QPCR (quantitative polymerase chain reaction) freeze-drying reagent for triple detection of influenza A, influenza B and novel coronavirus as well as preparation method and application of qPCR freeze-drying reagent

Abstract

The invention discloses a qPCR (quantitative polymerase chain reaction) freeze-drying reagent for triple detection of influenza A, influenza B and novel coronavirus as well as a preparation method and application of the qPCR freeze-drying reagent. The reagent is prepared by freeze-drying a liquid premixed solution containing an enzyme, a primer probe, dNTPs, Mg, a freeze-drying protective agent and an additive. According to the invention, a formula of a specific additive consisting of betaine and dextran sulfate is adopted, and a conventional freeze-drying protective agent (trehalose, polysucrose and BSA) is cooperated, so that the technical problems of easy enzyme activity inactivation, poor storage stability, non-uniform multi-reaction efficiency after redissolution and the like of a multiple qPCR reagent in the freeze-drying process are effectively solved. The reagent disclosed by the invention can be stably stored and transported at normal temperature, can realize high-sensitivity, high-specificity and high-reproducibility detection of three pathogens only by one-step sample adding operation during use, and is particularly suitable for on-site rapid detection and large-scale screening.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, and in particular to a qPCR lyophilized reagent for triple detection of influenza A, influenza B and novel coronavirus, its preparation method and application. Background Technology

[0002] Quantitative real-time PCR (qPCR) technology has become the gold standard for pathogen detection. Multiplex qPCR detection can detect multiple targets simultaneously in a single reaction system, greatly improving detection efficiency and reducing costs and sample volume, making it of great value in infectious disease control and clinical diagnosis.

[0003] Traditional qPCR reactions typically rely on cold chain transportation and frozen storage of liquid reagents. Users need to perform multiple pipetting operations to prepare the reaction system, which is cumbersome and increases the risk of aerosol contamination and operational errors. This greatly limits its application in scenarios such as rapid on-site testing (e.g., clinics, airports, community clinics).

[0004] Lyophilization technology dehydrates active substances in aqueous solutions at low temperatures, forming a solid, dried product that remains stable at room temperature. While existing qPCR lyophilized reagents have some applications, they face significant challenges in multiplex detection (especially triplex and higher). 1. Poor stability: During freeze-drying and storage, enzyme activity is easily damaged, leading to a decrease in reagent efficiency.

[0005] 2. Inconsistent sensitivity and efficiency: In multiplex detection, there is competition and interference between different primer-probe combinations, which can easily lead to a significant decrease in the detection sensitivity of some targets and inconsistent amplification efficiency.

[0006] 3. Poor reconstitution effect: After the lyophilized reagent is reconstituted, the components cannot be quickly and completely reconstituted into a uniform and efficient reaction system, which affects the repeatability of the detection.

[0007] Therefore, there is an urgent need in this field to develop a qPCR lyophilized reagent formulation and preparation method that is specifically designed for multiplex detection, has excellent abnormal temperature stability and balanced and efficient detection performance. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a qPCR lyophilized reagent for triple detection of influenza A, influenza B, and novel coronavirus, along with its preparation method and applications. The lyophilized reagent of this invention contains a specific additive formulation that effectively protects the activity of enzymes and nucleic acids during the lyophilization process, solving the compatibility and stability problems of multiplex reaction systems. This reagent can be stably stored and transported at room temperature. When used, only water and RNA template need to be added to perform triple target detection of influenza A virus, influenza B virus, and novel coronavirus (COVID-19), achieving rapid, room-temperature detection in a single tube.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a qPCR lyophilized reagent for triple detection of influenza A, influenza B and novel coronavirus is provided, which is prepared by lyophilization of a premix containing the following components: hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, lyophilization protectant, additives, and a specific primer and probe set for influenza A virus, influenza B virus and novel coronavirus. The additives include betaine and dextran sulfate.

[0010] Preferably, the freeze-drying protectant includes trehalose, sucrose, and bovine serum albumin.

[0011] Preferably, in the premixed solution, the concentrations of each component of the additive and the lyophilization protectant are as follows: Trehalose: 0.1-0.5M; Sucrose: 1-5% (w / v); Bovine serum albumin: 0.1-1 mg / mL; Betaine: 0.5-2M; Dextran sulfate: 0.01-0.1% (w / v).

[0012] Preferably, in the liquid premix, the concentrations of each component of the additive and the lyophilization protectant are as follows: Trehalose: 0.3M; Sucrose: 3% (w / v); Bovine serum albumin: 0.5 mg / mL; Betaine: 1M; Dextran sulfate: 0.05% (w / v).

[0013] Preferably, the specific primer-probe set comprises: Primer pairs such as SEQ ID NO: 1 and SEQ ID NO: 2, and probes such as SEQ ID NO: 3, targeting conserved sequences of influenza A virus (INVA); Primer pairs such as SEQ ID NO:4 and SEQ ID NO:5 targeting conserved sequences of influenza B virus (INVB), and probes such as SEQ ID NO:6; Primer pairs as shown in SEQ ID NO: 7 and SEQ ID NO: 8, and probes as shown in SEQ ID NO: 9, targeting the N gene of the novel coronavirus (COVID19); Primer pairs as shown in SEQ ID NO: 10 and SEQ ID NO: 11, and probes as shown in SEQ ID NO: 12, targeting the Orf1ab gene of the novel coronavirus (COVID19); Primer pairs as shown in SEQ ID NO: 13 and SEQ ID NO: 14, and probe as shown in SEQ ID NO: 15, targeting the human internal reference RnaseP gene.

[0014] Each probe is labeled with a distinguishable fluorescent reporter group and a quencher group.

[0015] Among them, the human internal reference RnaseP gene is used as a control to exclude false negatives.

[0016] A second aspect of the present invention provides a method for preparing the qPCR lyophilized reagent as described above, comprising the following steps: (1) Mix hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, lyophilization protectant, additives, and specific primer and probe sets to prepare a premix solution; (2) Dispense the premixed solution into reaction tubes; (3) The reaction tubes after dispensing are pre-frozen, dried once and dried twice to obtain lyophilized reagents, and then sealed.

[0017] Preferably, the pre-freezing temperature in step (3) is below -40°C, the temperature of the first drying is between -20°C and -10°C, and the temperature of the second drying is between 20°C and 30°C.

[0018] A third aspect of the present invention provides the application of the qPCR lyophilized reagent described above in the triple detection of influenza A, influenza B and novel coronavirus.

[0019] In a fourth aspect, the present invention provides a detection kit comprising the qPCR lyophilized reagents as described above.

[0020] Preferably, the test kit further includes one or more of enzyme-free water, a positive control, and a negative control.

[0021] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following advantages: 1. Excellent room temperature stability: The lyophilized reagents prepared can be stored stably at 37°C for at least 4 weeks, or at 25°C for more than 6 months, without significant performance degradation, greatly reducing storage and transportation costs.

[0022] 2. High sensitivity and excellent amplification efficiency: The unique additive formula ensures efficient reconstruction of the system after reconstitution. The detection limit for all three viruses can reach below 200 cps / ml, and the amplification efficiency of each reconstitution is between 90% and 110%, without interference between them.

[0023] 3. Simple operation and pollution prevention: Users only need to do one step, add the extracted RNA sample and water, vortex centrifuge and it can be put into operation, which greatly reduces operation time and pollution risk.

[0024] 4. Good reproducibility: small intra-batch and inter-batch differences, reliable results, suitable for standardized production and large-scale application. Attached Figure Description

[0025] Figure 1 The image shows the appearance of the lyophilized reagent prepared in Example 1 of this invention. Figure 2 This is a triple qPCR amplification curve of the lyophilized reagent of the present invention against influenza A, influenza B, and COVID-19 pseudovirus standards (gradient dilution); Figure 3 This is a comparison chart of the Ct values ​​of the lyophilized reagent of this invention and the conventional liquid reagent. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0027] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0029] This invention provides a qPCR lyophilized reagent for triple detection of influenza A, influenza B and novel coronavirus, which is prepared by lyophilization of a premix containing the following components: hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, lyophilization protectant, additives, and a specific primer and probe set for influenza A virus, influenza B virus and novel coronavirus. The additives include betaine and dextran sulfate.

[0030] Betaine, as a compatible solute, can alleviate osmotic stress and ice crystal damage during freeze-drying, and stabilize enzyme protein structures after reconstitution, promoting primer annealing specificity. Dextran sulfate can form a protective network at the microscopic level during freeze-drying, "fixing" macromolecules such as enzymes and primers within it, minimizing their exposure and denaturation. Simultaneously, after reconstitution, it can locally increase the effective concentration of macromolecules in the reaction system, enhancing reaction kinetics. This is crucial for overcoming the competitive inhibition effect caused by excessive primers and probes in multiplex PCR.

[0031] In a preferred embodiment, the freeze-drying protectant includes trehalose, sucrose, and bovine serum albumin.

[0032] In a preferred embodiment, the concentrations of each component of the additive and lyophilization protectant in the premix are as follows: Trehalose: 0.1-0.5M; Sucrose: 1-5% (w / v); Bovine serum albumin: 0.1-1 mg / mL; Betaine: 0.5-2M; Dextran sulfate: 0.01-0.1% (w / v).

[0033] In a preferred embodiment, the concentrations of each component of the additive and lyophilization protectant in the liquid premix are as follows: Trehalose: 0.3M; Sucrose: 3% (w / v); Bovine serum albumin: 0.5 mg / mL; Betaine: 1M; Dextran sulfate: 0.05% (w / v).

[0034] Preferably, the specific primer-probe set comprises: Primer pairs such as SEQ ID NO: 1 and SEQ ID NO: 2, and probes such as SEQ ID NO: 3, targeting influenza A virus; Primer pairs such as SEQ ID NO: 4 and SEQ ID NO: 5, and probes such as SEQ ID NO: 6, targeting influenza B virus; Primer pairs such as SEQ ID NO: 7 and SEQ ID NO: 8, and probes such as SEQ ID NO: 9, targeting the N gene of the novel coronavirus; Primer pairs such as SEQ ID NO: 10 and SEQ ID NO: 11, and probes such as SEQ ID NO: 12, targeting the Orf1ab gene of the novel coronavirus; Primer pairs as shown in SEQ ID NO: 13 and SEQ ID NO: 14, and probe as shown in SEQ ID NO: 15, targeting the human internal reference RnaseP gene.

[0035] Each probe is labeled with a distinguishable fluorescent reporter group and a quencher group.

[0036] Among them, the human internal reference RnaseP gene is used as a control to exclude false negatives.

[0037] This invention also provides a method for preparing the above-mentioned qPCR lyophilized reagent, comprising the following steps: (1) Mix hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, lyophilization protectant, additives, and specific primer and probe sets to prepare a premix solution; (2) Dispense the premixed solution into reaction tubes; (3) The reaction tubes after dispensing are pre-frozen, dried once and dried twice to obtain lyophilized reagents, and then sealed.

[0038] In a preferred embodiment, the pre-freezing temperature in step (3) is below -40°C, the temperature for the first drying is from -20°C to -10°C, and the temperature for the second drying is from 20°C to 30°C.

[0039] In a preferred embodiment, the method for preparing the qPCR lyophilized reagent includes the following steps: (1) Mix hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, lyophilization protectant, additives, and specific primer and probe sets to prepare a premix solution; (2) Dispense the premixed solution into reaction tubes; (3) Place the dispensed reaction tubes in a freeze dryer and rapidly cool them to below -40°C and maintain the temperature for 1-2 hours; Primary drying: Under vacuum conditions, the shelf temperature is gradually raised to -20°C to -10°C and maintained at this temperature for several hours to remove most of the free water; Secondary drying: The shelf temperature is further raised to 20°C to 30°C, and vacuum drying is continued to remove bound water, finally obtaining the dried reagent; Tighten the cap under vacuum or inert gas (such as nitrogen) protection and seal for storage.

[0040] A third aspect of the present invention provides the application of the above-mentioned qPCR lyophilized reagent in the triple detection of influenza A, influenza B and novel coronavirus.

[0041] In a fourth aspect, the present invention provides a detection kit comprising the qPCR lyophilized reagents described above.

[0042] In a preferred embodiment, the test kit further includes one or more of enzyme-free water, a positive control, and a negative control.

[0043] The above is the general concept of the present invention. Based on this, detailed embodiments and comparative examples are provided below to further illustrate the present invention.

[0044] Example 1: Preparation of lyophilized reagents 1. Prepare the liquid premix (taking a 1mL system as an example): (1) Buffer solution (pH 8.5) (2) dNTPs: 400 μM (3) MgCl2: 5mM (4) Hot-start Taq enzyme: 1.5 U / reaction (5) Reverse transcriptase: 0.5 U / reaction (6) Lyophilization protectant: Trehalose: 0.3M Sucrose: 3% (w / v) BSA: 0.5 mg / mL (7) Specific additives: Betaine: 1M Dextran sulfate: 0.05% (w / v) (8) Primers and probes (sequences validated, concentrations optimized): INVA-F: SEQ ID NO: 1, 0.4 μM; INVA-R: SEQ ID NO: 2, 0.4 μM; INVA-P: [CY5] SEQ ID NO: 3[BHQ2], 0.2 μM; INVB-F: SEQ ID NO: 4, 0.4 μM; INVB-R: SEQ ID NO: 5, 0.4 μM; INVB-P: [VIC] SEQ ID NO: 6 [BHQ1], 0.2μM; COVID19-NF: SEQ ID NO: 7, 0.4μM; COVID19-N-R: SEQ ID NO: 8, 0.4μM; COVID19-N-P: [FAM] SEQ ID NO: 9[BHQ1], 0.2μM; COVID19-Orf1ab-F: SEQ ID NO: 10, 0.4μM; COVID19- Orf1ab-R: SEQ ID NO: 11, 0.4μM; COVID19- Orf1ab -P: [FAM] SEQ ID NO: 12 [BHQ1], 0.2μM; RnaseP-F: SEQ ID NO: 13, 0.4 μM; RnaseP-R: SEQ ID NO: 14, 0.4 μM; RnaseP-P: [ROX] SEQ ID NO: 15 [BHQ1], 0.2 μM.

[0045] 2. After thoroughly mixing the above components, dispense them (25 μL equivalent per tube), pre-freeze, dry once, and dry twice to obtain a milky white and dense reagent, which is then sealed with nitrogen gas.

[0046] The specific sequences are shown in Table 1 below: Table 1 Example 2: Performance Verification The lyophilized reagent prepared in Example 1 was compared with a conventional liquid reagent containing the same core components.

[0047] Methods: Standards containing RNA from influenza A, influenza B, and SARS-CoV-2 pseudoviruses (concentration gradients: 10⁸, 10⁷, 10⁶, 10⁵, 10⁴ copies / mL) were used for testing. For the lyophilized reagent group, 20 μL of enzyme-free water and 5 μL of template RNA were added to each tube. The liquid reagent group was prepared according to the standard system. The same qPCR program was run on an ABI 7500 instrument. Taking influenza A as an example, there was no significant difference in CT values ​​before and after lyophilization, indicating good linearity.

[0048] Result: As Figure 2As shown, the lyophilized reagent of this invention exhibited excellent amplification curves for all three targets, with a sensitivity of 200 cps / ml. The calculated amplification efficiencies were: INVA 107%, INVB 104%, Covid-19 108%, and the internal control RNase P 113%, meeting the requirements for high-efficiency amplification. Compared with the liquid reagent, the Ct value showed no significant difference (P>0.05), indicating that the lyophilization process did not cause performance loss.

[0049] The specific data is shown in Table 2 below: Table 2 Example 3: Stability Test. The lyophilized reagent prepared in Example 1 was stored at 4°C, 25°C, and 37°C. Samples were taken on days 0, 7, 14, and 28, and tested using the same medium concentration (200 copies / mL) of triple-positive samples. Changes in Ct values ​​were compared.

[0050] Results: As shown in Table 3, after 4 weeks of storage under accelerated degradation testing at 37℃, the Ct values ​​of all three targets shifted by less than 1.5, indicating that the reagents have excellent room temperature stability. It is speculated that they can be stably stored at 4℃ or 25℃ for at least 1 year.

[0051] Table 3 Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A qPCR lyophilized reagent for triple detection of influenza A, influenza B, and novel coronavirus, characterized in that, It is made by freeze-drying a premix solution containing the following components: hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, freeze-drying protectants, additives, and specific primer probe sets for influenza A virus, influenza B virus, and novel coronavirus; The additives include betaine and dextran sulfate.

2. The qPCR lyophilized reagent of claim 1, wherein, The freeze-drying protectants include trehalose, polysucrose, and bovine serum albumin.

3. The qPCR lyophilized reagent of claim 2, wherein, In the premix solution, the concentration of each component of the additives and freeze-drying protectants is: Trehalose: 0.1-0.5M; Polysucrose: 1-5%(w / v); Bovine serum albumin: 0.1-1mg / mL; Betaine: 0.5-2M; Dextran sulfate: 0.01-0.1%(w / v).

4. The qPCR lyophilized reagent of claim 3, wherein, In the liquid premix solution, the concentration of each component of the additives and freeze-drying protectants is: Trehalose: 0.3M; Polysucrose: 3%(w / v); Bovine serum albumin: 0.5mg / mL; Betaine: 1M; Dextran sulfate: 0.05%(w / v).

5. The qPCR lyophilized reagent according to any one of claims 1 to 4, characterized in that, The specific primer probe set contains: For influenza A virus, a primer pair as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3; For influenza B virus, a primer pair as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a probe as shown in SEQ ID NO: 6; For novel coronavirus N gene, a primer pair as shown in SEQ ID NO: 7 and SEQ ID NO: 8, and a probe as shown in SEQ ID NO: 9; For novel coronavirus Orf1ab gene, a primer pair as shown in SEQ ID NO: 10 and SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12; For human internal reference RnaseP gene, a primer pair as shown in SEQ ID NO: 13 and SEQ ID NO: 14, and a probe as shown in SEQ ID NO:

15.

6. A method of preparing a qPCR lyophilized reagent according to any one of claims 1-5, characterized in that, It comprises the following steps: (1) Mix hot-start Taq DNA polymerase, reverse transcriptase, dNTPs, magnesium ions, freeze-drying protectants, additives, and specific primer probe sets to prepare a premix solution; (2) Dispense the premix solution into reaction tubes; (3) Pre-freeze, primary dry, and secondary dry the dispensed reaction tubes to obtain freeze-dried reagents, which are sealed.

7. The method of claim 6, wherein, In step (3), the pre-freezing temperature is below -40℃, the primary drying temperature is -20℃ to -10℃, and the secondary drying temperature is 20℃ to 30℃.

8. Use of the qPCR freeze-dried reagent of any one of claims 1-5 in triple detection of influenza A, influenza B, and novel coronavirus.

9. A test kit comprising, It comprises the qPCR freeze-dried reagent of any one of claims 1-5.

10. The test kit according to claim 9, characterized in that The detection kit also comprises one or more of enzyme-free water, positive control, and negative control.

Images