Freeze-drying protective agent and freeze-drying method of HCV molecular detection reagent

Abstract

The invention provides a freeze-drying protective agent and a freeze-drying method of an HCV molecular detection reagent, and particularly relates to an appropriate formula of the freeze-drying protective agent of the HCV molecular detection reagent, which comprises trehalose, dextran, cane sugar and cyclodextrin, so that the prepared freeze-drying reagent for HCV nucleic acid detection is stable in form, good in stability and good in stability. The amplification performance of the freeze-dried reagent is basically the same as that of a liquid amplification reagent, and the freeze-dried reagent has excellent stability at normal temperature or even high temperature.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular, relates to a freeze-drying protective agent and a freeze-drying method for an HCV molecular detection reagent. Background Art

[0002] Hepatitis C virus (HCV) is a hepatotropic RNA virus that can cause progressive liver damage, which may lead to cirrhosis and hepatocellular carcinoma. With the continuous development of direct antiviral drugs (DAA), most hepatitis C patients can be cured, and simplified HCV infection detection and diagnosis processes can shorten the turnaround time from consultation to treatment, so that hepatitis C patients can receive treatment as soon as possible.

[0003] However, HCV biological diagnostic reagents are active substances, mainly protein products, which are particularly sensitive to temperature, very unstable, and easily react with each other. Due to the above characteristics, biological agents have a short life cycle, require low-temperature storage, cold chain transportation throughout the process, and a very short storage life, so the cost of use is very high and the risks cannot be controlled. The stability of biological reagents has become a key factor in this industry. Therefore, those skilled in the art are committed to developing preservation technologies that can improve the stability of detection reagents. Summary of the invention

[0004] The purpose of the present invention is to provide a freeze-drying protective agent for HCV molecular detection reagents to solve the problem of poor stability of HCV biological diagnostic reagents.

[0005] The first aspect of the present invention provides a protective agent for freeze-drying HCV molecular detection reagents, the protective agent comprising: 1-3% trehalose, 8-10% dextran, 1-3% sucrose, 0.7-1.2% cyclodextrin and 0.3-0.7% PVP, the balance being water.

[0006] In another preferred embodiment, the protective agent includes: 2% trehalose, 9% dextran, 2% sucrose, 0.7% cyclodextrin, and 0.5% PVP.

[0007] In another preferred embodiment, the dextran may be dextran 3000, dextran 10000, dextran 40000 or dextran 65000, preferably dextran 40000.

[0008] In another preferred example, the PVP may be PVP10000 or PVP40000, preferably PVP 40000.

[0009] In another preferred embodiment, the protective agent further comprises a surfactant; the surfactant is preferably Tween 80; preferably, the concentration of Tween 80 is 0.05-0.2%.

[0010] In another preferred embodiment, in the protective agent, the concentration ratio of trehalose to dextran is 1:3-5.

[0011] The second aspect of the present invention provides a lyophilized reagent for detecting HCV molecules, wherein the lyophilized reagent comprises the protective agent described in the first aspect of the present invention and the HCV reaction system.

[0012] In another preferred embodiment, the HCV reaction system includes HCV nucleic acid-specific PCR amplification primers and probes, RT-PCR reaction enzyme system and RT-PCR reaction reagents.

[0013] In another preferred embodiment, the RT-PCR reaction enzyme system includes hot-start Taq enzyme, reverse transcriptase, dNTPs and RNasin.

[0014] In another preferred embodiment, the RT-PCR reaction reagents include Tris-HCl, MgCl2, KCl, and (NH4)2SO4.

[0015] The third aspect of the present invention provides a kit for HCV nucleic acid detection, the kit comprising the lyophilized reagent described in the second aspect of the present invention.

[0016] A fourth aspect of the present invention provides a method for preparing a lyophilized reagent for HCV nucleic acid detection, the method comprising the steps of:

[0017] (1) Preparation of liquid HCV reaction system

[0018] The HCV reaction system includes HCV nucleic acid-specific PCR amplification primers and probes, RT-PCR reaction enzyme system and RT-PCR reaction reagents;

[0019] (2) Preparation of freeze-drying protective agent:

[0020] preparing the lyoprotectant according to the first aspect of the present invention;

[0021] (3) Preparation of pre-freezing solution

[0022] The lyophilization protective agent and the liquid HCV reaction system are mixed to prepare a pre-frozen solution;

[0023] (4) Freeze-drying

[0024] The pre-frozen solution provided in step (3) is freeze-dried to obtain the freeze-dried reagent.

[0025] In another preferred embodiment, in step (4), the freeze-drying procedure is:

[0026] 1) Precool the freeze dryer shelf to -45-50°C in advance, keep it for 10-30 minutes, and then put it into the prefreezing solution;

[0027] 2) Turn off the pre-cooling setting, control the shelf temperature at -45 to -50°C, the cooling rate is 0°C / min, and maintain it for 120 to 180 minutes;

[0028] 3) Annealing stage: the shelf temperature is raised to -25--30°C, the heating time is 20 minutes, the average heating rate is 0.75-1.25°C / min, and it is kept at -25--30°C for 120-180 minutes; then the temperature is lowered to -45--50°C, the cooling time is 20 minutes, the average cooling rate is 0.75-1.25°C / min, and it is kept at -45--50°C for 180-220 minutes;

[0029] 4) Primary sublimation stage: turn on the vacuum, set the vacuum degree to 20-50 mTorr, keep the shelf temperature at -30--35°C, the heating time is 120 min, the average heating rate is 0.08-0.16°C / min, and keep at -30--35°C for 800-1000 min;

[0030] 5) Secondary sublimation stage: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -25--30°C, the heating time is 30 min, the average heating rate is 0.10-0.33°C / min, and maintained at -25--30°C for 100-200 min;

[0031] 6) Three sublimation stages: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -5--10°C, the heating time is 60 min, the average heating rate is 0.25-0.41°C / min, and maintained at -5--10°C for 50-100 min;

[0032] 7) Drying stage: set the vacuum degree to 20-30 mTorr, raise the shelf temperature to 25-30°C, the heating time is 60 min, the average heating rate is 0.50-0.67°C / min, and maintain at 25-30°C for 400-600 min;

[0033] 8) Product storage stage: After the freeze-drying process is completed, the product enters the temporary storage stage, and the vacuum degree is set to 800mTorr and the shelf temperature is set to 28°C.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1.Results of 45 days of 37℃ assessment (blue is the control group, red is the freeze-dried balls assessed at 37℃).

[0036] Figure 2 .Results of 78 days of 37℃ assessment (blue is the control group, red is the freeze-dried balls assessed at 37℃).

[0037] Figure 3 .Results of 100 days of 37℃ assessment (blue is the control group, red is the 37℃ assessment freeze-dried balls).

[0038] Figure 4 .Results of 243 days of assessment at 37℃ (blue is the control group, red is the freeze-dried balls assessed at 37℃).

[0039] Figure 5 .Results of 4-day test at 45℃ (blue is the control group, red is the freeze-dried balls tested at 45℃).

[0040] Figure 6 .Results of 21 days of test at 45℃ (blue is the control group, red is the freeze-dried balls tested at 45℃).

[0041] Figure 7 .Results of 35 days of test at 45℃ (blue is the control group, red is the freeze-dried balls tested at 45℃).

[0042] Figure 8 .Results of 56 days of testing at 45℃ (blue is the control group, red is the freeze-dried balls tested at 45℃). DETAILED DESCRIPTION

[0043] The present invention provides a freeze-dried reagent and a kit for HCV nucleic acid detection. Specifically, after extensive research, the present invention obtains a freeze-dried reagent for HCV nucleic acid detection with extremely high stability, which solves the problems of complicated preparation process of HCV nucleic acid detection reagent and high cost of product storage and transportation.

[0044] The main advantages of the present invention are:

[0045] (1) The present invention optimizes and selects a suitable freeze-dried protective agent formula, so that the prepared freeze-dried PCR reagent for HCV nucleic acid detection has a stable morphology, and the amplification performance of the freeze-dried reagent is basically the same as that of the liquid amplification reagent.

[0046] (2) The freeze-dried PCR reagent for HCV nucleic acid detection prepared by the present invention has excellent stability at room temperature or even high temperature, and the prepared freeze-dried PCR reagent still maintains its detection effect after being transported at room temperature.

[0047] (3) The HCV nucleic acid detection freeze-dried reagent prepared by the present invention greatly reduces the production, transportation and storage costs of the detection reagent, while reducing the use operation steps, improving the reagent stability, extending the shelf life and increasing the flexibility of the sample volume, thereby improving the detection sensitivity.

[0048] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0049] Example 1

[0050] Preparation of HCV reaction system:

[0051] HCV Reaction Solution A:

[0052] The primer concentration was 0.01mmol / L, and the probe concentration was 0.002mmol / L;

[0053] The sequences of the forward and reverse primers are:

[0054] 5'-CGTCAAGTTCCCGGGTGGCGGT-3' (SEQ ID NO.: 1) and

[0055] 5'-GCCAACCTGCCCACCCACAGCC-3' (SEQ ID NO.:2);

[0056] The sequence of the probe is:

[0057] 5'-CCATAAAGAGGCCAAGGG-3' (SEQ ID NO.: 3), the two ends of the probe are respectively bound to the fluorescence generating group FAM and the fluorescence quenching group Eclipse;

[0058] The sequences of the forward and reverse primers for the internal reference are:

[0059] 5'-CGAGGGACTTCTGATTTGAT-3' (SEQ ID NO.: 4) and

[0060] 5'-ATTGAAGAAGAGAGTGGCAT-3' (SEQ ID NO.:5),

[0061] The sequence of the internal reference probe is:

[0062] 5'-ATCATCACCAGGCACAACCCGAAGC-3' (SEQ ID NO.: 6), the two ends of the probe are respectively bound to the fluorescence generating group YELLOW and the fluorescence quenching group BHQ1.

[0063] HCV Reaction Solution B:

[0064] It includes hot start Taq enzyme, reverse transcriptase, dNTPs and RNasin; the amount of hot start Taq enzyme in each HCV reaction solution B is 5U; the amount of reverse transcriptase is 5U; the amount of dNTPs is 10mmol, and the amount of RNasin is 30U.

[0065] HCV Reaction Solution C:

[0066] Prepared with pure water, pH value is 8.8-9.0, containing 50mmol / L Tris-HCl, 10mmol / L MgCl2, 300mmol / L KCl, 20mmol / L (NH4)2SO4, and 0.1% Tween-20.

[0067] The HCV reaction solution A, HCV reaction solution B, and HCV reaction solution C were mixed to prepare the HCV reaction system, as shown in the following table:

[0068] Table 1

[0069]

[0070] Lyoprotectant:

[0071] Ultrapure water preparation: trehalose 2%, dextran 40000 9%, sucrose 2%, cyclodextrin 0.7%, PVP400000 0.5%, Tween 80 0.1%;

[0072] Preparation of lyophilized pellets:

[0073] 1) Preparation of freeze-dried protective agent: Take the amount of trehalose, dextran, sucrose, cyclodextrin, PVP, Tween 80 and ultrapure water in the formula and mix them evenly:

[0074] 2) Preparation of reaction reagents: Prepare a liquid HCV reaction system;

[0075] 3) Mix the lyophilized protective agent and the HCV reaction reagent in a volume ratio of 5:4, and mix them evenly. The total volume is 45 μL. At this time, the solution is a pre-frozen solution;

[0076] 4) Transfer the prepared pre-frozen solution to the bead dropping machine and set the bead dropping volume to 45 μL / bead;

[0077] 5) The prepared freeze-dried balls (lyophilized beads) are temporarily stored in liquid nitrogen to prevent melting, and then transferred to a freeze dryer for freeze drying.

[0078] Lyophilization steps:

[0079] 1) Precool the freeze dryer shelf to -45-50°C in advance, maintain for 10-30 minutes to ensure the uniformity of the freeze dryer shelf temperature, and then place the prepared freeze-dried balls;

[0080] 2) Turn off the pre-cooling setting, control the shelf temperature at -45 to -50°C, the cooling rate is 0°C / min, and maintain it for 120 to 180 minutes;

[0081] 3) Annealing stage: the shelf temperature is raised to -25--30°C, the heating time is 20 minutes, the average heating rate is 0.75-1.25°C / min, and it is kept at -25--30°C for 120-180 minutes; then the temperature is lowered to -45--50°C, the cooling time is 20 minutes, the average cooling rate is 0.75-1.25°C / min, and it is kept at -45--50°C for 180-220 minutes;

[0082] 4) Primary sublimation stage: turn on the vacuum, set the vacuum degree to 20-50 mTorr, keep the shelf temperature at -30--35°C, the heating time is 120 min, the average heating rate is 0.08-0.16°C / min, and keep at -30--35°C for 800-1000 min;

[0083] 5) Secondary sublimation stage: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -25--30°C, the heating time is 30 min, the average heating rate is 0.10-0.33°C / min, and maintained at -25--30°C for 100-200 min;

[0084] 6) Three sublimation stages: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -5--10°C, the heating time is 60 min, the average heating rate is 0.25-0.41°C / min, and maintained at -5--10°C for 50-100 min;

[0085] 7) Drying stage: set the vacuum degree to 20-30 mTorr, raise the shelf temperature to 25-30°C, the heating time is 60 min, the average heating rate is 0.50-0.67°C / min, and maintain at 25-30°C for 400-600 min;

[0086] 8) Product storage stage: After the freeze-drying process is completed, the product enters the temporary storage stage, and the vacuum degree is set to 800mTorr and the shelf temperature is set to 28°C.

[0087] HCV testing program:

[0088] Add 40 μl of the sample nucleic acid to be tested into the HCV reaction tube containing the above freeze-dried pellet, and perform the test after it is fully dissolved.

[0089] Place the reaction tube into the instrument sample slot. ABIPrism 7500 instrument settings (refer to this operation for ABIPrism 7300 instrument, see the manual for specific operations). Open the "Setup" window, set the negative quality control (NTC), positive quality control, unknown sample (Unknow), and positive quantitative reference (Standard) in the corresponding order of the samples, and set the sample name in the "Sample Name" column; the probe detection mode is set to: Reporter Dye1: FAM, Quencher Dye1: TAMRA, ReporterDye2: VIC, Quencher Dye2: none, Passive Reference: NONE. Open the instrument window and set the cycle conditions as follows:

[0090] 50°C for 15 min, 1 cycle;

[0091] 95°C for 15 min, 1 cycle;

[0092] 94°C for 15 seconds → 55°C for 45 seconds (collecting fluorescence), 45 cycles.

[0093] After the settings are completed, save the file and run the program.

[0094] After the reaction is completed, the results are automatically saved. According to the analyzed image, the Start value, End value and Threshold value of the Baseline are adjusted (users can adjust according to actual conditions. The Start value can be set between 3 and 15, and the End value can be set between 5 and 20. In the Log graph window, the Threshold Value is set so that the baseline is in the exponential phase of the amplification curve. The amplification curve of the negative control product is adjusted to be flat or below the threshold line). Click Analysis to automatically obtain the analysis results. View the results in the Report interface and record the unknown sample values ​​(C).

[0095] Stability test:

[0096] The HCV nucleic acid detection reagent freeze-dried pellets prepared above were stored at 37°C and 45°C to test the stability of the freeze-dried reagent. The newly prepared HCV nucleic acid detection reagent was used as the control group. The test samples were quantitatively concentrated at 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0097] Real-time stability at 37°C:

[0098] Figure 1 .37℃ test results for 45 days (blue is the control group, red is the 37℃ test freeze-dried balls), from left to right 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4 IU / mL, 1.0×10 3 IU / mL,

[0099] 1.0×10 2 IU / mL of HCV RNA virus-like particles.

[0100] Figure 2 .37℃ test results for 78 days (blue is the control group, red is the 37℃ test freeze-dried balls), from left to right 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4 IU / mL, 1.0×10 3 IU / mL,

[0101] 1.0×10 2 IU / mL of HCV RNA virus-like particles.

[0102] Figure 3 .37℃ test results for 100 days (blue is the control group, red is the 37℃ test freeze-dried balls), from left to right 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4 IU / mL.

[0103] Figure 4 .37℃ test results for 243 days (blue is the control group, red is the 37℃ test freeze-dried pellet), from left to right 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4 IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0104] Real-time stability at 45°C

[0105] Figure 5.45℃ test results for 4 days (blue is the control group, red is the 45℃ test freeze-dried balls), from left to right 1.0×10 4 IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0106] Figure 6 .45℃ test results for 21 days (blue is the control group, red is the 45℃ test freeze-dried balls), from left to right 1.0×10 4 IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0107] Figure 7 .45℃ test results for 35 days (blue is the control group, red is the 45℃ test freeze-dried balls), from left to right 1.0×10 4 IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0108] Figure 8 .45℃ test results for 56 days (blue is the control group, red is the 45℃ test freeze-dried balls), from left to right 1.0×10 4 IU / mL, 1.0×10 3 IU / mL of HCV RNA virus-like particles.

[0109] Some existing HCV testing reagent freeze-dried balls on the market have poor stability and a short shelf life, and cannot be stored and transported at room temperature in the true sense. Their performance will be significantly reduced after being left at 37°C or even at room temperature for a month.

[0110] The HCV detection reagent freeze-dried pellets prepared by the protective agent formula and freeze-drying process of the present invention not only have a smooth appearance, strong moisture resistance, but also have good stable performance. Not only does the performance not decrease in the 14-day room temperature transportation test, but also there is no obvious decrease in the performance test under the high temperature condition of 45°C for 3 months.

[0111] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A protective agent for freeze-drying of HCV molecular detection reagents, characterized in that: The protective agent comprises: 1-3% trehalose, 8-10% dextran, 1-3% sucrose, 0.7-1.2% cyclodextrin and 0.3-0.7% PVP, and the balance is water.

2. The protective agent according to claim 1, characterized in that The protective agent comprises: 2% trehalose, 9% dextran, 2% sucrose, 0.7% cyclodextrin and 0.5% PVP.

3. The protective agent according to claim 1, characterized in that The dextran may be dextran 3000, dextran 10000, dextran 40000 or dextran 65000, preferably dextran 40000; preferably, the PVP may be PVP10000 or PVP40000, preferably PVP 40000.

4. The protective agent according to claim 1, characterized in that In the protective agent, the concentration ratio of trehalose to dextran is 1:3-5.

5. A freeze-dried reagent for HCV molecular detection, characterized in that: The freeze-dried reagent comprises the protective agent according to claim 1 and the HCV reaction system.

6. The freeze-dried reagent according to claim 5, characterized in that The HCV reaction system comprises HCV nucleic acid-specific PCR amplification primers and probes, RT-PCR reaction enzyme system and RT-PCR reaction reagents.

7. The lyophilized reagent according to claim 6, characterized in that The RT-PCR reaction enzyme system includes hot-start Taq enzyme, reverse transcriptase, dNTPs and RNasin.

8. A kit for HCV nucleic acid detection, comprising the lyophilized reagent described in the second aspect of the present invention.

9. A method for preparing a freeze-dried reagent for HCV nucleic acid detection, the method comprising the steps of: (1) Preparation of liquid HCV reaction system The HCV reaction system includes HCV nucleic acid-specific PCR amplification primers and probes, RT-PCR reaction enzyme system and RT-PCR reaction reagents; (2) Preparation of freeze-drying protective agent: preparing the lyoprotectant according to the first aspect of the present invention; (3) Preparation of pre-freezing solution The lyophilization protective agent and the liquid HCV reaction system are mixed to prepare a pre-frozen solution; (4) Freeze-drying The pre-frozen solution provided in step (3) is freeze-dried to obtain the freeze-dried reagent.

10. The method according to claim 9, characterized in that In the step (4), the freeze-drying procedure is: 1) Precool the freeze dryer shelf to -45-50°C in advance, keep it for 10-30 minutes, and then put it into the prefreezing solution; 2) Turn off the pre-cooling setting, control the shelf temperature at -45 to -50°C, the cooling rate is 0°C / min, and maintain it for 120 to 180 minutes; 3) Annealing stage: the shelf temperature is raised to -25--30°C, the heating time is 20 minutes, the average heating rate is 0.75-1.25°C / min, and it is kept at -25--30°C for 120-180 minutes; then the temperature is lowered to -45--50°C, the cooling time is 20 minutes, the average cooling rate is 0.75-1.25°C / min, and it is kept at -45--50°C for 180-220 minutes; 4) Primary sublimation stage: turn on the vacuum, set the vacuum degree to 20-50 mTorr, keep the shelf temperature at -30--35°C, the heating time is 120 min, the average heating rate is 0.08-0.16°C / min, and keep at -30--35°C for 800-1000 min; 5) Secondary sublimation stage: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -25--30°C, the heating time is 30 min, the average heating rate is 0.10-0.33°C / min, and maintained at -25--30°C for 100-200 min; 6) Three sublimation stages: the vacuum degree remains unchanged, maintained at 20-50 mTorr, the shelf temperature is raised to -5--10°C, the heating time is 60 min, the average heating rate is 0.25-0.41°C / min, and maintained at -5--10°C for 50-100 min; 7) Drying stage: set the vacuum degree to 20-30 mTorr, raise the shelf temperature to 25-30°C, the heating time is 60 min, the average heating rate is 0.50-0.67°C / min, and maintain at 25-30°C for 400-600 min; 8) Product storage stage: After the freeze-drying process is completed, the product enters the temporary storage stage, and the vacuum degree is set to 800mTorr and the shelf temperature is set to 28°C.

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