was tested 3 times in each reagent, and the average value of 3 times was taken for statistics. The results are shown in Table 7. The amplification effect of nanoparticles with a particle size of 10nm-20nm added to the amplification system is better than that of 50nm and better than that of 100nm. Among them, the amplification effect of nanoparticles with a particle size of 20nm is the best.

[0147] Table 7 Effects of nanoparticles of different sizes on PCR detection (Ct value)

[0148]

[0149] Note: The control group is the freeze-dried protective agent group without nanoparticles.

[0150] Example 10

[0151] Comparison of lyoprotectant containing nanoparticles, lyoprotectant without nanoparticles, and no lyoprotectant.

[0152] The lyophilization protectant containing nanoparticles (the final concentration of each component of the lyophilization protectant in the lyophilization reagent is the same as in Example 1), the lyophilization protectant without nanoparticles, and the lyophilization system without lyophilization protectant were added to the hepatitis B virus (HBV) PCR detection system, and lyophilization was performed. The lyophilization system is shown in Table 8. After lyophilization, 30 μl of water was used for reconstitution and comparative detection. Each concentration was repeated 3 times in each reagent, and the average value of 3 times was taken for statistical analysis. The results are shown in Table 9. The lyophilized form of the lyophilized reagent is as shown in Table 8. Figure 1-3 shown.

[0153] Table 8 HBV freeze-drying system

[0154]

[0155] Table 9 Comparison of PCR test results (Ct values) of three different freeze-dried systems

[0156]

[0157] Note: FAM represents target, and VIC represents internal standard.

[0158] Embodiment 11

[0159] This example is a fully premixed lyophilized reagent (qPCR) for the hepatitis B virus (HBV) PCR detection system.

[0160] A lyophilized protective agent was added to the HBV system for lyophilization. The final concentration of each component of the lyophilized protective agent in the lyophilized reagent was the same as in Example 1. After lyophilization and reconstitution, a comparative test was performed with the liquid reagent (control). Each concentration was tested 3 times in each reagent, and the average of the 3 times was taken for statistical analysis. The results are shown in Table 10. The results show that the detection performance of the reagent after lyophilization is not affected. The lyophilized form of the lyophilized reagent is as shown in Figure 4 shown.

[0161] Table 10 Comparison of HBV PCR test results (Ct value) between freeze-dried reagent and liquid reagent

[0162]

[0163] Note: FAM represents target, and VIC represents internal standard.

[0164] Example 12

[0165] This example is a fully premixed lyophilized reagent (RT-qPCR) for the hepatitis C virus (HCV) PCR detection system.

[0166] A lyophilized protective agent was added to the HCV high-sensitivity system for lyophilization. The final concentration of each component of the lyophilized protective agent in the lyophilized reagent was the same as in Example 2. The lyophilized system was shown in Table 11. After lyophilization, it was reconstituted with 30 μl of water and compared with the liquid reagent (control). Each concentration was repeated 3 times in each reagent, and the average value of 3 times was taken for statistics. The results are shown in Table 12. The results show that the detection performance of the reagent after lyophilization is not affected. The lyophilized form of the lyophilized reagent is as shown in Figure 5 shown.

[0167] Table 11 HCV freeze-drying system

[0168]

[0169] Table 12 Comparison of PCR test results (Ct value) of HCV freeze-dried reagent and liquid reagent

[0170]

[0171] Note: FAM represents target, and VIC represents internal standard.

[0172] Example 13

[0173] This example is a fully premixed lyophilized reagent for the apolipoprotein E (Apolipoprotein E, APOE) genotyping PCR detection system (enzyme digestion-qPCR).

[0174] A lyophilized protective agent was added to the APOE PCR system for lyophilization. The final concentration of each component of the lyophilized protective agent in the lyophilized reagent was the same as in Example 3. The lyophilized system was shown in Table 13. After lyophilization, 35 μl of water was used to reconstitute and then compared with the liquid reagent (control). Each concentration was repeated 3 times in each reagent, and the average value of 3 times was taken for statistics. The results are shown in Table 14. The results show that the detection performance of the reagent after lyophilization is not affected. The lyophilized form of the lyophilized reagent is as shown in Figure 6 shown.

[0175] Table 13 APOE genotyping freeze-dried system

[0176]

[0177] Table 14 Comparison of PCR test results (Ct value) of APOE genotyping freeze-dried reagent and liquid reagent

[0178]

[0179] Note: FAM represents target, and VIC represents internal standard.

[0180] Embodiment 14

[0181] This example is a fully premixed lyophilized reagent for a super-multiplex PCR detection system for 31 genotyping types of human papillomavirus (HPV) (melting curve qPCR).

[0182] A lyophilized protective agent was added to the HPV31 typing system for lyophilization. The final concentration of each component of the lyophilized protective agent in the lyophilized reagent was the same as in Example 4. The lyophilized system was as shown in Table 15. After lyophilization, it was reconstituted with 15 μl of water and compared with the liquid reagent (control). A portion of the enterprise reference product was taken for gradient dilution and then compared. The results are shown in Table 16.

[0183] The freeze-drying effect of freeze-dried reagents is as follows Figure 7 The dissolution peak diagram of the HPV 31 genotyping liquid reagent is shown in Figure 8 The peak pattern of the HPV 31 genotyping freeze-dried reagent dissolution is shown in Fig. 9 shown.

[0184] Table 15 HPV 31 genotyping freeze-dried reagents

[0185]

[0186] Table 16 Comparison of PCR test results (Ct values) of lyophilized reagents and liquid reagents for HPV 31 genotyping

[0187]

[0188] Note: FAM represents target 1, VIC represents target 2, ROX represents target 3, and CY5 represents internal standard.

[0189] Embodiment 15

[0190] Accelerated stability testing of lyophilized reagents with and without nanoparticle lyoprotectants.

[0191] The lyophilized reagents containing nanoparticle lyophilized protective agents and those without nanoparticle lyophilized protective agents were placed at 50°C for accelerated stability testing. The test was conducted once a week, and three sub-tubes were tested at the same concentration each time. The monitoring was continued for 4 weeks, and the average Ct value of each test was taken for statistics. The stability results are shown in Fig.10 As shown. Among them, the freeze-dried protective agent containing nanoparticles is the same as that in Example 13. The difference between the freeze-dried protective agent without nanoparticles and the freeze-dried protective agent containing nanoparticles is that the nanoparticles are removed.

[0192] Example 16

[0193] This example is a real-time stability monitoring of the lyophilized reagent containing the lyophilization protection in Example 13 at 30°C.

[0194] The batch of freeze-dried reagents was sealed and placed in a 30°C constant temperature box. It was tested every 3 months. Each time, the company's reference products P3 and P4 were used for gradient dilution testing for real-time stability monitoring. The statistical results are as follows: Fig.11 .

[0195] Embodiment 17

[0196] This example is a precision test of the freeze-dried reagent containing nanoparticles in Example 10.

[0197] The HBV national secondary standard material (GBW(E)090664) produced by Bondeson was diluted to 20 IU / ml with negative serum and tested 20 times. The coefficient of variation (CV) of the Ct value of the test result was calculated to see whether the CV value met the requirement of <5%.

[0198] The results are shown in Table 17 and Fig.12 shown.

[0199] Table 17 Lyophilized reagent precision test data

[0200]

[0201] Embodiment 18

[0202] This example compares the shaking effects of freeze-dried reagents with and without defoaming agents after reconstitution.

[0203] Taking the lyophilized reagent in Example 10 as an example, the states of the lyophilized reagent containing and not containing the defoaming agent after reconstitution and shaking are respectively as follows: Fig.14 and Fig.13 shown.

[0204] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0205] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A freeze-dried protective agent for a complete PCR premix, characterized in that: The lyoprotectant includes the following components at working concentrations: 0.01% v / v ~ 0.05% v / v lipid nanoparticles, 4% w / v ~ 10% w / v trehalose, 2% w / v ~ 5.36% w / v dextran, 0.10% w / v ~ 0.30% w / v BSA, 0.10% w / v ~ 0.30% w / v PEG8000, 0.01% v / v ~ 0.05% v / v surfactant and 0.01% v / v ~ 0.03% v / v defoaming agent.

2. The lyoprotectant according to claim 1, characterized in that The lyoprotectant meets one or more of the following conditions: (1) The surfactant includes Tween-20; (2) The dextran includes dextran-40; (3) The defoaming agent includes a non-ionic TF composite fermentation defoaming agent.

3. The lyoprotectant according to claim 1 or 2, characterized in that The lipid nanoparticles are 10nm to 50nm.

4. A freeze-dried reagent of a complete PCR premix, characterized in that: The method comprises the freeze-dried protective agent according to any one of claims 1 to 3 and a PCR reaction system.

5. The freeze-dried reagent according to claim 4, characterized in that The PCR reaction system includes PCR Buffer, enzyme, primers, probe, dATP, dCTP, dGTP, dUTP, Mg 2+ and one or more of an RNase inhibitor.

6. The freeze-dried reagent according to claim 5, characterized in that The enzymes include DNA polymerase and / or reverse transcriptase; Optionally, the enzyme further comprises UNG enzyme and / or UDG enzyme.

7. The freeze-dried reagent according to any one of claims 4 to 6, characterized in that The freeze-dried reagent of the PCR premix includes one or more of the freeze-dried reagent of the common PCR premix, the freeze-dried reagent of the qPCR premix, the freeze-dried reagent of the RT-qPCR premix, the freeze-dried reagent of the enzyme cutting method PCR premix and the freeze-dried reagent of the single-tube super multiplex PCR melting curve method premix.

8. The method for preparing a freeze-dried reagent according to any one of claims 4 to 7, characterized in that: The steps include: Mixing the lyophilized protective agent with the PCR reaction system to prepare a PCR premix solution; and The PCR complete premix is ​​freeze-dried to prepare a freeze-dried reagent.

9. The preparation method according to claim 8, characterized in that: Freeze drying includes pre-freezing process and sublimation process in sequence; The parameters of the pre-freezing process are: Set the temperature to 10℃~-10℃, the temperature rise and fall time is 30min~90min, and the holding time is 0.5h~1h; Set the temperature to -20℃~-50℃, the heating and cooling time to 30min~90min, and the holding time to 0.5h~1h; Set the temperature from 0℃ to -20℃, the heating and cooling time from 30min to 90min, and the holding time from 0.5h to 1h; Set the temperature to -20℃~-50℃, the temperature rise and fall time is 30min~90min, and the holding time is 1h~3h; The parameters of the sublimation process are: Set the temperature to -20℃~-50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 3h~10h; Set the temperature to -35℃~-50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 0.5h~2h; Set the temperature to -5℃~-20℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 10Pa~50Pa, and the holding time to 0.5h~1h; Set the temperature to -10℃~-5℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 1Pa~20Pa, and the holding time to 0.5h~1h; Set the temperature to 10℃~25℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 0Pa~1Pa, and the holding time to 0.5h~1h; Set the temperature to 25℃~50℃, the temperature rise and fall time to 30min~90min, the vacuum degree to 0Pa~1Pa, and the holding time to 1h~3h; Set the temperature to 25℃~30℃, the heating and cooling time to 0min~30min, the vacuum degree to 0Pa~1Pa, and the holding time to 0.5h~1h.

10. Use of the freeze-dried reagent according to any one of claims 4 to 7 in PCR detection.

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