A crab meat DNA extraction kit and extraction method

Abstract

The application discloses a crab meat DNA extraction kit and an extraction method, and belongs to the field of molecular biology. The kit comprises a lysis solution, and the lysis solution comprises SDS, protease K and RNase A. The kit is simple in components, easy to prepare, low in cost and free of toxicity. The extraction method is simple and easy to operate, and crab meat DNA can be quickly extracted, thereby promoting the high-quality and sustainable development of the aquaculture industry.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a crab meat DNA extraction kit and extraction method. Background Technology

[0002] As a major aquaculture nation, China accounts for one-third of the world's total aquatic product output and approximately 60% of global aquaculture production. The rapid development of the aquaculture industry relies heavily on biotechnology. Molecular biology methods, by tracing the nucleotide sequences of organisms' genes and studying their structure and function, help explore aspects such as aquatic product breeding and reproduction, diseases, and immunity. In particular, people's fondness for aquatic foods has led to an increasing focus of research on the breeding and cultivation of aquatic organisms. Currently, aquaculture faces several challenges, including a limited number of genetically modified varieties, severe diseases, and low breeding rates.

[0003] Qualified sample DNA should possess the integrity of the primary nucleic acid structure and minimize contamination from RNA, protein, and polysaccharides. In aquatic organisms, such as crabs, the muscle is loose and fragile, and contains high levels of protein. Due to the different cellular and tissue characteristics compared to terrestrial organisms, conventional DNA extraction methods cannot yield high-quality crab meat DNA. Summary of the Invention

[0004] To solve at least one of the above-mentioned technical problems, the present invention adopts the following technical solution:

[0005] The first aspect of the present invention provides a crab meat DNA extraction kit, comprising a lysis buffer, wherein the lysis buffer comprises SDS, proteinase K and RNase A.

[0006] In some embodiments of the present invention, the lysis buffer comprises 0.6-2.4% SDS, 2% proteinase K and 1% RNase A.

[0007] In some preferred embodiments of the present invention, the lysis buffer comprises 1% SDS, 2% proteinase K and 1% RNase A.

[0008] In some embodiments of the present invention, the kit further includes a rinsing solution and an elution solution.

[0009] In some embodiments of the present invention, the rinsing solution is an organic alcohol solution. In some preferred embodiments of the present invention, the rinsing solution is 80% ethanol.

[0010] In some embodiments of the present invention, the eluent is selected from one of TE buffer, Tris-HCl buffer, and water. In some preferred embodiments of the present invention, the eluent is 10 mM Tris-HCl.

[0011] In some embodiments of the present invention, the kit further includes proteinase K.

[0012] A second aspect of this invention provides a method for extracting crab meat DNA using a crab meat DNA extraction kit, comprising the following steps:

[0013] S1, add the lysis solution described in the first aspect of the present invention to a grinding tube containing steel balls, take an appropriate amount of crab meat into the grinding tube, and grind it for 1 to 3 minutes using a grinder at 50 Hz;

[0014] S2, place the sample tube in a 65℃ water bath for 13-15 minutes;

[0015] S3, place the sample tube in a 70℃ water bath for 13-15 minutes;

[0016] S4. Place the sample tube in a centrifuge at 8000-10000g for 10-15 minutes, and transfer the supernatant to a new centrifuge tube.

[0017] S5, add 0.5 times the volume of chloroform, mix by inverting until the solution is completely emulsified into white, then centrifuge at 12000-15000×g at 4℃ for 8-15 min;

[0018] S6, aspirate the supernatant and transfer it to a new sterile centrifuge tube;

[0019] S7, add 0.6 times the volume of the well-mixed magnetic beads to the clear liquid, mix by blowing and stirring, and let stand at room temperature for 3-8 minutes;

[0020] S8. Place the centrifuge tube on the magnetic rack until the solution is clear, then discard the supernatant.

[0021] S9. Keep the centrifuge tube on the magnetic rack, add 200 μL of washing solution, let it stand at room temperature for 20-40 seconds, and then discard the supernatant.

[0022] S10, instantaneous centrifugation, discard excess rinsing solution in centrifuge tube, and dry magnetic beads at room temperature for 2-5 minutes;

[0023] S11, remove the centrifuge tube from the magnetic rack and let it stand at room temperature for 2-5 minutes; add 100 μL of elution buffer, gently mix with a pipette, and let it stand at room temperature for 3-8 minutes.

[0024] S12, place the centrifuge tube on a magnetic rack and let it stand at room temperature for 3-8 minutes until the solution becomes clear. Transfer the supernatant to the corresponding new sample preservation tube to obtain the purified crab meat DNA.

[0025] In some embodiments of the present invention, step S9 is repeated before step S10.

[0026] In some embodiments of the present invention, the rinsing solution is 80% ethanol.

[0027] In some embodiments of the present invention, the eluent is selected from one of TE buffer, Tris-HCl buffer, and water.

[0028] Beneficial effects of the present invention

[0029] Compared with the prior art, the present invention achieves the following beneficial effects:

[0030] The reagent kit of the present invention has simple components, is easy to prepare, low in cost, and non-toxic.

[0031] The extraction method of the present invention is simple, easy to operate, and can quickly extract crab meat DNA.

[0032] This invention provides a highly efficient and high-quality extraction method for crab meat DNA, which can promote the high-quality and sustainable development of the aquaculture industry. Attached Figure Description

[0033] Figure 1 The agarose gel electrophoresis results of crab meat DNA extracted using lysis buffer 1 prepared in Example 1 and the extraction method of Example 2 are shown. M: DNA Marker, 1-3: Sample numbers.

[0034] Figure 2 The agarose gel electrophoresis results of crab meat DNA extracted using lysis buffer 2 prepared in Example 1 and the extraction method of Example 2 are shown. M: DNA Marker, 1-3: Sample numbers.

[0035] Figure 3 The image shows the agarose gel electrophoresis results of crab meat DNA extracted according to the method provided in the Tiangen "Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304)". M: DNA Marker, 1-3: Sample Number.

[0036] Figure 4 The agarose gel electrophoresis results of crab meat DNA extracted using lysis buffers containing different concentrations of SDS in conjunction with the extraction method of Example 2 are shown. M: DNA Marker; 1: SDS concentration 0.2%; 2: SDS concentration 0.6%; 3: SDS concentration 2.4%; 4: SDS concentration 3.2%. Detailed Implementation

[0037] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0038] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0039] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0040] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0041] Example

[0042] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0044] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0046] Example 1: Crab Meat DNA Extraction Reagent

[0047] The extraction reagents include lysis buffer, washing buffer, and elution buffer, as follows:

[0048] 1. Lysis solution

[0049] In this embodiment, two lysis buffers were prepared in order to obtain the best extraction effect.

[0050] Lysis buffer 1: 1 mL 2.4% SDS, 20 μL proteinase K, 10 μL RNase A;

[0051] Lysis buffer 2: 1 mL 2.4% CTAB, 20 μL proteinase K, 10 μL RNase A.

[0052] 2. Rinse solution

[0053] 80% ethanol.

[0054] 3. Elution solution

[0055] 10mM Tris-HCl.

[0056] Example 2: Method for extracting DNA from crab meat

[0057] First, prepare a 2mL sample tube and add one sterilized steel ball each with a diameter of 6mm and 3mm.

[0058] (1) Add 1 mL of lysis buffer (lysis buffer 1 or lysis buffer 2 prepared in Example 1) to the grinding tube, and use scissors and tweezers to take a certain amount (about 500 mg) of Chinese mitten crab meat into the grinding tube, and use a grinder at 50 Hz for 3 min.

[0059] (2) Place the sample tube in a 65℃ water bath for 15 minutes;

[0060] (3) Place the sample tube in a 70℃ water bath for 15 minutes;

[0061] (4) Place the sample tube in a centrifuge at 8000g for 15 minutes, and transfer the supernatant to a new 2mL centrifuge tube;

[0062] (5) Add about 0.5 times the volume of the supernatant to the centrifuge tube and mix by inverting or vortexing until the solution is completely emulsified and turns white.

[0063] (6) Place the centrifuge tube in a centrifuge and centrifuge at 13000g for 10 min at 4℃. Transfer the supernatant to a new 1.5mL centrifuge tube.

[0064] (7) Add about 0.6 times the volume of supernatant to the centrifuge tube and mix well with magnetic beads (Hangzhou Lianchuan Biotechnology Co., Ltd.), mix well by blowing and let stand at room temperature for 5 minutes;

[0065] (8) Place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the solution is clear. Discard the supernatant.

[0066] (9) Keep the centrifuge tube on the magnetic rack, add 200 μL of rinsing solution, let it stand at room temperature for 30 seconds, and then discard the supernatant;

[0067] (10) Repeat the above steps once;

[0068] (11) Remove the centrifuge tubes from the magnetic rack and allow the magnetic beads to dry at room temperature for 5 minutes;

[0069] (12) Add 100 μL of elution buffer, mix well by pipetting, resuspend the magnetic beads, and let stand at room temperature for 5 min.

[0070] (13) Place the centrifuge tube on a magnetic rack and let it stand for 5 minutes until the solution is clear. Then, transfer the supernatant to the corresponding new sample storage tube and store it at -20°C.

[0071] DNA was extracted from three crab meat samples using lysis buffer 1 and lysis buffer 2, respectively. The agarose gel electrophoresis results are as follows: Figure 1 and Figure 2As shown. Simultaneously, the inventors also used the Tiangen "Blood / Cell / Tissue Genomic DNA Extraction Kit (DP304)" to extract DNA from three crab meat samples according to the method provided in the kit. The agarose gel electrophoresis results are as follows. Figure 3 As shown.

[0072] Therefore, it can be seen that using lysis buffer 1 and the method of this embodiment to extract DNA from crab meat yields the largest amount of DNA sample.

[0073] The inventors further measured the concentration and OD260 / OD280 of each DNA sample. The results are shown in Table 1.

[0074] Table 1. Concentration and OD260 / OD280 of each DNA sample

[0075]

[0076] As shown in Table 1, the DNA sample obtained by using the lysis buffer 1 prepared in Example 1 in combination with the extraction method of this example has the highest concentration, reaching 212.2 ng / μL, and the highest purity.

[0077] Example 3: Effect of lysis buffer formulation on DNA extraction from crab meat

[0078] To further improve the extraction efficiency of crab meat DNA, the inventors further optimized the formulation of the lysis buffer, specifically by adjusting the SDS concentration and increasing proteinase K. The detailed scheme is as follows:

[0079] Lysis buffers containing 0.2%, 0.6%, 2.4%, and 3.2% SDS were prepared, and crab meat DNA was extracted according to the method described above. The final DNA samples were analyzed for sample concentration and OD260 / OD280 ratios, and agarose gel electrophoresis was performed for detection.

[0080] The results are as follows Figure 4 As shown in Table 2,

[0081] Table 2. Concentration of DNA samples and OD260 / OD280 ratio

[0082] SDS concentration Concentration (ng / μL) OD260 / OD280 0.2％ 75.8 2.50 0.6％ 83.2 2.64 2.4％ 129.5 2.83 3.2％ 76.3 2.66

[0083] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for extracting DNA from crab meat, characterized in that, Includes the following steps: S1, add lysis buffer to a grinding tube containing steel balls, take an appropriate amount of crab meat into the grinding tube, and grind it for 1-3 minutes using a grinder at 50 Hz. The lysis buffer is obtained by adding 2% proteinase K and 1% RNase A to a 2.4% SDS solution. S2, place the sample tube in a 65℃ water bath for 13~15 minutes; S3, place the sample tube in a 70℃ water bath for 13~15 minutes; S4. Place the sample tube in a centrifuge at 8000~10000g for 10~15min, and transfer the supernatant to a new centrifuge tube; S5, add 0.5 times the volume of chloroform, mix by inverting until the solution is completely emulsified into white, then centrifuge at 12000~15000×g, 4℃ for 8~15min; S6, aspirate the supernatant and transfer it to a new sterile centrifuge tube; S7, add 0.6 times the volume of the well-mixed magnetic beads to the clear liquid, mix by blowing and stirring, and let stand at room temperature for 3~8 minutes; S8. Place the centrifuge tube on the magnetic rack until the solution is clear, then discard the supernatant. S9. Keep the centrifuge tube on the magnetic rack, add 200µL of rinsing solution, let it stand at room temperature for 20~40s, then discard the supernatant. The rinsing solution is 80% ethanol. Repeat this step once. S10, instantaneous centrifugation, discarding excess rinsing solution in the centrifuge tube, and drying the magnetic beads at room temperature for 2-5 minutes; S11, remove the centrifuge tube from the magnetic rack and let it stand at room temperature for 2-5 minutes; add 100µL of elution buffer, gently mix with a pipette, and let it stand at room temperature for 3-8 minutes. The elution buffer is selected from one of TE buffer, Tris-HCl buffer and water. S12, place the centrifuge tube on a magnetic rack and let it stand at room temperature for 3-8 minutes until the solution becomes clear. Transfer the supernatant to the corresponding new sample storage tube to obtain the purified crab meat DNA.

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