Rapid nucleic acid release reagent, kit, method for animal tissue genotyping and application

Abstract

This invention relates to the field of nucleic acid release, specifically disclosing a rapid nucleic acid release reagent, kit, method, and application for animal tissue genotyping. The release reagent comprises a weakly alkaline reaction buffer, an enzyme-catalyzed reaction enhancer, a nonionic surfactant, a nucleic acid amplification enhancer, a first component composed of histidine, trehalose, and glutathione in a specific ratio, and a second component with a visual indicator function; a matching thermosensitive proteinase K formation kit is also included. The kit can be pre-packaged in multi-well reaction plates for improved ease of use. The release method of this invention achieves rapid release of nucleic acids from animal tissues through pre-incubation activation, mild-temperature lysis, and high-temperature enzyme inactivation steps. Simultaneously, the first component exhibits a strong synergistic effect with the thermosensitive proteinase K, effectively protecting the integrity of the nucleic acids.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid release, and more specifically, to a rapid nucleic acid release reagent, kit, method, and application for animal tissue genotyping. Background Technology

[0002] Rat tail samples are the most commonly used and critical tissue source for gene identification in transgenic mice in laboratories. With the rapid development of gene editing technology in biomedicine, agriculture, and disease research, numerous mouse animal models, including gene knockout and insertion models, are constantly being constructed, making rat tail genotyping a routine and often tedious procedure in laboratories. Faced with the ever-increasing sample volume, laboratories urgently need a high-throughput, rapid, and low-cost method for extracting nucleic acids from rat tail tissue to meet the demands of large-scale screening, quality control, and shortened research and development cycles.

[0003] Currently, common methods for extracting nucleic acid from rat tail tissue mainly include alkaline lysis, proteinase K digestion combined with salting-out / organic extraction, and proteinase K digestion combined with high-temperature inactivation. Alkaline lysis is extremely fast, enzyme-free, and low-cost, but its disadvantages are also obvious: the extracted DNA is severely fragmented and contains many impurities, adversely affecting subsequent amplification reactions such as PCR. Proteinase K is used to digest the tissue overnight, followed by salting-out, multiple washes, and centrifugation to purify the nucleic acid. This method offers high yield, high purity, and stable quality, but it is time-consuming (usually requiring overnight digestion), cumbersome, and generates large amounts of organic solvents, which is not environmentally friendly or conducive to rapid detection.

[0004] In recent years, although there have been reports of using combinations of specific nucleic acid release reagents and thermosensitive proteinase K to achieve rapid nucleic acid release, existing methods typically only focus on the lysis and inactivation steps. Further improvements are needed to enhance lysis efficiency, ensure reagent activity, and achieve process visualization without increasing operational complexity. In particular, during rapid lysis, the large amount of endogenous nucleases and oxidizing substances released from tissues can degrade and damage the released nucleic acids, affecting the sensitivity and accuracy of downstream detection. Current technologies lack effective solutions to this problem.

[0005] In summary, there is an urgent need to develop a method for releasing nucleic acids from animal tissues that can rapidly release nucleic acids while effectively protecting their integrity. Summary of the Invention

[0006] This invention provides a rapid nucleic acid release reagent, kit, method, and application for animal tissue genotyping, enabling rapid, mild, and high-quality acquisition of nucleic acid templates suitable for downstream genotyping, while simultaneously solving the technical problem of easy degradation of nucleic acids during rapid lysis.

[0007] Based on the above research, the inventors, through a series of studies, innovatively introduced the first component and verified its significant synergistic effect with the thermosensitive proteinase K. At the same time, by combining the "pre-incubation activation" step and the "visualized second component", the reaction process was further optimized, and the stability of the reagents and the convenience of operation were improved, thus completing the present invention.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a rapid nucleic acid release reagent for animal tissue genotyping is provided, comprising a weakly alkaline reaction buffer, an enzyme-catalyzed reaction enhancer, a nonionic surfactant, a nucleic acid amplification enhancer, and a first component; the first component is composed of histidine, trehalose, and glutathione, wherein the concentration of histidine is 10-100 mM, the concentration of trehalose is 50-500 mM, the concentration of glutathione is 1-20 mM, and the mass concentration ratio of histidine, trehalose, and glutathione is 1:(5-10):(0.1-0.5).

[0009] Preferably, the weakly alkaline reaction buffer is selected from one or more of Tris-HCl, HEPES, Bis-TrisPropane, and Bicine, with a pH range of 7.5 to 9.2, preferably Tris-HCl with a pH of 8.5, to provide a suitable weakly alkaline environment for nucleic acid release and enzymatic reactions.

[0010] Preferably, the enzyme reaction enhancer is selected from one or more of Ca²⁺, Mg²⁺, and K⁺, which can improve the enzyme reaction efficiency of the heat-sensitive proteinase K and promote the digestion of tissue proteins.

[0011] Preferably, the nonionic surfactant is selected from one or more of NP-40, Tween20, Tween80, and Brij30, which can disrupt the cell membrane structure and improve cell lysis efficiency. The preferred combination is 0.5% NP-40, 0.5% Tween20, 1.0% Tween80, and 0.1% Brij30.

[0012] Preferably, the nucleic acid amplification enhancer is selected from one or more of gelatin, betaine, tetramethylammonium chloride, and spermidine, which can improve the efficiency of subsequent nucleic acid PCR amplification. The preferred combination is 0.25% gelatin, 450mM betaine, 500mM tetramethylammonium chloride, and 50μg / mL spermidine.

[0013] Preferably, the nucleic acid release reagent further comprises a second component, which is selected from one or more of phenol red, bromothymol blue, and bromocresol purple. The second component can produce a specific color change according to the pH value change, which is used to indicate whether the pH value of the nucleic acid release reagent is within the effective range, and at the same time to determine whether the reaction process is normal after the sample is added, so as to realize the visual monitoring of the reaction process.

[0014] The first component of this invention protects nucleic acids through multiple mechanisms: histidine, as a pH buffer and free radical scavenger, neutralizes acidic metabolites released from cleavage and scavenges oxidative free radicals; trehalose, as a non-reducing disaccharide, forms a hydration layer on the surface of nucleic acid molecules, stabilizing their native conformation and preventing thermal denaturation damage; glutathione, as a reducing agent, directly reduces oxidizing substances, protecting nucleic acid bases from oxidative damage. Furthermore, this first component exhibits a significant and strong synergistic effect with the thermosensitive proteinase K, with a co-existence index (CI) value less than 0.7.

[0015] In a second aspect, a rapid nucleic acid release kit for animal tissue genotyping is provided, comprising the nucleic acid release reagent of the first aspect and a thermosensitive proteinase K; wherein the final concentration of the thermosensitive proteinase K in the nucleic acid release system is 1-10 mg / mL, preferably 5 mg / mL.

[0016] Preferably, the nucleic acid release reagent and the thermosensitive proteinase K reagent are pre-allocated in a multi-well plate (such as a 96-well plate) to meet the needs of high-throughput detection; the thermosensitive proteinase K is independently aliquoted into the bottom of the wells of the multi-well plate in lyophilized form, and the nucleic acid release reagent is aliquoted into liquid form separately from the thermosensitive proteinase K lyophilized cake, which can improve the storage stability of the kit and simplify the operation steps.

[0017] The kit of the present invention can be stably stored at room temperature for more than 6 months, and can still maintain good nucleic acid release effect after storage at 4℃ and 37℃, thus solving the problem of poor stability of existing kits.

[0018] Thirdly, a rapid nucleic acid release method for animal tissue genotyping is provided, comprising the following steps: Animal tissue samples were mixed with the above-mentioned nucleic acid release reagent and thermosensitive proteinase K reagent to obtain a mixed system, wherein the final concentration of thermosensitive proteinase K in the mixed system was 1-10 mg / mL; Optionally, the mixture is pre-incubated at room temperature or below a first temperature, using a nucleic acid release reagent to dissolve the heat-sensitive proteinase K, thereby fully hydrating it and restoring its activity, while simultaneously allowing the nucleic acid release reagent components to fully infiltrate the tissue sample; preferably, the pre-incubation temperature is 25°C and the time is 3 minutes. The mixture is incubated at a first temperature of 25-35°C for 5-10 minutes to lyse the tissue cells and release nucleic acids, while the heat-sensitive proteinase K digests and releases the proteins in the supernatant; preferably, the first temperature is 30°C and the incubation time is 5 minutes. The mixture is incubated at a second temperature of 45-65°C for 5-10 minutes to inactivate the heat-sensitive proteinase K, thereby obtaining a release product containing nucleic acid, wherein the second temperature is higher than the first temperature; preferably, the second temperature is 55°C and the incubation time is 5 minutes.

[0019] Preferably, the animal tissue sample is selected from one or more of the following: mouse tail, mouse ear, mouse lung, mouse spleen, mouse liver, and mouse toe. The method of the present invention has a good nucleic acid release effect on different animal tissues and has wide applicability. The animal is preferably a mouse, especially suitable for genotyping of transgenic mice.

[0020] Preferably, the downstream genotyping detection is a PCR amplification detection. The nucleic acid released by the method of the present invention has good integrity, and the length of the amplifiable nucleic acid fragment is more than 5kb. Some samples can amplify 8kb fragments, and the PCR sensitivity is improved by about 10 times.

[0021] The method of this invention fully reconstitutes the lyophilized thermosensitive proteinase K through a pre-incubation activation step, thereby enhancing enzyme activity; it uses a mild temperature of 25-35°C for lysis to avoid nucleic acid damage caused by high temperature; at the same time, the strong synergistic effect between the first component and the thermosensitive proteinase K effectively protects the integrity of nucleic acid while rapidly lysing. The entire method, from sample processing to obtaining the nucleic acid template, only takes 10-20 minutes, which greatly improves the nucleic acid release efficiency.

[0022] Fourthly, the invention provides the application of the above-mentioned rapid nucleic acid release reagent in nucleic acid release from animal tissues; the application of the above-mentioned rapid nucleic acid release kit in nucleic acid release from animal tissues; and the application of the above-mentioned rapid nucleic acid release reagent, kit, and method in animal genotyping, preferably in the genotype identification of transgenic mice, and is particularly suitable for high-throughput genotype screening of transgenic mice.

[0023] The reagents, kits, and methods of this invention can achieve rapid and high-quality release of nucleic acids from animal tissues, with a nucleic acid recovery rate that is more than 30% higher than that of existing technologies. They are easy to operate, have high throughput, and are low cost, and can be widely used in animal genotyping detection in fields such as biomedicine, agriculture, and disease research, with significant practical value.

[0024] The beneficial effects of this invention are as follows: This invention innovatively introduces a first component composed of histidine, trehalose, and glutathione into the nucleic acid release reagent. This combination achieves protection of nucleic acids through multiple mechanisms: histidine, as a pH buffer and free radical scavenger, can effectively neutralize acidic metabolites released by cleavage and scavenge oxidative free radicals; trehalose, as a non-reducing disaccharide, can form a hydration layer on the surface of nucleic acid molecules, stabilizing their native conformation and preventing damage caused by thermal denaturation; glutathione, as the most important reducing agent in cells, can directly reduce oxidizing substances and protect nucleic acid bases from oxidative damage.

[0025] On the other hand, this invention experimentally confirms a significant synergistic effect between the first component and the thermosensitive proteinase K. The Combination Index (CI) method was used to quantitatively evaluate the synergistic effect of the three components. The results showed that when histidine, trehalose, and glutathione were used in a specific ratio (1:5-10:0.1-0.5), the CI value was less than 0.7, indicating a strong synergistic effect. Using any one component alone or in a combination not specified in this invention could not achieve the same level of nucleic acid protection. The mechanism of this synergistic effect is that the peptides and free amino acids produced by the thermosensitive proteinase K during protein digestion may be synergistically stabilized by histidine and trehalose, while the reducing environment maintained by glutathione effectively protects the sulfhydryl groups at the enzyme's active site. The combined effect of these three components significantly improves the integrity of nucleic acid release and subsequent amplification efficiency.

[0026] Furthermore, this invention further enhances the convenience and reliability of operation by introducing a pre-incubation activation step and a visualized second component. Pre-incubation ensures thorough reconstitution and activation of the lyophilized enzyme, while the visualized second component enables quality monitoring of the reaction process.

[0027] Experiments have shown that, using the optimized scheme of this invention, the nucleic acid recovery rate is increased by more than 30% compared with the existing technology, fragments with a length of more than 5kb can be amplified, PCR sensitivity is increased by about 10 times, and the kit can be stably stored at room temperature for more than 6 months. Attached Figure Description

[0028] Figure 1 Electrophoresis diagram showing the comparison of nucleic acid release effects of different treatment methods (with and without pre-incubation steps).

[0029] Figure 2 Photographs showing the color changes of the nucleic acid release reagent under different pH conditions after the addition of the second component.

[0030] Figure 3 Electrophoresis diagrams comparing the protective effects of different first components on nucleic acid integrity.

[0031] Figure 4 This is a comparative electrophoresis diagram of thermosensitive proteinase K and ordinary proteinase K.

[0032] Figure 5 Electrophoresis results comparing the nucleic acid release effects of lysis buffers with different pH values.

[0033] Figure 6 Electrophoresis diagram showing the comparison of nucleic acid release efficiency at different incubation times during the two-step incubation process.

[0034] Figure 7Electrophoresis diagram showing the comparison of nucleic acid release efficiency at different incubation temperatures during the two-step incubation process.

[0035] Figure 8 Electrophoresis results of nucleic acid release in different animal tissue samples (rat tail, ear, lung, spleen, liver, and toe). Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the art to which this invention pertains. The terminology used herein is intended to describe particular embodiments only and is not intended to limit the scope of the invention.

[0038] In this invention, the terms "comprising," "including," and "having" are open-ended descriptions and do not exclude other unspecified steps or components.

[0039] This invention provides a nucleic acid release reagent comprising a first component. This first component is composed of histidine, trehalose, and glutathione in a specific ratio. Its mechanism of action includes: histidine scavenging free radicals and maintaining pH stability through its imidazole group; trehalose interacting with the nucleic acid phosphate backbone through hydrogen bonds to form a protective hydration layer; and glutathione maintaining a reducing environment and protecting nucleic acid bases and enzyme active sites. The synergistic effect of these three components significantly enhances the integrity of nucleic acids during rapid lysis.

[0040] This invention quantitatively evaluates the synergistic effect between the first component and the thermosensitive proteinase K using the Combination Index (CI). The CI value is calculated based on the median effect equation (Chou-Talalay method), where CI < 1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect. In this invention, a CI value less than 0.7 is defined as a strong synergistic effect.

[0041] Example 1: Screening and Synergistic Effect Evaluation of the First Component This embodiment aims to screen for the optimal first component and evaluate its synergistic effect with the thermosensitive proteinase K.

[0042] Following the basic nucleic acid release reagent formulation shown in Table 1 (50 mM Tris-HCl pH=8.5, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.5 mM KCl, 0.1% NP-40, 0.5% Tween 20, 1% Tween 80, 0.1% Brij 30, 0.3% gelatin, 600 mM betaine, 275 mM tetramethylammonium chloride, 100 μg / mL spermidine), different concentrations and proportions of the first component candidate (histidine, trehalose, glutathione, DTT, ascorbic acid, mannitol, etc.) were added. The final concentration of the heat-sensitive proteinase K was 5 mg / mL. The sample was a rat tail with a diameter of approximately 1 mm, incubated at 30℃ for 5 min, then at 55℃ for 5 min. After incubation, the supernatant was collected for the following assays:

[0043] 1) The nucleic acid recovery rate was quantitatively detected using a Qubit fluorometer; 2) Agarose gel electrophoresis was used to detect nucleic acid integrity; 3) Take equal amounts of template for long fragment (3kb and 5kb) PCR amplification, and evaluate the amplification efficiency using Ct value.

[0044] Meanwhile, the Chou-Talalay method was used to calculate the combination index (CI) of each component used alone and in combination. With the thermosensitive proteinase K concentration fixed at 5 mg / mL, various protective agents were added to the system in different concentration combinations, and their enhancing effect on nucleic acid recovery was measured. The CI value was calculated using the median effect equation.

[0045] Table 1. Evaluation of the synergistic effect of different first components As shown in Table 1, the use of histidine, trehalose, or glutathione (A1-A3) alone all have certain protective effects, but the improvement is limited. When the three are used in combination according to the preferred ratio of this invention (A4, A7), the nucleic acid recovery rate is significantly increased to over 28 ng / μL, approximately 3.5 times that of the blank control (A9), and the 3kb PCR Ct value is reduced by nearly 10 cycles, indicating a significant improvement in both template quality and quantity. The combined index calculation results show that the CI values ​​of A4 and A7 are 0.53 and 0.61, respectively, both less than 0.7, indicating a strong synergistic effect. When DTT is used to replace glutathione (A5) or the ratio of histidine and trehalose deviates from the preferred range of this invention (A6, A8), the CI value is >0.7, and the synergistic effect is significantly weakened.

[0046] The above results indicate that when the first component (histidine, trehalose, and glutathione) of this invention is used in combination in a specific ratio, it has a significant synergistic effect with the thermosensitive proteinase K, which can greatly improve the yield and quality of nucleic acid release.

[0047] Example 2: Effect of pre-incubation activation step on nucleic acid release efficiency This example compares the effect of having or not having a pre-incubation step on nucleic acid release efficiency. The nucleic acid release reagent formulation used was the A4 combination, which showed the best performance in Example 1. Thermosensitive proteinase K was pre-placed at the bottom of the PCR tube in lyophilized form. The sample was a rat tail with a diameter of approximately 1 mm.

[0048] Experimental group: Add nucleic acid release reagent to the tube, pre-incubate at 25℃ for 3 minutes, and then incubate at 30℃ for 5 minutes + 55℃ for 5 minutes.

[0049] Control group: After adding nucleic acid release reagent to the tube, no pre-incubation was performed, and the tube was directly incubated at 30℃ for 5 min + 55℃ for 5 min.

[0050] After incubation, 2 μL of the supernatant was used as a template for 3 kb fragment PCR amplification. The products were detected by gel electrophoresis, and the results are as follows. Figure 1 As shown.

[0051] from Figure 1 It can be seen that the amplification band in the experimental group (lane 2) is significantly brighter than that in the control group (lane 1). This indicates that adding a pre-incubation activation step can fully rehydrate and activate the lyophilized heat-sensitive proteinase K and pre-wet the tissue with the nucleic acid release reagent, thereby significantly improving the nucleic acid release efficiency.

[0052] Example 3: Color observation of nucleic acid release reagent containing the second component Based on the A4 combination nucleic acid release reagent formula, 0.001% (w / v) of phenol red, bromothymol blue, or bromocresol purple were added respectively. The pH of the nucleic acid release reagent was adjusted to 6.0, 7.0, 8.0, and 9.0 with HCl or NaOH, respectively. The color changes were observed and recorded, and the results are as follows: Figure 2 As shown.

[0053] For example, phenol red appears yellow at pH 6.0, orange-red at pH 7.0, and purplish-red at pH 8.0 and above. By comparing the colors, operators can visually determine the pH state of the nucleic acid release reagent. When a rat tail sample is added to a nucleic acid release reagent containing phenol red (pH 8.5), if the sample is clean, the color should remain a stable purplish-red; if the sample is contaminated with acidic substances, the color will turn yellow, indicating that the sample may have a problem, and the test results should be interpreted with caution.

[0054] Example 4: The protective effect of different first components on nucleic acid integrity This embodiment compares the protective effects of different combinations of protective agents on the integrity of released nucleic acids. Rat tail samples were treated with nucleic acid release reagents without protective agents (control group), those containing A1 (single component), those containing A4 (preferred in this invention), and those containing A5 (DTT substitute), respectively. Incubation conditions were the same as in Example 1. Equal volumes of supernatant were subjected to agarose gel electrophoresis to observe the integrity of the nucleic acid bands. The results are as follows: Figure 3 As shown.

[0055] from Figure 3 It can be seen that the nucleic acid bands in the control group (lane 1) are severely diffused, indicating a large amount of degradation; the A1 group (lane 2) shows some improvement but still shows tailing; the A5 group (lane 3) shows limited improvement; while the preferred group of this invention, A4 (lane 4), shows clear high molecular weight bands without obvious diffusion, indicating that the integrity of the nucleic acid is best protected.

[0056] Example 5: Comparison of Thermosensitive Proteinase K and Regular Proteinase K In this embodiment, the A4 combination nucleic acid release reagent was used to test the addition of heat-sensitive proteinase K (experimental group) and ordinary proteinase K (control group) to a final concentration of 5 mg / mL. The sample was a rat tail with a diameter of approximately 1 mm. Incubation was performed at 30°C for 5 min and then at 55°C for 5 min. Subsequent PCR and detection were the same as in Example 1, and the results are as follows. Figure 4 As shown.

[0057] from Figure 4 As can be seen, the amplification band of the experimental group using thermosensitive proteinase K (lane 2) was significantly brighter than that of the control group using ordinary proteinase K (lane 1). This indicates that, under the same mild lysis conditions, ordinary proteinase K is not completely inactivated at 55℃, and residual activity still interferes with subsequent PCR amplification, while thermosensitive proteinase K can be rapidly inactivated, yielding a high-quality amplification template.

[0058] Example 6 Comparison of buffer pH The pH of the Tris-HCl buffer in the nucleic acid release reagent was adjusted to 7.5, 8.5, and 9.2, respectively, and other components used the A4 combination. Sample processing, incubation (30℃ for 5 min, 55℃ for 5 min), PCR, and detection were the same as in Example 1, and the results are as follows. Figure 5 As shown.

[0059] from Figure 5 It can be seen that amplification bands can be obtained in the pH range of 7.5-9.2, with the brightest band and the best amplification effect at pH 8.5.

[0060] Example 7: Comparison of nucleic acid release effects at different incubation times in two-step incubation This embodiment selects the optimal time parameters for two-step incubation.

[0061] Reagent preparation: Prepare the selected A4 nucleic acid release reagent and protease components (1 mg / mL heat-sensitive proteinase K, 50 mM Tris-HCl pH=8.5, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.5 mM M KCl, 0.1% NP-40, 0.5% Tween 20, 1% Tween 80, 0.1% Brij 30, 0.3% gelatin, 600 mM betaine, 275 mM tetramethylammonium chloride, 100 μg / mL spermidine).

[0062] Sample preparation: Add rat tail samples with a diameter of about 1 mm and treat them according to different incubation time conditions: ① 30℃ 0 min + 55℃ 10 min; ② 30℃ 5 min + 55℃ 5 min; ③ 30℃ 10 min + 55℃ 5 min; ④ 30℃ 10 min + 55℃ 10 min; ⑤ 30℃ 10 min + 55℃ 0 min.

[0063] Detection method: PCR and electrophoresis were performed according to the method described in Example 5. The effect was semi-quantitatively evaluated by the brightness of the gel electrophoresis bands. The results are as follows: Figure 6 As shown.

[0064] The results showed that the bands were clear and the amplification effect was good under the conditions of 30℃ for 5 min + 55℃ for 5 min; appropriately extending the incubation time (30℃ for 10 min + 55℃ for 5 min, 30℃ for 10 min + 55℃ for 10 min) further improved the yield of amplified products; nucleic acid release was not good when incubated at 30℃ or 55℃ alone. The time range for the two-step incubation was determined to be 5-10 min at the first temperature and 5-10 min at the second temperature.

[0065] Example 8: Comparison of nucleic acid release effects at different incubation temperatures during two-step incubation This embodiment selects the optimal temperature parameters for two-step incubation.

[0066] Reagents and samples: Same as in Example 7, the sample is a rat tail sample with a diameter of about 1 mm.

[0067] Sample processing: Samples were processed according to different incubation temperature conditions, with an incubation time of 5 min: ① 25℃ + 45℃; ② 30℃ + 55℃; ③ 35℃ + 65℃.

[0068] Detection method: PCR and electrophoresis were performed according to the method described in Example 5. The effect was semi-quantitatively evaluated by the brightness of the gel electrophoresis bands. The results are as follows: Figure 7 As shown.

[0069] The results showed that effective amplification could be achieved within the range of 25-35℃ for the first incubation step and 45-65℃ for the second incubation step. Among them, the bands were brightest and the amplification effect was best when the first incubation step was 30℃ and the second incubation step was 55℃. Therefore, 25-35℃ was determined to be the first incubation temperature range, 45-65℃ to be the second incubation temperature range, and 30℃ and 55℃ to be the optimal temperature combination.

[0070] Example 9: Comparison of Enzyme Reaction Enhancer Combinations Based on the A4 combined nucleic acid release reagent, but with CaCl2, MgCl2, and KCl removed, different concentrations of CaCl2, MgCl2, and KCl were added according to the orthogonal experiments shown in Table 2. Sample processing, incubation, PCR, and detection were the same as in Example 1, and the band brightness was semi-quantitatively scored by gel electrophoresis (0-5 points, higher score means brighter), where 0 = no band, 1 = very weak, 2 = weak, 3 = medium, 4 = strong, and 5 = very strong. The results are shown in Table 2.

[0071] Table 2 Comparison of Enzyme Response Enhancer Combinations The results showed that, within the tested concentration range, the combinations with added enzyme reaction enhancers (combinations 1-9) were all superior to the blank control without added enzymes (combination 10), indicating that the combination of Ca²⁺, Mg²⁺, and K⁺ has an enhancing effect on nucleic acid release and subsequent amplification.

[0072] Example 10: Comparison of Nonionic Surfactant Combinations Table 3 Combinations of nonionic surfactants Based on the A4 combined nucleic acid release reagent, but with NP-40, Tween20, Tween80, and Brij30 removed, and four surfactants of different concentrations added according to the orthogonal experiments shown in Table 3. Sample processing, incubation, PCR, and detection were the same as in Example 7, and the results are shown in Table 3. The scoring criteria were the same as in Table 2.

[0073] The results showed that the combinations with added nonionic surfactants (combinations 1-9) were all better than the blank control (combination 10) without surfactants, indicating that the surfactant combinations enhanced nucleic acid release.

[0074] Example 11 Comparison of nucleic acid amplification enhancer combinations Based on the A4 combination nucleic acid release reagent, but with gelatin, betaine, tetramethylammonium chloride, and spermidine removed, four different concentrations of enhancers were added according to the orthogonal experiments shown in Table 4. Sample processing, incubation, PCR, and detection were the same as in Example 7, and the results are shown in Table 4, with the scoring criteria the same as in Table 2. The results showed that the combinations with added nucleic acid amplification enhancers (combinations 1-9) were all superior to the blank control without enhancement (combination 10), indicating that these enhancers help improve subsequent amplification efficiency.

[0075] Table 4 Combinations of Nucleic Acid Amplification Enhancers Example 12: Applicability of Different Animal Tissue Samples Using the A4 combination nucleic acid release reagent and a final concentration of 5 mg / mL of heat-sensitive proteinase K, approximately 1 mm pieces of mouse tail, ear, lung, spleen, liver, and two mouse toes were added as samples. Incubation was performed at 30°C for 5 minutes and then at 55°C for 5 minutes. Subsequent PCR and detection were the same as in Example 1, and the results are as follows. Figure 8 As shown.

[0076] from Figure 8 As can be seen, clear and bright amplification bands were obtained from all tested rat tail, ear, lung, spleen, liver, and toe samples, indicating that the method of the present invention has wide applicability to different animal tissue samples.

[0077] Example 13 Reagent Kit Stability Test Thermosensitive proteinase K was aliquoted into 96-well plates in lyophilized form, and the nucleic acid release reagent containing the A4 combination was individually packaged. The assembled kits were stored at 4°C, 25°C, and 37°C for 1 month, 3 months, and 6 months, respectively. Each month, the kits were removed, mouse tail samples were treated according to the method in Example 1, and their nucleic acid release efficiency (expressed as 3kb PCR Ct value) was measured.

[0078] The results showed that the Ct values ​​obtained after processing samples from kits stored at different temperatures for 6 months were all less than 1.5 different from those of freshly prepared kits, indicating that the kits of the present invention have good room temperature storage stability.

[0079] Example 14 Long Fragment Amplification Capability Test Rat tail samples were treated with the A4 combination nucleic acid release reagent and a final concentration of 5 mg / mL of thermosensitive proteinase K. The supernatant was used as a template for PCR amplification of 500 bp, 1 kb, 3 kb, 5 kb, and 8 kb fragments, respectively. The results showed that the nucleic acid template released using the method of this invention could stably amplify 5 kb fragments, and some samples could amplify 8 kb fragments, indicating excellent nucleic acid integrity, far superior to the traditional alkaline lysis method (which typically only amplifies <1 kb fragments).

[0080] Based on the above experimental results, this invention analyzes the synergistic mechanism of histidine, trehalose, glutathione, and thermosensitive proteinase K: 1) The imidazole group of histidine can effectively scavenge reactive oxygen species (ROS) released by cleavage, reduce damage to nucleic acids and system components, and help maintain the stability of the system's microenvironment; 2) Trehalose interacts with the nucleic acid phosphate backbone through hydrogen bonds to form a stable hydration layer, which reduces nucleic acid damage caused by thermal denaturation; 3) Glutathione maintains the reducing environment of the system, reducing oxidative damage to nucleic acid bases; 4) The synergistic effect of the three is that the heat-sensitive proteinase K maintains a high activity level during the lysis phase, improving the protein digestion efficiency; the released nucleic acid is more fully protected during lysis and heat treatment, and its integrity and amplifurity are significantly improved; ultimately, this results in improved nucleic acid recovery rate, increased amplifiable length, and improved PCR sensitivity.

[0081] In summary, the rapid nucleic acid release method and kit provided by this invention innovatively introduces a first component composed of histidine, trehalose, and glutathione in a specific ratio into the nucleic acid release reagent, and verifies its significant synergistic effect with the thermosensitive proteinase K (CI<0.7). At the same time, combined with pre-incubation activation and visualization indicators, it significantly improves nucleic acid release efficiency, nucleic acid integrity, ease of operation, and kit stability, making it very suitable for large-scale, high-throughput genotyping screening.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A rapid nucleic acid release reagent for animal tissue genotyping, characterized in that, It contains a weakly alkaline reaction buffer, an enzyme-catalyzed reaction enhancer, a nonionic surfactant, a nucleic acid amplification enhancer, and a first component; The first component consists of histidine, trehalose and glutathione, with histidine concentration of 10-100 mM, trehalose concentration of 50-500 mM and glutathione concentration of 1-20 mM, and the mass concentration ratio of histidine, trehalose and glutathione is 1:(5-10):(0.1-0.5).

2. The rapid nucleic acid release reagent according to claim 1, characterized in that, The weakly alkaline reaction buffer is selected from one or more of Tris-HCl, HEPES, Bis-TrisPropane, and Bicine, with a pH range of 8 to 9; The enzyme reaction enhancer is selected from one or more of Ca²⁺, Mg²⁺, and K⁺; The nonionic surfactant is selected from one or more of NP-40, Tween20, Tween80, and Brij30; The nucleic acid amplification enhancer is selected from one or more of gelatin, betaine, tetramethylammonium chloride, and spermidine.

3. The rapid nucleic acid release reagent according to claim 1, characterized in that, The nucleic acid release reagent further includes a second component selected from one or more of phenol red, bromothymol blue, and bromocresol violet, used to indicate the pH value of the nucleic acid release reagent and the reaction progress.

4. A rapid nucleic acid release kit for animal tissue genotyping, characterized in that, Includes the nucleic acid release reagent according to any one of claims 1-3, and the thermosensitive proteinase K reagent; The final concentration of the thermosensitive proteinase K in the nucleic acid release system is 1-10 mg / mL.

5. The rapid nucleic acid release kit according to claim 4, characterized in that, The nucleic acid release reagent and the thermosensitive proteinase K reagent are pre-allocated into multi-well reaction plates; The thermosensitive proteinase K reagent is individually dispensed into the bottom of the wells of the multi-well reaction plate in lyophilized form, and the nucleic acid release reagent is dispensed into liquid form separately from the lyophilized cake of the thermosensitive proteinase K reagent.

6. A rapid nucleic acid release method for animal tissue genotyping, characterized in that, Includes the following steps: Animal tissue samples are mixed with the nucleic acid release reagent and the thermosensitive proteinase K reagent according to any one of claims 1-3 to obtain a mixed system, wherein the final concentration of the thermosensitive proteinase K in the mixed system is 1-10 mg / mL; Optionally, the mixture is pre-incubated at room temperature or below a first temperature to allow the heat-sensitive proteinase K to fully hydrate and restore its activity, while simultaneously allowing the nucleic acid release reagent components to fully infiltrate the tissue sample. The mixture was incubated at a first temperature of 25-35°C for 5-10 minutes to induce cell lysis and release of nucleic acids, while heat-sensitive proteinase K digested the proteins in the supernatant. The mixture was incubated at a second temperature of 45-65°C for 5-10 minutes to inactivate the heat-sensitive proteinase K, resulting in a release product containing nucleic acid.

7. The rapid nucleic acid release method according to claim 6, characterized in that, The pre-incubation temperature is 25℃ and the pre-incubation time is 3 minutes.

8. The rapid nucleic acid release method according to claim 6, characterized in that, The animal tissue samples are selected from one or more of the following: mouse tail, mouse ear, mouse lung, mouse spleen, mouse liver, and mouse toe; the downstream genotyping detection is a PCR amplification detection, and the amplifiable nucleic acid fragments are longer than 5kb.

9. The use of the rapid nucleic acid release reagent according to any one of claims 1-3 and the rapid nucleic acid release kit according to any one of claims 4-5 in the release of nucleic acid from animal tissues.

10. The application of the rapid nucleic acid release reagent according to any one of claims 1-3, the rapid nucleic acid release kit according to any one of claims 4-5, and the rapid nucleic acid release method according to any one of claims 6-8 in animal genotyping, characterized in that, The animal is a mouse, and the genotyping refers to the genotyping of transgenic mice.

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