Composite freeze-drying protective agent for giant salamander iridovirus detection reagent and preparation method of reagent

Abstract

The invention relates to the technical field of epidemic disease detection of aquatic animals, in particular to a composite freeze-drying protective agent for a giant salamander iridovirus detection reagent and a preparation method of the reagent. The composite freeze-drying protective agent is mainly prepared from the following components: trehalose, glucan and hydroxyethyl starch according to the mass ratio of (3 to 5) to (0.8 to 1.2) to (0.3 to 0.7). The three-component composite protective agent is adopted, trehalose, dextran 40 and hydroxyethyl starch are mixed for use, protein is effectively protected, the glass transition temperature is prevented from being too high or too low, and redissolution and caking are inhibited. In the freeze-drying process of the detection reagent based on the protective agent, Q92R / E118K double mutation is performed on Taq enzyme, so that a dual protection mechanism for freeze-drying damage is formed; stepwise heating is introduced in the sublimation stage, and the freeze-drying time is shortened to 12 h; the detection reagent is treated through the gradient annealing freeze-drying process, so that the enzyme retention rate can reach 95% or above. The zero turbidity (OD600 is less than 0.05) of the redissolved reagent is ensured, and the interference of fluorescence detection is eliminated.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic animal disease detection, and in particular to a composite freeze-dried protective agent for a giant salamander iridescent virus detection reagent and a reagent preparation method. Background Art

[0002] Giant salamander iridovirus is a double-stranded DNA virus that can cause explosive mortality in giant salamanders. The mortality rate after infection is as high as over 40%, and can even reach 80-100%. Therefore, it is called "giant salamander cancer". Existing treatment methods can only treat the symptoms and cannot cure it. Therefore, early diagnosis and isolation treatment are crucial for the prevention and treatment of giant salamander iridovirus disease. PCR detection is used in existing detection technologies, but the detection reagents have the following problems: (1) The Taq enzyme in the liquid reagent is inactivated by more than 90% at 25°C within 48 hours, resulting in failure of detection in remote farms. (2) The reagent transportation requires a cold chain of -20°C throughout the entire process, and the cost accounts for more than 40% of the total detection cost. (3) After the reagent is opened, multiple freeze-thaw cycles cause the enzyme activity to drop by more than 30% (compared to the first use), causing false negatives.

[0003] Therefore, it is very important to develop a protective agent system with high activity retention, rapid re-dissolution, and no detection interference. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to address the deficiencies of the existing technology and provide a composite freeze-dried protective agent and reagent preparation method for giant salamander iridescent virus detection reagent that is stable at room temperature and can be dissolved and used immediately.

[0005] Technical solution: The present invention discloses a composite freeze-dried protective agent for giant salamander iridescent virus detection reagent, which is mainly composed of the following components: trehalose, dextran, and hydroxyethyl starch, with the mass ratio of the three being (3-5): (0.8-1.2): (0.3-0.7).

[0006] Furthermore, the mass ratio of trehalose, dextran and hydroxyethyl starch is 4:1:0.5. With this ratio, it is found that low-dose hydroxyethyl starch (HES) can eliminate the adhesiveness of dextran.

[0007] Furthermore, the dextran is dextran 40 with a molecular weight of 40 kDa, and the degree of substitution of hydroxyethyl starch is 0.7.

[0008] Another technical solution of the present invention is a method for preparing a giant salamander iridescent virus detection reagent based on the composite lyophilized protective agent, comprising the following steps:

[0009] (1) Mix primers, probes, dNTPs, buffer and lyophilization protectant;

[0010] (2) Adding HotStart Taq enzyme pre-coated with trehalose;

[0011] (3) After packaging, gradient annealing and freeze-drying are performed to obtain freeze-dried detection reagents.

[0012] The mechanism of action in step (2) of the present invention is the positive charge-enriched region: Q92R / E118K increases the positive charge density on the enzyme surface by 2.3 times, anchoring the negatively charged glucan chains (-COO- groups) through electrostatic attraction. Hydrogen bond-directed optimization: After the mutation, an "arginine-lysine bipolar pocket" is formed on the enzyme surface, which preferentially binds to the hydroxyl group of HES and inhibits re-dissolution and agglomeration.

[0013] Furthermore, the freeze-drying step in the present invention adopts a gradient freeze-drying process, and the specific steps are as follows:

[0014] Pre-freeze: -45℃, keep for 3h;

[0015] Sublimation drying: first keep at -25℃ for 1 hour, then keep at -15℃ for 1 hour, and finally keep at -5℃ for 1 hour; the vacuum degree of the whole process is ≤5Pa;

[0016] Drying: Keep at 20℃ for 2h.

[0017] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0018] 1. The three-component composite protective agent is a mixture of trehalose, dextran 40 and hydroxyethyl starch, which effectively protects the protein, prevents the glass transition temperature from being too high or too low, and inhibits re-dissolution and agglomeration.

[0019] 2. The Taq enzyme was subjected to a Q92R / E118K double mutation to increase the surface positive charge and enhance the hydrogen bond with the protective agent, forming a dual protection mechanism against freeze-drying damage.

[0020] 3. In the present invention, a step-by-step temperature increase from -25°C to -5°C is introduced during the sublimation stage, with each step being kept warm for 1 hour, shortening the freeze-drying time to 12 hours. The detection reagent is treated through this gradient annealing freeze-drying process, so that the enzyme retention rate can reach more than 95%.

[0021] 4. Ensure that the reagent has zero turbidity (OD600 < 0.05) after reconstitution to eliminate interference with fluorescence detection. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention are described in detail below through examples, but the protection scope of the present invention is not limited to the examples.

[0023] Example 1

[0024] A composite lyophilized protective agent for use in a giant salamander iridescent virus detection reagent is primarily composed of the following components: trehalose, dextran, and hydroxyethyl starch, with a mass ratio of 4:1:0.5. The dextran is Dextran 40 with a molecular weight of 40 kDa, and the hydroxyethyl starch has a degree of substitution of 0.7.

[0025] A method for preparing a giant salamander iridescent virus detection reagent based on the above-mentioned composite lyophilized protective agent comprises the following steps:

[0026] (1) Mixing primers, probes, dNTPs, buffer and lyophilization protectant; wherein:

[0027] Primer sequences:

[0028] GSIV-RPA-F:5'-CGTACCTGGTATGATCGACCC-3' (SEQ ID NO:1);

[0029] GSIV-RPA-R: 5'-Biotin-GCAGTTGTCGGTGTTGTTGTC-3' (SEQ ID NO: 2);

[0030] Probe: 5'-FAM-CCGTACCT[THF]CGGTAT-BHQ1-3' (SEQ ID NO: 3);

[0031] The buffer was lysis buffer containing 0.5% Triton X-100 and 10 mM EDTA;

[0032] (2) Adding HotStart Taq enzyme with Q92R / E118K mutation;

[0033] (3) After packaging, gradient annealing and freeze-drying are performed to obtain freeze-dried detection reagents; wherein the steps of gradient annealing and freeze-drying are as follows:

[0034] Pre-freeze: -45℃, maintain for 3h;

[0035] Sublimation drying: first keep at -25℃ for 1 hour, then keep at -15℃ for 1 hour, and finally keep at -5℃ for 1 hour; the vacuum degree of the whole process is ≤5Pa;

[0036] Drying: Keep at 20℃ for 2h.

[0037] After the resulting freeze-dried reagent was stored at 45°C for 14 days, the Ct value shift of qPCR detection was only 0.3.

[0038] Example 2

[0039] The method is basically the same as Example 1, except that the mass ratio of trehalose, dextran and hydroxyethyl starch is 3:0.8:0.3.

[0040] Example 3

[0041] The method is basically the same as Example 1, except that the mass ratio of trehalose, dextran and hydroxyethyl starch is 5:1.2:0.7.

[0042] Comparative Example 1:

[0043] A freeze-dried protective agent for a giant salamander iridescent virus detection reagent mainly consists of the following components: 10 parts of trehalose.

[0044] Comparative Example 2:

[0045] A composite freeze-dried protective agent for a giant salamander iridescent virus detection reagent mainly consists of the following components: trehalose and dextran in a mass ratio of 4:1.

[0046] Comparative Example 3:

[0047] The method is basically the same as that of embodiment 1, except that the freeze-drying step in step (3) adopts the traditional process, i.e., constant sublimation at -30°C for 12 hours and a freeze-drying cycle of 28 hours.

[0048] Comparing the embodiment with the comparative example, the experimental results are as follows:

[0049]

[0050] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A composite freeze-dried protective agent for a giant salamander iridescent virus detection reagent, characterized in that: Mainly composed of the following ingredients Composition: Trehalose, dextran, hydroxyethyl starch, the mass ratio of the three is (3-5): (0.8-1.2): (0.3-0.7).

2. The composite freeze-dried protective agent for the detection reagent of giant salamander iridescent virus according to claim 1, characterized in that: The mass ratio of trehalose, dextran and hydroxyethyl starch is 4:1:0.

5.

3. The composite lyophilized protective agent for the detection reagent of giant salamander iridescent virus according to claim 1, characterized in that: The dextran is Dextran 40 with a molecular weight of 40 kDa, and the degree of substitution of hydroxyethyl starch is 0.

7.

4. A method for preparing a detection reagent for giant salamander iridescent virus based on the composite lyophilized protective agent according to any one of claims 1 to 3, characterized in that: The steps include: (1) Mix primers, probes, dNTPs, buffer, and lyophilization protectant; (2) Adding HotStart Taq enzyme with Q92R / E118K mutation; (3) After packaging, gradient annealing and freeze-drying are performed to obtain freeze-dried detection reagents.

5. The method according to claim 4, wherein In step (3), the freeze-drying step includes: Pre-freeze: -45°C, maintain for 3 hours; Sublimation drying: first keep at -25℃ for 1 hour, then keep at -15℃ for 1 hour, and finally keep at -5℃ for 1 hour; the vacuum degree of the whole process is ≤5Pa; Drying: Keep at 20℃ for 2h.

6. The method according to claim 4, wherein The water content after freeze-drying is ≤2.5%, and the reconstitution time is ≤10 seconds.

Images