Flow ploidy analysis kit

Abstract

The invention discloses a flow ploidy analysis kit, and relates to the technical field of biological detection. According to the flow ploidy analysis kit provided by the invention, the DAPI staining solution is specifically combined with the DNA double-helix minor groove, so that accurate marking of the genetic material of the cell nucleus is realized, and the flow ploidy analysis kit can be directly used for flow ploidy analysis and detection of animal cells. By optimizing the working concentration of the DAPI staining solution, the toxic effect of the dye on cells is reduced to the maximum extent while the signal intensity is guaranteed. The kit is suitable for trace living samples, ploidy analysis can be completed without killing animals, and lossless monitoring support is provided for polyploidy breeding. The kit provided by the invention adopts DAPI dyeing, does not need an illumination activation step, can complete the dyeing process within 5 minutes of incubation at room temperature, has the advantage that a fluorescence signal is slightly influenced by pH fluctuation, has the advantage of'detection while dyeing ', and is especially suitable for rapid screening in a field or resource limited scene.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a flow cytometry ploidy analysis kit. Background Technology

[0002] In the process of cultivating wild-type plants and animals that are difficult to adapt to aquaculture environments into superior farmed varieties through biotechnology, every step relies on rapid and accurate genetic typing identification. For plant genetic breeding, chromosome ploidy identification is a crucial step, forming the basis for a deep understanding of their genetic background and subsequent applications. In the animal field, particularly in basic marine biological research and the genetic improvement of economically important animals (such as fish and shellfish), efficient ploidy analysis is equally valuable.

[0003] Traditional methods for chromosome ploidy identification primarily rely on microscopic chromosome counting. This method is cumbersome, time-consuming, and difficult to perform high-throughput analysis of large numbers of cells, with relatively low reproducibility (data consistency). In contrast, flow cytometry ploidy analysis exhibits significant advantages: a relatively simplified procedure, shorter processing time, rapid analysis of large numbers of cells in a single operation, high data reproducibility, and the ability to acquire multiple cellular parameters simultaneously. Especially for valuable triploid organisms, flow cytometry requires very small sample volumes (such as trace amounts of blood or tissue fragments) and can obtain ploidy information without euthanizing the individual being tested. This is crucial for scenarios requiring the organism to remain alive for subsequent polyploid induction or research.

[0004] However, there is currently a lack of mature and reliable flow cytometry ploidy analysis kits in the domestic market. Domestic research and applications mainly rely on imported reagents, which not only increases costs but also poses potential risks in the supply chain. Therefore, developing domestically produced flow cytometry ploidy analysis kits with independent intellectual property rights is of practical necessity to meet the growing domestic research and breeding needs and break the market dominance of foreign products. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a flow cytometry ploidy analysis kit.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] In a first aspect, the present invention provides a flow cytometry ploidy analysis kit, comprising a cell staining solution, wherein the cell staining solution is a DAPI staining solution with a concentration of 3-20 μg / mL.

[0008] Furthermore, the cell staining solution is manufactured by Thermo Fisher Scientific, Sangon Biotech (Shanghai) Co., Ltd., or Sigma-Aldrich.

[0009] Furthermore, the cell staining solution is DAPI staining solution produced by Thermo Fisher Scientific, with a concentration of 3-8 μg / mL.

[0010] Flow cytometry ploidy detection offers significant advantages over traditional microscopy in determining the cell cycle status of eukaryotic cells, being exceptionally rapid, accurate, convenient, and sensitive. However, it requires preparing the sample as a single-cell suspension. Therefore, the flow cytometry ploidy analysis kit provided by this invention contains cell staining solution and can be directly applied to ploidy analysis in animals. This is because some animal cell types exist in vivo in single-cell form, and animal tissues can also be converted into single-cell suspensions through protein hydrolysis. The resulting single-cell suspension sample can then be added to the cell staining solution for ploidy analysis.

[0011] Based on the aforementioned flow cytometry ploidy analysis kit, this invention provides a flow cytometry ploidy analysis kit for plant cell detection, further comprising a nuclear extract, wherein the nuclear extract comprises the following components:

[0012] Nuclear stabilizer 0.5–8 mmol / L;

[0013] Chelating agent 2–35 mmol / L;

[0014] Na + K + Regulator 80–100 mmol / L;

[0015] Buffer solution 15–250 mmol / L, pH 7.5;

[0016] Triton X-1001 - 2% v / v;

[0017] Sodium metabisulfite 8-12 mmol / L;

[0018] PVP-101-2% v / v.

[0019] Furthermore, the nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer solution is selected from Tris-HCl or MOPS.

[0020] Furthermore, the cell nuclear extract is composed of the following components;

[0021] Spermine 4HCl 0.5–1.0 mmol / L;

[0022] Na2EDTA 1.0–10.0 mmol / L;

[0023] Na + K + Regulator 75–110 mmol / L;

[0024] Tris-HCl buffer 150–250 mmol / L, pH 7.5;

[0025] Triton X-1001 - 2% v / v;

[0026] Sodium metabisulfite 8-12 mmol / L;

[0027] PVP-101-2% v / v.

[0028] Furthermore, the cell nuclear extract is composed of the following components;

[0029] MgCl2·6H2O 2.0–6.0mmol / L;

[0030] Sodium citrate 25–35 mmol / L;

[0031] Na + K + Regulator 75–110 mmol / L;

[0032] Tris-HCl buffer 150–250 mmol / L, pH 7.5;

[0033] Triton X-1001 - 2% v / v;

[0034] Sodium metabisulfite 8-12 mmol / L;

[0035] PVP-101-2% v / v.

[0036] Furthermore, the cell nuclear extract is composed of the following components;

[0037] MgCl2·6H2O 2.0–6.0mmol / L;

[0038] Na2EDTA 1.0–3.0 mmol / L;

[0039] Na + K + Regulator 75–110 mmol / L;

[0040] Tris-HCl buffer 150–250 mmol / L, pH 7.5;

[0041] Triton X-1001 - 2% v / v;

[0042] Sodium metabisulfite 8-12 mmol / L;

[0043] PVP-101-2% v / v.

[0044] Furthermore, the cell nuclear extract is composed of the following components;

[0045] MgCl2·6H2O 2.0–6.0mmol / L;

[0046] Na2EDTA 1.0–3.0 mmol / L;

[0047] Na + K + Regulator 75–110 mmol / L;

[0048] MOPS buffer 15–25 mmol / L, pH 7.5;

[0049] Triton X-1001 - 2% v / v;

[0050] Sodium metabisulfite 8-12 mmol / L;

[0051] PVP-101-2% v / v.

[0052] Higher plant cells typically exist in complex three-dimensional tissue structures. In order to meet the requirement that the test sample is a single-cell suspension, the plant tissue needs to be mechanically homogenized, the cell nuclei extracted, and then cell staining solution added for ploidy analysis.

[0053] The present invention also provides the application of a flow cytometry ploidy analysis kit containing cell staining solution in the detection and analysis of ploidy in animal cells, or the application of a flow cytometry ploidy analysis kit containing cell staining solution and cell nuclear extract in the detection and analysis of ploidy in plant cells.

[0054] Compared with the prior art, the technical effects achieved by the present invention include:

[0055] The flow cytometry ploidy analysis kit provided by this invention utilizes DAPI staining solution to specifically bind to the minor groove of the DNA double helix, achieving precise labeling of nuclear genetic material. It can be directly used for flow cytometry ploidy analysis of animal cell samples. DAPI can efficiently embed into DNA even at low concentrations, generating a strong blue fluorescent signal with minimal background fluorescence interference. This characteristic ensures high specificity for DNA content detection, making G0 / G1 and G2 / M phase cell populations clearly distinguishable in flow cytometry, significantly improving the ability to identify diploid, tetraploid, and other polyploid cells.

[0056] This invention optimizes the working concentration of DAPI staining solution to minimize the toxic effects of the dye on cells while ensuring signal strength. This makes the kit suitable for use with small amounts of live samples (such as fish fin puncture tissue and shellfish gill filaments), allowing ploidy analysis to be performed without euthanizing the animal, thus providing non-destructive monitoring support for polyploid breeding.

[0057] The flow cytometry ploidy analysis kit provided by this invention uses DAPI staining, eliminating the need for a light activation step. The staining process can be completed within 5 minutes of incubation at room temperature, and the fluorescence signal is minimally affected by pH fluctuations. This allows for "instant staining and testing," significantly shortening the detection cycle and reducing result deviations caused by operational delays, making it particularly suitable for rapid screening in field or resource-limited scenarios.

[0058] Furthermore, considering the requirement for nuclear extraction to obtain single-cell suspensions from plant cells, the flow cytometry ploidy analysis kit provided in this invention, based on DAPI staining solution, significantly improves the detection accuracy and universality of plant samples by optimizing the nuclear extraction solution formulation. Simultaneously, the new formulation exhibits significant storage stability and requires only 1–2 cm of sample. 2 Leaf tissue analysis can be completed. The flow cytometry ploidy analysis kit of this invention provides a highly stable and broad-spectrum ploidy analysis solution for complex samples such as woody crops and medicinal plants, supporting rapid germplasm screening in large-scale breeding. Attached Figure Description

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 The results of the tomato leaves on day 0 in the stability test of Example 1 are shown.

[0061] Figure 2 The results of the test on oysters in the generalization test of Example 2 are shown. Detailed Implementation

[0062] The invention is further illustrated below by specific application examples, but these examples do not limit the invention to the scope of the specific applications described. Experimental methods not specifying particular conditions in the following specific application examples should be performed according to conventional methods and conditions, or as selected in the product instructions. Unless otherwise specified, temperature generally refers to reactions conducted at room temperature; in this invention, room temperature refers to 16°C to 30°C.

[0063] Unless otherwise specified, the experimental methods used in the following application examples are conventional methods.

[0064] Unless otherwise specified, all materials and reagents used in the following application examples are commercially available.

[0065] Example 1

[0066] This embodiment provides a flow cytometry ploidy analysis kit for plant cell ploidy detection and analysis, hereinafter referred to as: Flow Cytometry ploidy Analysis Kit (Plant). The kit includes nuclear extraction solution and cell staining solution.

[0067] Since plant cells typically exist in a complex three-dimensional tissue structure, it is necessary to first extract the cell nuclei from the plant tissue to obtain a single-cell suspension before flow cytometry ploidy analysis can be performed.

[0068] The flow cytometry detection methods for plant samples are described below:

[0069] 1. Place 1-2cm of [something] in the petri dish. 2 Leaf tissue or other plant material;

[0070] 2. Add 0.5 mL of cell nuclear extract;

[0071] 3. Use a sharp blade to chop the sample for 1-2 minutes;

[0072] 4. Incubate at room temperature for 1-5 minutes;

[0073] 5. Filter the sample using a 35μm-50μm filter;

[0074] 6. Add 1.5 mL of cell staining solution and incubate briefly in the dark for 30-60 seconds;

[0075] 7. Analyze using flow cytometry.

[0076] Note: The required sample volume, weight, incubation time, and other conditions may vary depending on the plant species or different plant parts.

[0077] In some embodiments, the nuclear extract consists of the following components:

[0078] Nuclear stabilizer 0.5–8 mmol / L;

[0079] Chelating agent 2–35 mmol / L;

[0080] Na + K + Regulator 80–100 mmol / L;

[0081] Buffer solution 15–250 mmol / L, pH 7.5;

[0082] Triton X-1001 - 2% v / v;

[0083] Sodium metabisulfite 8-12 mmol / L;

[0084] PVP-101-2% v / v.

[0085] In some embodiments, the nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer is selected from Tris-HCl or MOPS.

[0086] Specifically, embodiments of the present invention provide the following cell nuclear extract formulations, as shown in Table 1 below:

[0087] Table 1 Formulas of different cell nucleus extracts

[0088]

[0089] The above four formulations were used to extract nuclei from plant tissue cells, and the ploidy of the extracted cell sap was detected by flow cytometry and compared with that of SYSMEX CyStain UV Precise P. The cell staining solution used in the test was the fluorescent staining solution in SYSMEX CyStain UV Precise P. The results are shown in Table 2 below.

[0090] Table 2. Ploidiness test results obtained from different cell nucleus extracts.

[0091]

[0092] The results showed that the Test-4 formulation had the lowest mean CV value, and the Test-4 formulation was selected as the cell nuclear extraction solution in the following examples.

[0093] In some embodiments, the cell staining solution is DAPI staining solution manufactured by Thermo Fisher Scientific, Sangon Biotech, or Sigma, with a concentration of 3-20 μg / mL.

[0094] The following experiments used Test-4 formulation as the nuclear extraction medium to extract nuclear cells from tomato leaf tissues, and the extracted cell sap was analyzed by flow cytometry for ploidy detection. The results were compared with SYSMEX CyStainUV Precise P. The final solutions were prepared with DAPI staining solutions from Thermo Fisher Scientific, Sangon Biotech, and Sigma at concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, for experimental testing. The results are shown in Table 3.

[0095] Table 3. Detection results of different cell staining solutions

[0096]

[0097]

[0098] The results showed that Thermo Fisher Scientific DAPI 5 μg / mL and Sigma 20 μg / mL had the lowest CV values, but for cost considerations, Thermo Fisher Scientific DAPI 5 μg / mL was chosen as the cell staining solution.

[0099] In the following tests, the nuclear extract of the flow cytometry ploidy analysis kit (plant) was the extract of the Test-4 formulation, and the cell staining solution was Thermo Fisher Scientific DAPI 5μg / mL.

[0100] (1) Universality test

[0101] The flow cytometry ploidy analysis kit (plant) of this invention was used as the experimental group (3 replicates), and CyStain UVPrecise P was used as the control group (1 replicate). Different plant samples were tested, and the mean values ​​of the three replicates in the experimental group were calculated. The results are shown in Table 4.

[0102] Table 4. Universality test results of the flow cytometry ploidy analysis kit (plant).

[0103]

[0104]

[0105] The results showed that, in seven families of plants including Crassulaceae (Sedum morganianum), Actinidiaceae (Actinidia chinensis), and Moraceae (Ficus microcarpa), the mean CV value of the kit of this invention remained stable at 3.11%–5.31%, significantly lower than that of the imported control SYSMEX (5.32%–8.90%). Furthermore, the CV values ​​were significantly lower for woody plants (Ficus microcarpa CV 3.75%) and high-polysaccharide samples (banana CV 3.92%).

[0106] (2) Stability test

[0107] Three different batches of the flow cytometry ploidy analysis kit (plant) of the present invention were stored in an environment of 2-8℃, and samples were taken for testing according to the time in Table 5. The test sample was tomato leaves, and the test results are shown in Table 5.

[0108] Table 5. Stability test results of the flow cytometry ploidy analysis kit (plant).

[0109]

[0110]

[0111] Figure 1The test results for tomato leaves on day 0 are shown. The results show that the CV value is still less than 8% after 20 months of storage, indicating that the quality control shelf life of the flow cytometry ploidy analysis kit (plant) of this invention can reach 20 months, and its stability surpasses that of mainstream imported reagents (usually with a shelf life of ≤12 months), reducing users' storage costs.

[0112] Example 2

[0113] This embodiment provides a flow cytometry ploidy analysis kit for analyzing the ploidy of animal cells. This kit is referred to as: Flow Cytometry ploidy Analysis Kit (Animal). The kit includes cell staining solution.

[0114] Since animal cells often exist in vivo in the form of single cells, or some animal tissues can be digested into single cells through protein hydrolysis, the flow cytometry ploidy analysis kit (animal) provided by this invention contains cell staining solution and can be used to perform ploidy analysis of animal cells.

[0115] The flow cytometry detection methods for different animal tissue samples are described below:

[0116] I. Animal tissue samples

[0117] 1. Place approximately 20 mg of animal tissue in a petri dish;

[0118] 2. Add 0.5 mL of cell staining solution;

[0119] 3. Use a sharp blade to chop the sample for 30-60 seconds;

[0120] 4. Add 1.5 mL of cell staining solution and incubate at room temperature for 5 min;

[0121] 5. After filtering with a 35μm-50μm filter, analyze using a flow cytometer.

[0122] II. Blood Samples

[0123] 1. Dilute the blood 5-10 times with physiological saline and take 50 μL;

[0124] 2. Add 1-2 mL (depending on the number of nucleated cells) of cell staining solution and incubate at room temperature or 4°C for 1-2 min;

[0125] 3. Analyze using flow cytometry.

[0126] III. Cell Sample Preparation

[0127] 1. Collect 1.0 × 10 6 -1.0×10 7 Each cell was washed twice with 1×PBS, centrifuged at 300g for 5 min, and the supernatant was discarded.

[0128] 2. (Optional) Add 0.5 mL of 70% ice-cold ethanol, fix at 4°C for at least 2 hours, centrifuge at 300 g for 5 minutes and discard the supernatant;

[0129] 3. Add 1-2 mL (depending on the amount of cells collected) of cell staining solution and incubate at room temperature or 4°C for 5 min;

[0130] 4. After filtering with a 35μm-50μm filter, analyze using a flow cytometer.

[0131] In this embodiment of the invention, DAPI staining solutions from manufacturers such as Bismerace, Sangon Biotech, and Sigma were prepared into final solutions of 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, and flow cytometry was used to test the ploidy of chicken blood samples. The results were compared with those obtained using SYSMEX CyStainUV Ploidy. The experimental results are shown in Table 6 below.

[0132] Table 6. Test results of DAPI staining solutions from different manufacturers.

[0133]

[0134] The results showed that Thermo Fisher Scientific's DAPI had the lowest CV value at 5 μg / mL and had the least impact on cell viability, meeting the in vivo detection requirements for precious aquatic animals (such as triploid oysters).

[0135] In the following tests, Thermo Fisher Scientific DAPI 5μg / mL was selected as the cell staining solution for the flow cytometry ploidy analysis kit (animal).

[0136] (1) Universality test

[0137] The flow cytometry ploidy analysis kit (animal) of this invention was used as the experimental group (3 replicates), and CyStain UVPrecise P was used as the control group (1 replicate). Different animal samples were tested, and the mean values ​​of the three replicates in the experimental group were calculated. The results are shown in Table 7.

[0138] Table 7. Universality test results of the flow cytometry ploidy analysis kit (animals)

[0139]

[0140] Figure 2 The test results for oysters (Ostreidae) are presented. The results show that in representative aquatic animals such as oysters (Ostreidae), *Litopenaeus vannamei* (Penaeus spp.), loach (Cobitidae), and carp (Cyprinididae), the CV value of this invention's kit is consistently between 5.26% and 6.79%, close to the accuracy of imported reagents (SYSMEX: 3.25%–5.43%). It is also suitable for complex samples with mucus, such as crustaceans (prawns) and fish.

[0141] (2) Stability test

[0142] Three different batches of the flow cytometry ploidy analysis kit (animal) of the present invention were stored in an environment of 2-8°C, and samples were taken for testing according to the time specified in Table 8. The test sample was chicken blood, and the test results are shown in Table 8.

[0143] Table 8. Stability test results of the flow cytometry ploidy analysis kit (animal).

[0144]

[0145] The results showed that the CV value was still less than 8% after 20 months of storage, indicating that the quality control shelf life of the flow cytometry ploidy analysis kit (animal) of the present invention can reach 20 months, and its stability surpasses that of mainstream imported reagents (usually with a shelf life of ≤12 months), reducing users' storage costs.

[0146] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0147] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A flow cytometric ploidy analysis kit, characterized by, The cell staining solution is a DAPI staining solution with a concentration of 3-20 μg / mL.

2. The flow cytometric ploidy analysis kit of claim 1, wherein, The cell staining solution is produced by Thermo, Shenguo or Sigma.

3. The flow cytometric ploidy analysis kit of claim 1, wherein The cell staining solution is a DAPI staining solution produced by Thermo with a concentration of 3-8 μg / mL.

4. The flow cytometric ploidy analysis kit according to any one of claims 1 to 3, wherein The cell nucleus extraction solution is composed of the following components: Nuclear stabilizer 0.5-8 mmol / L; Chelating agent 2-35 mmol / L; Na + , K + Modulator 80-100 mmol / L; Buffer 15-250 mmol / L, pH 7.5; Triton X-100 1-2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-10 1-2% v / v.

5. The flow cytometric ploidy analysis kit of claim 4, wherein The nuclear stabilizer is selected from spermine 4HCl or MgCl2·6H2O; the chelating agent is selected from Na2EDTA or sodium citrate; and the buffer is selected from Tris-HCl or MOPS.

6. The flow cytometric ploidy analysis kit of claim 5, wherein The cell nucleus extraction solution is composed of the following components: Spermine 4HCl 0.5-1.0 mmol / L; Na2EDTA 1.0-10.0 mmol / L; Na + , K + Modulator 75-110 mmol / L; Tris-HCl buffer 150-250 mmol / L, pH 7.5; Triton X-100 1-2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-10 1-2% v / v.

7. The flow cytometric ploidy analysis kit of claim 5, wherein The cell nucleus extraction solution is composed of the following components: MgCl2·6H2O 2.0-6.0 mmol / L; Sodium citrate 25-35 mmol / L; Na + , K + Modulator 75-110 mmol / L; Tris-HCl buffer 150-250 mmol / L, pH 7.5; Triton X-100 1-2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-10 1-2% v / v.

8. The flow cytometric ploidy analysis kit of claim 5, wherein, The cell nucleus extraction solution is composed of the following components: MgCl2·6H2O 2.0-6.0 mmol / L; Na2EDTA 1.0-3.0 mmol / L; Na + , K + Modulator 75-110 mmol / L; Tris-HCl buffer 150-250 mmol / L, pH 7.5; Triton X-100 1-2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-10 1-2% v / v.

9. The flow cytometric ploidy analysis kit of claim 5, wherein The cell nucleus extraction solution is composed of the following components: MgCl2·6H2O 2.0-6.0 mmol / L; Na2EDTA 1.0-3.0 mmol / L; Na + , K + Modulator 75-110 mmol / L; MOPS buffer 15-25 mmol / L, pH 7.5; Triton X-100 1-2% v / v; Sodium metabisulfite 8-12 mmol / L; PVP-10 1-2% v / v.

10. Use of the flow ploidy analysis kit according to any one of claims 1-3 in the analysis of animal cell ploidy or use of the flow ploidy analysis kit according to any one of claims 4-9 in the analysis of plant cell ploidy.

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