Pathogenic nucleic acid preservation tube with wall breaking effect

Abstract

The invention relates to the technical field of biological sample preservation, and provides a pathogen nucleic acid preservation tube with a wall breaking effect, the pathogen nucleic acid preservation tube comprises a preservation solution and grinding beads, the preservation solution comprises 8-12 wt% of guanidine isothiocyanate, 0.3-0.8 wt% of trihydroxymethyl aminomethane, 0.2-0.6 wt% of sodium chloride, 0.2-0.5 wt% of ethylenediamine tetraacetic acid, and the balance purified water. According to the invention, the components are innovatively and optimally proportioned, and grinding beads arranged in the storage tube are combined, so that triple technical effects are realized: a sample can be stably stored for 7 days at 37 DEG C, and the integrity of nucleic acid is kept; the growth of bacteria is effectively inhibited and pathogenic microorganisms are inactivated; wall breaking of pathogenic microorganisms is achieved through chemical and physical synergistic effects, and nucleic acid release is remarkably promoted. The preservation tube is an integrated pathogenic nucleic acid preservation tube with wall breaking, nucleic acid stabilization, inactivation and bacteriostasis functions, is particularly suitable for collection, transportation and storage of clinical samples, and solves the technical problems that in the prior art, nucleic acid is easy to degrade, pathogenic microorganisms are not thoroughly inactivated and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological sample preservation, in particular to a pathogenic nucleic acid preservation tube with a cell wall breaking function, and more particularly to an integrated pathogenic nucleic acid preservation tube with cell wall breaking, nucleic acid stabilizing, inactivation and bacteriostatic functions. BACKGROUND

[0002] In the fields of pathogenic microorganism detection, gene sequencing and molecular diagnosis, the collection and preservation of pathogenic nucleic acid samples are crucial. Traditional pathogenic nucleic acid preservation tubes are mainly used to maintain the stability of nucleic acids, but in actual applications, samples often need to be subjected to additional cell wall breaking treatment (such as mechanical crushing, chemical lysis, etc.) to release nucleic acids, which not only increases the operation steps, but also may lead to nucleic acid degradation or contamination risk.

[0003] In the prior art, traditional nucleic acid preservation tubes usually only contain a preservation solution (such as guanidine salt, EDTA, etc.), which can stabilize nucleic acids, but need to be combined with an external cell wall breaking step, resulting in a complicated process and increased sample loss. The performance of traditional nucleic acid preservation tubes in virus inactivation and microbial contamination inhibition is insufficient, and there is a biological safety risk.

[0004] To solve the above problems, there is an urgent need for a pathogenic nucleic acid preservation tube with integrated cell wall breaking function, which can complete cell wall breaking while preserving samples, and ensure nucleic acid stability, inhibit contamination and inactivation. SUMMARY

[0005] To achieve the above technical purposes, the technical solution adopted by the present application is as follows:

[0006] A pathogenic nucleic acid preservation tube with a cell wall breaking function, comprising:

[0007] A preservation solution containing the following substances in mass percentage: 8-12wt% guanidine isothiocyanate, 0.2-0.8wt% tris-hydroxymethyl aminomethane, 0.2-0.6wt% sodium chloride, 0.2-0.5wt% ethylenediaminetetraacetic acid, and the balance being purified water;

[0008] Grinding beads, the material of the grinding beads is zirconium oxide, silicon dioxide or glass, the diameter is 0.1-2mm and the density is 3.5-6.0g / cm 3 ;

[0009] Further, the pH value of the preservation solution is 7.5-8.5.

[0010] Further, the preservation solution contains the following substances in mass percentage: 10wt% guanidine isothiocyanate, 0.5wt% tris-hydroxymethyl aminomethane, 0.4wt% sodium chloride, and 0.32wt% ethylenediaminetetraacetic acid.

[0011] Further, the preparation method of the preservation solution is as follows:

[0012] S1. Take guanidinium isothiocyanate, tris-hydroxymethyl aminomethane, sodium chloride, ethylenediaminetetraacetic acid, and dissolve with purified water;

[0013] S2. Stir for 10-30 minutes with a magnetic stirrer until mixed evenly;

[0014] S3. Adjust the pH to 7.5-8.5;

[0015] S4. Filter with a 0.22 μm filter membrane to obtain a preservation solution.

[0016] Further, the preservation tube is a standard 2ml cryogenic tube, the filling volume of the preservation solution is 1ml, and the mass of the grinding beads is 0.8-1.2g.

[0017] Beneficial effects:

[0018] The main purpose of the present application is to provide a pathogenic nucleic acid preservation tube with a cell wall breaking effect, which realizes three technical effects: the sample can be stably preserved for 7 days at 37℃ and the nucleic acid integrity is maintained; effective inactivation and bacteriostasis; and the cell wall of pathogenic microorganisms is broken through chemical and physical synergistic effect, which significantly promotes the release of nucleic acid. The preservation tube is an integrated pathogenic nucleic acid preservation tube with the functions of cell wall breaking, nucleic acid stabilization, inactivation and bacteriostasis, and is particularly suitable for the collection, transportation and storage of clinical samples, and solves the technical problems of easy degradation of nucleic acid and incomplete inactivation of pathogenic microorganisms in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Structure diagram of the preservation tube.

[0020] Figure 2 Photo of E. coli added to the pathogenic nucleic acid preservation tube of Example 1 for 5 minutes and then inoculated in LB medium for growth.

[0021] Figure 3 Photo of E. coli added to the pathogenic nucleic acid preservation tube of Example 2 for 5 minutes and then inoculated in LB medium for growth.

[0022] Figure 4 Photo of E. coli added to the pathogenic nucleic acid preservation tube of Example 3 for 5 minutes and then inoculated in LB medium for growth. DETAILED DESCRIPTION

[0023] In order to enable the personnel in the technical field to better understand the technical solutions in the present application, the present application will be further described in detail below in combination with examples.

[0024] Example 1

[0025] 1. A pathogenic nucleic acid preservative solution with cell wall breaking effect, comprising the following substances in mass percentage: 8wt% guanidine isothiocyanate, 0.2wt% tris-hydroxymethyl aminomethane, 0.2wt% sodium chloride, 0.2wt% ethylenediamine tetraacetic acid, and the balance being purified water.

[0026] 2. The preparation method of the preservative solution is as follows: guanidine isothiocyanate, tris-hydroxymethyl aminomethane, sodium chloride, and ethylenediamine tetraacetic acid are taken and dissolved in purified water, stirred for 10 minutes by a magnetic stirrer, uniformly mixed, adjusted to pH 8, filtered by a 0.22μm filter membrane, and a preservative solution is obtained.

[0027] 3. A pathogenic nucleic acid preservation tube with cell wall breaking effect is a standard 2ml cryogenic tube, which comprises 1ml of the preservative solution and 1g of grinding beads, and the material of the grinding beads is zirconium oxide with a diameter of 1mm.

[0028] Example 2

[0029] 1. A pathogenic nucleic acid preservative solution with cell wall breaking effect, comprising the following substances in mass percentage: 10wt% guanidine isothiocyanate, 0.5wt% tris-hydroxymethyl aminomethane, 0.4wt% sodium chloride, 0.32wt% ethylenediamine tetraacetic acid, and the balance being purified water.

[0030] 2. The preparation method of the preservative solution is as follows: guanidine isothiocyanate, tris-hydroxymethyl aminomethane, sodium chloride, and ethylenediamine tetraacetic acid are taken and dissolved in purified water, stirred for 10 minutes by a magnetic stirrer, uniformly mixed, adjusted to pH 8, filtered by a 0.22μm filter membrane, and a preservative solution is obtained.

[0031] 3. A pathogenic nucleic acid preservation tube with cell wall breaking effect is a standard 2ml cryogenic tube, which comprises 1ml of the preservative solution and 1g of grinding beads, and the material of the grinding beads is zirconium oxide with a diameter of 1mm.

[0032] Example 3

[0033] 1. A pathogenic nucleic acid preservative solution with cell wall breaking effect, comprising the following substances in mass percentage: 12wt% guanidine isothiocyanate, 0.8wt% tris-hydroxymethyl aminomethane, 0.6wt% sodium chloride, 0.5wt% ethylenediamine tetraacetic acid, and the balance being purified water.

[0034] 2. The preparation method of the preservative solution is as follows: guanidine isothiocyanate, tris-hydroxymethyl aminomethane, sodium chloride, and ethylenediamine tetraacetic acid are taken and dissolved in purified water, stirred for 10 minutes by a magnetic stirrer, uniformly mixed, adjusted to pH 8, filtered by a 0.22μm filter membrane, and a preservative solution is obtained.

[0035] 3. A pathogenic nucleic acid preservation tube with cell wall breaking effect is a standard 2ml cryogenic tube, which comprises 1ml of the preservative solution and 1g of grinding beads, and the material of the grinding beads is zirconium oxide with a diameter of 1mm.

[0036] In Example 1, Example 2, and Example 3, the preservation tubes differ only in the mass percentage of each component in the preservation solution.

[0037] Verification Test 1: Nucleic acid stability detection

[0038] 1. H. pylori and S. aureus were diluted by 10 times in three gradients.

[0039] 2. The diluted H. pylori and S. aureus samples were added to the pathogenic nucleic acid preservation tubes (as shown in Table 1).

[0040] Table 1

[0041]

[0042] 3. The samples were stored at 37°C, and sampled at 0d and 7d, respectively. At the time of sampling, 200 μl was taken from each group, and nucleic acid extraction was performed directly (200 μl was extracted each time, and the extraction was repeated once). After extraction, the samples were detected using a fluorescent quantitative PCR kit with specific primers. The detection results of H. pylori are shown in Table 2, and the detection results of S. aureus are shown in Table 3.

[0043] Table 2

[0044]

[0045] Table 3

[0046]

[0047] 4. Result analysis

[0048] The detection results show that after 7 days of storage at 37°C, the Ct values of H. pylori and S. aureus of different dilution gradients (0.1, 0.01, 0.001) change little (ΔCt < 1), indicating that the nucleic acid degradation is not obvious. The Ct values of the samples with high dilution (0.001) fluctuate slightly, which may be related to the detection limit of low-concentration nucleic acid, but are still within an acceptable range. The nucleic acid stability of Example 2 is the best.

[0049] Verification Test 2: Inactivation and bacteriostatic property detection

[0050] 1. E. coli was added to the pathogenic nucleic acid preservation tubes for 5 min, and then inoculated in LB medium. The preservation tubes of different examples were subjected to the pre-inoculation treatment as shown in Table 4.

[0051] Table 4

[0052]

[0053] 2. Result analysis

[0054] From Figure 2 , Figure 3 , Figure 4 it can be seen that no turbidity was observed in the storage tubes numbered 3, 6, and 9, and turbidity was observed in the storage tubes numbered 2, 5, and 8, indicating that E. coli in them was active; but no turbidity was observed in the storage tubes numbered 1, 4, and 7, indicating that the pathogenic nucleic acid storage tubes in Example 1, Example 2, and Example 3 can effectively inhibit the growth of inactivated E. coli.

[0055] Verification test 3: verification of the cell wall breaking effect

[0056] 1. After diluting the S. aureus culture solution 10 times, divide it into four parts, and add it to the pathogenic nucleic acid storage tubes of Example 1, Example 2, and Example 3, respectively, and add another part to the same amount of sterile water as the storage solution in the pathogenic nucleic acid storage tube as a control. After storing at room temperature for 1 hour, perform nucleic acid extraction (200 μl each time, repeated 1 time). After extraction, use a fluorescent quantitative PCR kit with specific primers for detection. Record the detection results in Table 5.

[0057] Table 5

[0058] Processing Ct value Example 1 23.46 Example 2 22.32 Example 3 23.15 Control group 24.73

[0059] 2. Result analysis

[0060] From Table 5, it can be seen that the Ct values of Example 1, Example 2, and Example 3 are lower than the control group (24.73), indicating that the pathogenic nucleic acid storage tube can effectively break the cell wall of S. aureus, thereby promoting the release of S. aureus nucleic acid, and Example 2 has the best cell wall breaking effect.

[0061] According to the above test results, the pathogenic nucleic acid storage tubes of Example 1, Example 2, and Example 3 can all store samples for 7 days at 37°C without significant effects on the integrity of pathogenic microorganism nucleic acid; can effectively inhibit the growth of inactivated bacteria; and have a certain cell wall breaking effect on pathogenic microorganisms, promoting the release of nucleic acid. Among them, Example 2 has the best overall performance in nucleic acid stability, inactivation effect, and cell wall breaking ability, and is suitable as the preferred formula of the pathogenic nucleic acid storage tube. The storage tube of the present application is an integrated pathogenic nucleic acid storage tube with the functions of cell wall breaking, nucleic acid stabilization, inactivation, and antibacterial function.

Claims

1. A pathogenic nucleic acid storage tube with a cell wall breaking effect, characterized by, The application relates to a preservative solution and a preservative tube. The preservative solution comprises the following mass percentages of substances: 8-12 wt% guanidine isothiocyanate, 0.2-0.8 wt% tris-hydroxymethyl aminomethane, 0.2-0.5 wt% sodium chloride and 0.2-0.5 wt% ethylenediaminetetraacetic acid, and the rest is purified water. Abrasive beads made of zirconium oxide, silicon dioxide or glass, having a diameter of 0.1 to 2 mm and a density of 3.5 to 6.0 g / cm3 3 ; The pH value of the preservative solution is 7.5-8.

5.

2. The pathogenic nucleic acid storage tube with cell wall breaking effect according to claim 1, characterized in that, The preservative solution comprises the following mass percentages of substances: 10 wt% guanidine isothiocyanate, 0.5 wt% tris-hydroxymethyl aminomethane, 0.4 wt% sodium chloride and 0.32 wt% ethylenediaminetetraacetic acid.

3. The pathogenic nucleic acid storage tube with cell wall breaking effect according to claim 1, characterized in that, The preparation method of the preservative solution is as follows: S1. Dissolving guanidine isothiocyanate, tris-hydroxymethyl aminomethane, sodium chloride and ethylenediaminetetraacetic acid in purified water; S2. Stirring for 10-30 minutes by a magnetic stirrer until the mixture is uniform; S3. Adjusting the pH value to 7.5-8.5; S4. Filtering by a 0.22-micron filter membrane to obtain the preservative solution.

4. The pathogenic nucleic acid storage tube with cell wall breaking function according to claim 1, characterized in that, The preservative tube is a standard 2-ml cryopreservation tube, the filling volume of the preservative solution is 1 ml, and the mass of the grinding beads is 0.8-1.2 g.

5. The pathogenic nucleic acid storage tube with cell wall breaking function according to any one of claims 1-4, characterized in that, The preservative tube has the functions of breaking the cell wall, stabilizing nucleic acid, inactivation and bacteriostasis.

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