A freeze-dried multiple nucleic acid detection kit that can be stored at room temperature and a preparation method thereof

Abstract

The application discloses a freeze-dried multiple nucleic acid detection kit capable of normal-temperature storage and a preparation method, relates to the technical field of freeze-dried multiple nucleic acid detection kits, and specifically discloses a freeze-dried multiple nucleic acid detection kit capable of normal-temperature storage, which comprises a freeze-dried reaction unit, nucleic acid eluent, negative control product, positive control product and sealed moisture-proof outer packaging; the freeze-dried reaction unit is a freeze-dried powder preparation packaged in a PCR reaction tube. When the split equipment of the application performs split operation on the kit, through the cooperation of the liquid supply assembly and the central positioning assembly, the liquid supply is transported while the liquid supply pipe 204 and the PCR reaction tube in the descending process are subjected to concentric positioning treatment. Through the concentric positioning treatment, the liquid flow is prevented from being inclined to impact the conical bottom slope of the PCR reaction tube, so that turbulence, vortex and large fluid shear force are prevented from being generated, the active structure of the heat-resistant DNA polymerase, reverse transcriptase and fluorescent probe in the split liquid is protected, and the activity loss of the reagent is reduced.

Description

Technical Field

[0001] This invention relates to the field of lyophilized multiplex nucleic acid detection kits, specifically to a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature and its preparation method. Background Technology

[0002] Multiplex nucleic acid detection technology can simultaneously amplify and identify multiple target nucleic acids at one time. Compared with single-target detection, it has advantages such as high detection efficiency, small sample volume, low detection cost, and convenient operation. It is widely used in clinical diagnosis, environmental monitoring, food traceability, animal disease detection, and public health emergency prevention and control.

[0003] Currently, most commercially available multiplex nucleic acid detection kits are liquid systems, with reaction components (such as enzymes, primers, probes, dNTPs, etc.) stored in liquid buffer solutions. This presents several technical drawbacks: First, the core active components (thermostable DNA polymerase, reverse transcriptase, fluorescent probes, etc.) are temperature-sensitive, requiring a -20°C cold chain for transportation and storage throughout the process. Breakage of the cold chain can easily lead to enzyme inactivation and probe degradation, resulting in detection failure and significantly increasing transportation and storage costs. Second, components in liquid systems are prone to sedimentation and interactions. Long-term storage can lead to primer dimers and non-specific amplification, resulting in decreased detection sensitivity and specificity. Third, existing lyophilized nucleic acid detection kits are mostly designed for single targets, while multiplex detection systems have complex components. Interference can easily occur between primers, probes, and buffer ions. Conventional lyophilization processes can easily cause enzyme activity loss and probe conformational damage, making it difficult to balance the stability and accuracy of multiplex detection. Fourth, existing lyophilized kits have poor system uniformity after reconstitution, with significant batch-to-batch variations, and the reconstitution process is cumbersome, failing to meet the needs of rapid on-site testing.

[0004] To address the aforementioned technical challenges, there is an urgent need to design a lyophilized formulation, layered lyophilization process, and sealed packaging structure adapted to multiplex nucleic acid amplification systems. This would enable long-term storage of the kit at room temperature while ensuring the specificity, sensitivity, and stability of multiplex target detection, eliminating the need for a cold chain and expanding its applicability. To this end, we propose a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature and its preparation method. Summary of the Invention

[0005] The purpose of this invention is to provide a freeze-dried multiplex nucleic acid detection kit that can be stored at room temperature and its preparation method, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a freeze-dried multiplex nucleic acid detection kit that can be stored at room temperature, comprising a freeze-drying reaction unit, nucleic acid elution buffer, negative control, positive control, and sealed moisture-proof outer packaging; The lyophilized reaction unit is a lyophilized powder formulation packaged in a PCR reaction tube, and is composed of the following components in parts by weight: 0.3-0.8 parts of thermostable DNA polymerase, 0.1-0.3 parts of reverse transcriptase, 1.2-2.5 parts of a mixture of multiple specific primers, 0.8-1.6 parts of a mixture of fluorescently labeled probes, 2.0-3.5 parts of a mixed substrate of dNTPs, 4.0-6.5 parts of a buffer salt system, 8.0-12.0 parts of a lyophilization complex protectant, and 0.5-1.2 parts of an anti-interference agent.

[0007] Preferably, the multiple specific primer mixture contains 2 to 6 sets of upstream and downstream specific primers for different targets, with a Tm value difference of ≥3°C between each set of primers. By using differentiated concentrations, primer cross-binding and primer dimer formation are avoided.

[0008] Preferably, in the fluorescently labeled probe mixture, the probes corresponding to different targets are labeled with different fluorescent groups, and the fluorescent groups are at least two of FAM, VIC, ROX, and CY5, so as to realize multi-channel and multi-signal synchronous recognition.

[0009] Preferably, the freeze-drying composite protectant is composed of trehalose, mannitol, bovine serum albumin and dextran in a mass ratio of (3-5):(2-3):(1-2):(1-2).

[0010] Preferably, the anti-interference agent is a mixture of polyethylene glycol 400 and sodium pyrophosphate in a mass ratio of 2:1, used to suppress sample impurities and non-specific amplification.

[0011] Preferably, the buffer salt system is a Tris-HCl buffer with a pH of 8.0 to 8.5; the nucleic acid elution buffer is an enzyme-free, sterile, weakly alkaline buffer with a pH of 7.2 to 7.8, which can completely reconstitute the lyophilized powder within 3 to 8 seconds.

[0012] Preferably, the sealed and moisture-proof outer packaging is an aluminum foil sealed bag with a silica gel desiccant inside. The PCR reaction tube is sealed with an aluminum foil sealed cap. The water content of the lyophilized powder is ≤3%. The kit has a shelf life of ≥12 months under sealed storage conditions at room temperature (25°C). After accelerated storage at 37°C for 30 days, the amplification fluorescence signal attenuation rate is <3%, and the enzyme activity residue is ≥92%.

[0013] A method for preparing a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature includes the following steps: S1. Under sterile and light-protected conditions, mix the buffer salt system, dNTP mixed substrate and anti-interference agent, and stir at low speed for 5-10 min to prepare the basic buffer solution; Under low temperature conditions of S2.4℃, add the mixture of multiple specific primers and fluorescently labeled probes to the basic buffer, mix well, and incubate at room temperature in the dark for 5 min; S3. Add heat-resistant DNA polymerase and reverse transcriptase in sequence, then add lyophilization complex protectant, and incubate at 4°C in the dark for 10-20 minutes; S4. Dispense 25 μL of the single reaction system into PCR reaction tubes using a dispensing device, and pre-cool at -20℃ for 25–35 min. S5. The freeze-dried powder is obtained by using a segmented gradient vacuum freeze-drying process. S6. After freeze-drying, seal quickly and package with nucleic acid eluent, negative control, positive control and silica gel desiccant. Control the ambient temperature at 2-8℃ and humidity at <30% throughout the process.

[0014] Preferably, the dispensing equipment described in S4 includes a frame, on which a conveyor for assisting in the transport of PCR reaction tubes is provided. Multiple sets of placement cylinders for placing PCR reaction tubes are fixed in an arranged manner on the conveyor. A storage box for storing dispensing liquid is provided on the frame. A liquid supply component for supplying liquid is provided on the storage box. A centering positioning component for keeping the liquid supply concentric with the PCR reaction tubes during the liquid supply process is provided on the liquid supply component. The liquid supply assembly includes a mounting frame, on which a lifting assembly for assisting the lifting and lowering of the mounting frame is provided. An installation pipe is fixed on the mounting frame. The upper end of the installation pipe is connected to the bottom of the storage tank through a flexible hose. A liquid supply pipe for assisting liquid supply is installed at the lower end of the installation pipe. The centering positioning component includes positioning plates evenly distributed around the liquid supply tube. An elastic component for assisting the elastic connection is provided between the positioning plate and the liquid supply tube. One side of the positioning plate has an arc-shaped surface for abutting against the inner side of the PCR reaction tube, and the lower end of the positioning plate has an inclined surface for abutting against the end of the PCR reaction tube. The elastic component includes multiple sets of sleeves fixed to the outside of the liquid supply pipe. A sliding rod is slidably connected to the sleeve. One end of the sliding rod is fixed to the positioning plate. A spring is sleeved on the outside of the sleeve. The two ends of the spring are respectively abutted against the positioning plate and the liquid supply pipe.

[0015] The lifting assembly includes a lifting platform fixed to the frame, a lifting plate slidably connected to the lifting platform, a mounting bracket fixed to the lifting plate, a side plate mounted on the lifting platform, and a cylinder for driving the lifting plate to move up and down mounted on the side plate.

[0016] Compared with the prior art, the beneficial effects of the present invention are: The kit of this invention achieves long-term storage at room temperature, eliminating the need for cold chain transportation and storage at -20℃. It has a shelf life of ≥12 months when stored at room temperature with a sealed container at 25℃, and 30 days when stored at 37℃ with accelerated storage. The amplification fluorescence signal decay rate is <3%, and the enzyme activity residue is ≥92%, significantly reducing transportation and storage costs and making it suitable for scenarios without a cold chain.

[0017] When the dispensing device of the present invention dispenses the reagent kit, the liquid supply component and the centering positioning component work together to perform concentric positioning treatment on the liquid supply tube 204 and the PCR reaction tube during the descent process while the liquid is being delivered. Through concentric positioning treatment, the liquid flow is prevented from deviating and impacting the conical bottom slope of the PCR reaction tube, thus preventing turbulence, eddies and large fluid shear forces, protecting the active structures of heat-resistant DNA polymerase, reverse transcriptase and fluorescent probe in the dispensing solution, and reducing reagent activity loss. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation process steps of the present invention; Figure 2 This is a schematic diagram of the overall external structure of the present invention; Figure 3 This is a schematic diagram of the liquid supply component structure of the present invention; Figure 4 This is a schematic diagram of the lifting component structure of the present invention; Figure 5 This is a schematic diagram of the centering positioning component and elastic component of the present invention; Figure 6 This is a schematic diagram of the state before the liquid supply tube and the PCR reaction tube are centered and positioned according to the present invention; Figure 7 This is a schematic diagram showing the state after the liquid supply tube and the PCR reaction tube of the present invention are centered.

[0019] In the diagram: 101, frame; 102, conveyor; 103, storage box; 104, placement cylinder; 201, mounting frame; 202, mounting pipe; 203, hose; 204, liquid supply pipe; 301, positioning plate; 302, inclined plane; 303, arc-shaped surface; 401, sleeve; 402, slide bar; 403, spring; 501, lifting platform; 502, lifting plate; 503, side plate; 504, cylinder. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0021] Please see Figure 1 The illustration shows a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature, which includes a lyophilized reaction unit, nucleic acid elution buffer, negative control, positive control, and sealed moisture-proof outer packaging. The lyophilized reaction unit is a lyophilized powder formulation packaged in a PCR reaction tube, and is composed of the following components in parts by weight: 0.3-0.8 parts of thermostable DNA polymerase, 0.1-0.3 parts of reverse transcriptase, 1.2-2.5 parts of a mixture of multiple specific primers, 0.8-1.6 parts of a mixture of fluorescently labeled probes, 2.0-3.5 parts of a mixed substrate of dNTPs, 4.0-6.5 parts of a buffer salt system, 8.0-12.0 parts of a lyophilization complex protectant, and 0.5-1.2 parts of an anti-interference agent.

[0022] Preferably, the multiple specific primer mixture contains 2 to 6 sets of upstream and downstream specific primers for different targets, with a Tm value difference of ≥3°C between each set of primers. By using differentiated concentrations, primer cross-binding and primer dimer formation are avoided.

[0023] Preferably, in the fluorescently labeled probe mixture, the probes corresponding to different targets are labeled with different fluorescent groups, and the fluorescent groups are at least two of FAM, VIC, ROX, and CY5, so as to realize multi-channel and multi-signal synchronous recognition.

[0024] Preferably, the freeze-drying composite protectant is composed of trehalose, mannitol, bovine serum albumin and dextran in a mass ratio of (3-5):(2-3):(1-2):(1-2).

[0025] Preferably, the anti-interference agent is a mixture of polyethylene glycol 400 and sodium pyrophosphate in a mass ratio of 2:1, used to suppress sample impurities and non-specific amplification.

[0026] Preferably, the buffer salt system is a Tris-HCl buffer with a pH of 8.0 to 8.5; the nucleic acid elution buffer is an enzyme-free, sterile, weakly alkaline buffer with a pH of 7.2 to 7.8, which can completely reconstitute the lyophilized powder within 3 to 8 seconds.

[0027] Preferably, the sealed and moisture-proof outer packaging is an aluminum foil sealed bag with a silica gel desiccant inside. The PCR reaction tube is sealed with an aluminum foil sealed cap. The water content of the lyophilized powder is ≤3%. The kit has a shelf life of ≥12 months under sealed storage conditions at room temperature (25°C). After accelerated storage at 37°C for 30 days, the amplification fluorescence signal attenuation rate is <3%, and the enzyme activity residue is ≥92%.

[0028] This solution describes a method for preparing a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature, comprising the following steps: S1. Under sterile and light-protected conditions, mix the buffer salt system, dNTP mixed substrate and anti-interference agent, and stir at low speed for 5-10 min to prepare the basic buffer solution; Under low temperature conditions of S2.4℃, add the mixture of multiple specific primers and fluorescently labeled probes to the basic buffer, mix well, and incubate at room temperature in the dark for 5 min; S3. Add heat-resistant DNA polymerase and reverse transcriptase in sequence, then add lyophilization complex protectant, and incubate at 4°C in the dark for 10-20 minutes; S4. Dispense 25 μL of the single reaction system into PCR reaction tubes using a dispensing device, and pre-cool at -20℃ for 25–35 min. S5. The freeze-dried powder is obtained by using a segmented gradient vacuum freeze-drying process. S6. After freeze-drying, seal quickly and package with nucleic acid eluent, negative control, positive control and silica gel desiccant. Control the ambient temperature at 2-8℃ and humidity at <30% throughout the process.

[0029] The working principle of this invention's reagent kit mainly consists of four core parts: multiplex amplification anti-interference principle, lyophilization at room temperature preservation principle, rapid reconstitution and detection principle, and quality control calibration principle, as detailed below: 1. Multiplex Amplification Anti-interference Principle: Specific upstream and downstream primers and fluorescently labeled probes are designed for 2 to 6 target nucleic acids. By optimizing the Tm value and concentration ratio of each primer group, cross-binding and primer dimer formation between different target primers are avoided. At the same time, an anti-interference agent (polyethylene glycol 400 and sodium pyrophosphate compound) is added to adsorb impurities in the sample (such as proteins and polysaccharides), inhibit nuclease activity, reduce non-specific amplification, and ensure specific amplification of each target nucleic acid, achieving simultaneous detection of multiple targets without interference.

[0030] 2. Principle of lyophilization at room temperature: The compound lyophilization protective agent (trehalose, mannitol, BSA, dextran) can form a dense protective film on the surface of active components such as heat-resistant DNA polymerase, reverse transcriptase, primers, and probes, isolating them from the damage to the component structure caused by low temperature and vacuum environment; in the segmented gradient vacuum lyophilization process, the system is first completely frozen by low temperature pre-freezing, and then the free water and bound water in the system are slowly removed by gradient temperature increase and vacuum environment, so that each active component is in an anhydrous dormant state, reducing the molecular motion rate and inhibiting oxidative degradation; the sealed moisture-proof outer packaging (aluminum foil bag + desiccant) can effectively isolate moisture and oxygen in the air, avoid moisture absorption of lyophilized powder and inactivation of active components, thereby achieving long-term storage of the kit at room temperature of 25℃.

[0031] 3. Rapid Reconstitution and Detection Principle: During on-site testing, add a quantitative amount (25 μL) of nucleic acid elution buffer to the lyophilized reaction unit (PCR reaction tube), gently shake for 3–5 seconds, and the lyophilized powder will be completely reconstituted. The reconstituted system can be directly used for nucleic acid amplification. Add the nucleic acid sample to be tested to the reconstituted system, place the reaction tube in a real-time PCR instrument or an isothermal amplification instrument, and under suitable temperature conditions (real-time PCR: 95℃ pre-denaturation for 3 min, then 95℃ denaturation for 15 s, 60℃ annealing extension for 30 s, cycle 40 times; isothermal amplification: 63℃ isothermal for 30 min), the thermostable DNA polymerase catalyzes the amplification of target nucleic acids, and the reverse transcriptase enables simultaneous reverse transcription and amplification of RNA targets. Different target amplification products can specifically bind to the corresponding fluorescently labeled probes, causing the probes to undergo conformational changes and release specific fluorescent signals. The instrument identifies different fluorescent signals through multiple channels (e.g., the FAM channel corresponds to target 1, and the VIC channel corresponds to target 2), realizing simultaneous qualitative and quantitative detection of multiple targets, and the detection results can be directly read.

[0032] 4. Quality Control Calibration Principle: The negative and positive controls provided with the kit are used in the detection process simultaneously with the test sample. The negative control (without target nucleic acid) is used to eliminate interference from reagent contamination, non-specific amplification, etc. If the negative control has no fluorescence signal, it indicates that the detection system is uncontaminated. The positive control (containing standard target nucleic acid) is used to verify the activity of the amplification system. If the positive control shows a corresponding fluorescence signal, it indicates that the detection system is normal and has good activity, thus ensuring the accuracy and reliability of the test results of the test sample.

[0033] Achieve long-term storage at room temperature: No need for -20℃ cold chain transportation and storage, ≥12 months of shelf life when sealed at 25℃, 30 days of accelerated storage at 37℃, amplification fluorescence signal decay rate <3%, enzyme activity residue ≥92%, significantly reducing transportation and warehousing costs, and suitable for scenarios without cold chain. Excellent detection performance: It can simultaneously detect 2 to 6 target nucleic acids, with a detection sensitivity of up to 10 copies / reaction, 100% specificity, intra-assay coefficient of variation <5%, inter-assay coefficient of variation <7%, and accurate and stable detection results; High freeze-drying stability: The compound freeze-drying protective agent can effectively protect active components such as enzymes and probes. The active components are minimally damaged during freeze-drying, and the system has good homogeneity after reconstitution with no obvious precipitation. No complicated preparation is required. Simple and quick to operate: It can be used immediately after opening. The nucleic acid elution buffer can quickly reconstitute the lyophilized powder. After reconstitution, it can be directly added to the sample for detection. The entire detection process (including reconstitution) can be completed within 40 to 60 minutes, making it suitable for grassroots field and emergency field testing. Strong anti-interference and anti-contamination capabilities: Anti-interference agents can suppress sample impurities, and the anhydrous freeze-drying system and sealed packaging can suppress bacterial and nuclease contamination. It has strong environmental adaptability and can be stably detected under different temperature and humidity conditions.

[0034] Please see Figures 2-7 The dispensing equipment described in S4 includes a frame 101, on which a conveyor 102 for assisting in the transport of PCR reaction tubes is provided. Multiple sets of placement cylinders 104 for placing PCR reaction tubes are fixed in an arranged manner on the conveyor 102. A storage box 103 for storing dispensing liquid is provided on the frame 101. A liquid supply component for supplying liquid is provided on the storage box 103. A centering positioning component for keeping the liquid supply concentric with the PCR reaction tubes during the liquid supply process is provided on the liquid supply component. It should be noted that when the dispensing equipment dispenses the reagent kits, the liquid supply component and the centering positioning component work together to concentrically position the liquid supply tube 204 and the PCR reaction tube during the descent process while the liquid is being delivered. This concentric positioning prevents the liquid flow from deviating and impacting the conical bottom slope of the PCR reaction tube, thus preventing turbulence, eddies, and large fluid shear forces. This protects the active structures of the heat-resistant DNA polymerase, reverse transcriptase, and fluorescent probes in the dispensed solution and reduces reagent activity loss.

[0035] The liquid supply assembly includes a mounting frame 201. A lifting assembly for assisting the lifting of the mounting frame 201 is provided on the frame 101. An installation pipe 202 is fixed on the mounting frame 201. The upper end of the installation pipe 202 is connected to the bottom of the storage tank 103 through a flexible hose 203. A liquid supply pipe 204 for assisting liquid supply is installed at the lower end of the installation pipe 202. It should be noted here that: the dispensing liquid inside the storage tank 103 is transported outward through the control valve inside the storage tank 103. During the transportation process, the dispensing liquid is transported into the PCR reaction tube through the interconnection of the hose 203, the installation tube 202 and the liquid supply tube 204, thus completing the liquid supply operation.

[0036] The centering positioning component includes positioning plates 301 evenly distributed around the liquid supply tube 204. An elastic component for assisting the elastic connection is provided between the positioning plate 301 and the liquid supply tube 204. An arc-shaped surface 303 is provided on one side of the positioning plate 301 for abutting against the inner side of the PCR reaction tube. An inclined surface 302 is provided at the lower end of the positioning plate 301 for abutting against the end of the PCR reaction tube. It should be noted that during the liquid supply process via the descending liquid supply tube 204, the inclined surfaces 302 on each group of positioning plates 301 abut against the upper end of the PCR reaction tube. During this abutment, each group of positioning plates 301 is forced to contract towards the liquid supply tube 204. During this contraction, each group of sliding rods 402 slides on each group of sleeves 401, causing the springs 403 to deform and generate elastic force. As the liquid supply tube 204 continues to descend, the elastic force of the springs 403 pushes each group of positioning plates 301 against the inner wall of the PCR reaction tube. Through this abutment and compression, the liquid supply tube 204 and the PCR reaction tube are concentrically positioned during the descent.

[0037] The elastic component includes multiple sets of sleeves 401 fixed to the outside of the liquid supply pipe 204. A slide rod 402 is slidably connected to the sleeve 401. One end of the slide rod 402 is fixed to the positioning plate 301. A spring 403 is sleeved on the outside of the sleeve 401. The two ends of the spring 403 are respectively abutted against the positioning plate 301 and the liquid supply pipe 204. It should be noted that the sleeve 401 and the slide rod 402 facilitate the guidance of the movement of the auxiliary positioning plate 301 after it is subjected to force, and the spring 403 facilitates the elastic push of the retracted positioning plate 301.

[0038] The lifting assembly includes a lifting platform 501 fixed on the frame 101, a lifting plate 502 slidably connected on the lifting platform 501, a mounting bracket 201 fixed on the lifting plate 502, a side plate 503 mounted on the lifting platform 501, and a cylinder 504 for lifting and lowering the lifting plate 502 mounted on the side plate 503. It should be noted that: the lifting plate 502 is driven by the cylinder 504 to move downward on the lifting platform 501. During the downward movement of the lifting plate 502, the mounting frame 201 and the mounting tube 202 on the mounting frame 201 are driven to move downward synchronously.

[0039] In this solution, the dispensing liquid is dispensed using dispensing equipment, including the following steps: The PCR reaction tubes to be dispensed are sequentially placed on the placement tubes 104 of the conveyor 102. After placement, the PCR reaction tubes are sequentially moved to the bottom of the liquid supply tube 204 by the conveyor 102. After the liquid supply tube 204 has moved, the lifting plate 502 is driven by the cylinder 504 to move downward on the lifting platform 501. During the downward movement of the lifting plate 502, the mounting frame 201 and the mounting tube 202 on the mounting frame 201 are driven to descend synchronously. Through the descent of the mounting tube 202, the front end of the liquid supply tube 204 at the lower end of the mounting tube 202 is inserted into the PCR reaction tube. After the liquid supply tube 204 has descended, the dispensing liquid inside the storage box 103 is transported outward through the control valve inside the storage box 103. During the transportation process, the dispensing liquid is transported into the PCR reaction tube through the interconnection of the hose 203, the mounting tube 202 and the liquid supply tube 204, thus completing the liquid supply operation. Because the PCR reaction tube is conical (narrow at the bottom), during the liquid supply process via the descending supply tube 204, the inclined surfaces 302 on each group of positioning plates 301 abut against the upper end of the PCR reaction tube. During this abutment, the positioning plates 301 are forced to contract towards the supply tube 204. This contraction pushes the sliding rods 402 to slide on the sleeves 401, causing the springs 403 to deform and generate elasticity. As the supply tube 204 continues to descend, the elasticity of the springs 403 pushes the positioning plates 301 against the inner wall of the PCR reaction tube. Through this abutment and compression, the supply tube 204 and the PCR reaction tube are concentrically positioned during the descent. This concentric positioning prevents the liquid flow from deviating and impacting the conical bottom inclined surface of the PCR reaction tube, preventing turbulence, eddies, and large fluid shear forces. It also protects the active structures of the heat-resistant DNA polymerase, reverse transcriptase, and fluorescent probes in the dispensed solution, reducing reagent activity loss.

[0040] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0041] Although embodiments of the invention have been 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 of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A lyophilized multiplex nucleic acid detection kit that can be stored at room temperature, characterized in that, include: Freeze-dried reaction unit, nucleic acid eluent, negative control, positive control, and sealed moisture-proof outer packaging; The lyophilized reaction unit is a lyophilized powder formulation packaged in a PCR reaction tube, and is composed of the following components in parts by weight: 0.3-0.8 parts of thermostable DNA polymerase, 0.1-0.3 parts of reverse transcriptase, 1.2-2.5 parts of a mixture of multiple specific primers, 0.8-1.6 parts of a mixture of fluorescently labeled probes, 2.0-3.5 parts of a mixed substrate of dNTPs, 4.0-6.5 parts of a buffer salt system, 8.0-12.0 parts of a lyophilization complex protectant, and 0.5-1.2 parts of an anti-interference agent.

2. The lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 1, characterized in that: The multiple specific primer mixture contains 2 to 6 sets of upstream and downstream specific primers for different targets. The Tm value difference between each set of primers is ≥3℃. By using differentiated concentrations, primer cross-binding and primer dimer formation are avoided.

3. The lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 2, characterized in that: In the fluorescently labeled probe mixture, probes corresponding to different targets are labeled with different fluorescent groups, and the fluorescent groups are at least two of FAM, VIC, ROX and CY5, so as to realize multi-channel and multi-signal synchronous recognition.

4. The lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 3, characterized in that: The freeze-drying composite protectant is composed of trehalose, mannitol, bovine serum albumin and dextran, with a mass ratio of (3-5):(2-3):(1-2):(1-2).

5. The lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 1, characterized in that: The anti-interference agent is a compound of polyethylene glycol 400 and sodium pyrophosphate in a mass ratio of 2:1, used to suppress sample impurities and non-specific amplification.

6. The lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 5, characterized in that: The buffer salt system is a Tris-HCl buffer with a pH of 8.0–8.5; the nucleic acid elution buffer is an enzyme-free, sterile, weakly alkaline buffer with a pH of 7.2–7.8, which can completely reconstitute the lyophilized powder within 3–8 seconds.

7. A lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 5, characterized in that: The sealed and moisture-proof outer packaging is an aluminum foil sealed bag with silica gel desiccant inside. The PCR reaction tube is sealed with an aluminum foil sealed cap. The water content of the lyophilized powder is ≤3%. The kit has a shelf life of ≥12 months under sealed storage conditions at room temperature of 25℃. After accelerated storage at 37℃ for 30 days, the amplification fluorescence signal attenuation rate is <3%, and the enzyme activity residue is ≥92%.

8. A method for preparing a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature, characterized in that, The preparation of the kit according to any one of claims 1-7 comprises the following steps: S1. Under sterile and light-protected conditions, mix the buffer salt system, dNTP mixed substrate and anti-interference agent, and stir at low speed for 5-10 minutes to prepare the basic buffer solution; S2. At a low temperature of 4℃, add the mixture of multiple specific primers and fluorescently labeled probes to the basic buffer, mix well, and incubate at room temperature in the dark for 5 min; S3. Add heat-resistant DNA polymerase and reverse transcriptase in sequence, then add lyophilization complex protectant, and incubate at 4°C in the dark for 10-20 minutes; S4. Dispense 25 μL of the single reaction system into PCR reaction tubes using a dispensing device, and pre-cool at -20℃ for 25–35 min; S5. A segmented gradient vacuum freeze-drying process is used to obtain freeze-dried powder; S6. After freeze-drying, seal quickly and package with nucleic acid eluent, negative control, positive control and silica gel desiccant. Control the ambient temperature at 2-8℃ and humidity at <30% throughout the process.

9. The method for preparing a freeze-dried multiplex nucleic acid detection kit that can be stored at room temperature according to claim 1, characterized in that, The dispensing equipment described in S4 includes a frame (101), on which a conveyor (102) for assisting in the transport of PCR reaction tubes is provided. Multiple sets of placement tubes (104) for placing PCR reaction tubes are fixed in an arranged manner on the conveyor (102). A storage box (103) for storing dispensing liquid is provided on the frame (101). A liquid supply component for supplying liquid is provided on the storage box (103). A centering positioning component for keeping the liquid concentric with the PCR reaction tubes during the liquid supply process is provided on the liquid supply component. The liquid supply assembly includes a mounting frame (201), and a lifting assembly for assisting the lifting of the mounting frame (201) is provided on the frame (101). An installation pipe (202) is fixed on the mounting frame (201). The upper end of the installation pipe (202) is connected to the bottom of the storage tank (103) through a flexible hose (203). A liquid supply pipe (204) for assisting liquid supply is installed at the lower end of the installation pipe (202). A control valve for liquid supply control is provided inside the storage tank (103). The centering positioning component includes positioning plates (301) evenly distributed around the liquid supply tube (204). An elastic component for assisting the elastic connection is provided between the positioning plate (301) and the liquid supply tube (204). An arc-shaped surface (303) is provided on one side of the positioning plate (301) for abutting against the inner side of the PCR reaction tube. An inclined surface (302) is provided at the lower end of the positioning plate (301) for abutting against the end of the PCR reaction tube. The elastic component includes multiple sets of sleeves (401) fixed to the outside of the liquid supply pipe (204). A slide rod (402) is slidably connected to the sleeve (401). One end of the slide rod (402) is fixed to the positioning plate (301). A spring (403) is sleeved on the outside of the sleeve (401). The two ends of the spring (403) are respectively abutted against the positioning plate (301) and the liquid supply pipe (204).

10. The method for preparing a lyophilized multiplex nucleic acid detection kit that can be stored at room temperature according to claim 9, characterized in that: The lifting assembly includes a lifting platform (501) fixed on a frame (101), a lifting plate (502) slidably connected to the lifting platform (501), a mounting bracket (201) fixed to the lifting plate (502), a side plate (503) mounted on the lifting platform (501), and a cylinder (504) for lifting and lowering the lifting plate (502) mounted on the side plate (503).

Images