A lysis buffer system for extracting total DNA from black tea and application thereof
Patent Information
- Application Number
- CN202611283683.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-24
- Publication Date
- 2026-09-25
AI Technical Summary
[0005]
| 常规CTAB法 | 高香红茶DNA得率低、纯度不足 | CTAB对多糖多酚复合物裂解不充分 |
| DTAB法 | 针对木质化茶叶效果一般 | 盐浓度未针对红茶优化 |
| 商业试剂盒 | 成本高、对微量微生物DNA回收率低 | 缺乏针对性裂解体系 |
[0030]1、DNA得率与纯度提升：通过对裂解体系中的表面活性剂的种类与浓度进行比较，发现OTAB比CTAB或DTAB具有更高的DNA得率与纯度，并且只有在OTAB浓度为0.2%~1.0%（尤其0.6%）时，DNA得率和纯度才同时达到最优水平；本发明提供的最优技术方案为采用0.6%OTAB、0.75 mol/L NaCl裂解缓冲液提取红茶总DNA，效果评分达89.64（显著高于2% CTAB的78.15及2% DTAB的74.50），蛋白质及酚类污染少。
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Figure CN122811332A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a DNA extraction lysis buffer system and its application, and more particularly to a lysis buffer system for total DNA extraction from black tea and its application. Background Technology
[0002] Some microorganisms in tea possess antibacterial, anti-inflammatory, or antioxidant functions, which help enhance the health value of tea. Since the microbial community of fermented teas such as black tea changes during fermentation, the detection and analysis of total DNA—black tea DNA and black tea microbial DNA—is beneficial for the research, development, production, and application of black tea.
[0003] DNA extraction from tea leaves often employs the CTAB method or a kit method. However, for black tea (such as Qimen black tea, Darjeeling black tea, and Ceylon highland black tea), due to the fermentation and drying processes, the cell wall is rich in polysaccharides, polyphenols, and secondary metabolites, resulting in low DNA purity and unstable yield from the traditional CTAB method; the SDS method is not effective in removing polysaccharides. According to the search, the shortcomings of the existing technology are shown in Table 1, including: (1) the choice of cationic surfactant is limited, often CTAB, but its lysis efficiency for difficult-to-treat tissues is limited; (2) there is a lack of optimized lysis systems for endophytic or ectophytic DNA in finished black tea products; (3) the salt concentration and surfactant concentration in the lysis buffer have not been systematically compared.
[0004] Table 1. Defects and Causes of Existing Technologies
[0005] Conventional CTAB method High-fragrant black tea has low DNA yield and insufficient purity. CTAB does not adequately cleave polysaccharide-polyphenol complexes. DTAB method Generally effective against lignified tea leaves Salt concentration not optimized for black tea Commercial reagent kits High cost and low recovery rate of trace microbial DNA Lack of targeted pyrolysis system
[0006] In summary, there is currently a lack of a lysis buffer system suitable for the extraction of total DNA from black tea. Summary of the Invention
[0007] Purpose of the invention: The purpose of this invention is to provide a lysis buffer system for total DNA extraction from black tea and its application.
[0008] Technical solution: The lysis buffer system for total DNA extraction from black tea contains surfactants, salts, chelating agents, a buffer system, and enzymes. The surfactant is 0.2%~1.0% (w / v) OTAB, and the concentration of the salts is 0.50~1.00 mol / L.
[0009] Replace CTAB with OTAB (octadecyltrimethylammonium bromide, C...) which has a longer alkyl hydrophobic chain. 18 OTAB (a surfactant) can significantly improve the extraction efficiency of total DNA from black tea as a surfactant in the lysis buffer system. The mechanism lies in the fact that OTAB's C... 18 The hydrophobic chain ratio of CTAB is higher than that of C. 16The addition of two methylene groups allows it to embed more deeply into the lipid bilayer and lignin-carbohydrate complex (LCC) of the black tea cell wall, effectively breaking down the highly cross-linked cell wall structure. At the same time, OTAB forms a more stable hydrophobic complex with nucleic acids under high-salt conditions (0.50~1.00 mol / L NaCl), while polysaccharides and polyphenols remain in the aqueous phase, which can be efficiently separated by chloroform extraction.
[0010] This invention uses OTAB as the core of its lysis buffer system, which significantly improves the concentration, purity, and amplification efficiency of total DNA extracted from high-aroma black tea leaves. Based on this, the invention also establishes a total DNA extraction process applicable to Qimen, Darjeeling, and Ceylon black teas.
[0011] The surfactant is preferably 0.6%~0.8% (w / v) OTAB, and most preferably 0.6% OTAB.
[0012] Preferably, the salt is NaCl, the chelating agent is EDTA, the buffer system is Tris-HCl, and the enzyme is lysozyme and / or protease.
[0013] The optimal lysis buffer system is: 0.6% (w / v) OTAB, 0.75 mol / L NaCl, 20 mmol / L EDTA, 100 mmol / L Tris-HCl (pH 8.0), 2 mg / mL lysozyme, and 0.8 mg / mL proteinase K. This system achieves a DNA extraction score of 89.64, significantly superior to CTAB or DTAB lysis buffer systems.
[0014] The present invention also provides the application of the lysis buffer system in the extraction of total DNA from black tea.
[0015] Preferably, the total DNA of the black tea includes black tea DNA and black tea microbial DNA.
[0016] Preferably, the black tea microorganisms include endophytic bacteria and ectophytic bacteria.
[0017] The preferred steps for extracting total DNA from black tea are as follows:
[0018] (1) Grinding tea samples with liquid nitrogen;
[0019] (2) Add the lysis buffer system;
[0020] (3) Water bath incubation;
[0021] (4) Add extraction buffer, centrifuge, and collect the supernatant;
[0022] (5) Add the precipitate to the supernatant, let stand, centrifuge, and discard the supernatant;
[0023] (6) Wash the precipitate, dry it and then redissolve it.
[0024] When extracting DNA from endophytic bacteria in black tea, it is preferable to disinfect the surface of the tea leaves before step (1).
[0025] In step (3), the incubation time is preferably 60±5 min and the temperature is preferably 60~70℃; most preferably 60 min and 65℃.
[0026] In step (6), the washing is preferably performed sequentially with 1.0~1.5 mol / L NaCl solution and 65%~75% ethanol aqueous solution; most preferably, 1.2 mol / L NaCl solution and 70% ethanol aqueous solution are used.
[0027] In step (5), the precipitate is preferably an aqueous solution containing 0.4%~0.6% CTAB and 0.03~0.05 mol / L NaCl; most preferably 0.5% CTAB and 0.04 mol / L NaCl.
[0028] Preferred extraction process: Tea sample is ground with liquid nitrogen → added to lysis buffer system and incubated at 65℃ → extracted with chloroform:isoamyl alcohol (24:1) → precipitated with CTAB precipitate → washed with high salt → washed with 70% ethanol → dissolved in TE.
[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
[0030] 1. Improved DNA yield and purity: By comparing the types and concentrations of surfactants in the lysis system, it was found that OTB has a higher DNA yield and purity than CTAB or DTAB. Moreover, the DNA yield and purity only reach the optimal level when the OTB concentration is 0.2%~1.0% (especially 0.6%). The optimal technical solution provided by this invention is to use 0.6% OTB and 0.75 mol / L NaCl lysis buffer to extract total DNA from black tea, with an effect score of 89.64 (significantly higher than 78.15 for 2% CTAB and 74.50 for 2% DTAB), and less protein and phenolic contamination.
[0031] 2. Enhanced PCR amplification efficiency: Agarose gel electrophoresis showed clear and bright bands, with reduced non-specific amplification; amplification using 16S rDNA / 18S rDNA primers achieved a 100% success rate, meeting the requirements of high-throughput sequencing.
[0032] 3. Wide applicability: It performs excellently in the extraction of endophytic and ectophytic DNA from Keemun black tea, Darjeeling black tea, and Ceylon black tea. Especially for difficult-to-extract samples such as Darjeeling black tea, the DNA extracted by the OTAB method has better integrity and is suitable for the study of the diversity and molecular identification of endophytic and ectophytic bacteria in black tea.
[0033] 4. Simplified operation: The salt concentration in the lysis system is optimized (0.75 mol / L NaCl) to avoid repeated freeze-thaw cycles. Combined with proteinase K, it can effectively remove histones and shorten the extraction time by about 20%. Attached Figure Description
[0034] Figure 1 Electrophoresis images of the replicate experiment of the optimal treatment (0.6% OTAB) of this invention (M is DNA Marker; 1~3 are the optimal treatment level experiments ①, ②, and ③ respectively).
[0035] Figure 2 Electrophoresis diagram of PCR products of endophytic bacteria using 18S primers (M is DNA Marker; 1~8 are samples G1~G8 respectively).
[0036] Figure 3 Electrophoresis diagram of 16S primer PCR products of endophytic bacteria (M is DNA Marker; 1~8 are samples G1~G8 respectively).
[0037] Figure 4 Electrophoresis diagram of PCR products of ectobacteria using 18S primers (M is DNA Marker; 1-8 are samples G1-G8 respectively).
[0038] Figure 5 Electrophoresis diagram of PCR products of ectobacteria using 16S primers (M is DNA Marker; 1-8 are samples G1-G8 respectively). Detailed Implementation
[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0040] Example 1
[0041] This embodiment compares 15 lysis buffers (see Table 2 for details, the main difference being the type and concentration of surfactants) and uses the total DNA concentration, purity (A260 / A280) of black tea and PCR amplification effect as indicators to determine the optimal combination.
[0042] Highly aromatic black tea sample: Darjeeling black tea G2 from India.
[0043] The extraction steps are as follows:
[0044] Step 1: Weigh 0.2 g of the high-fragrance black tea sample, grind it into powder with liquid nitrogen, and transfer it to a 2 mL centrifuge tube.
[0045] Step 2: Add 1.2 mL of lysis buffer preheated to 65°C: surfactant, salts (see Table 2), 20 mmol / L EDTA, 100 mmol / L Tris-HCl (pH 8.0), and simultaneously add lysozyme (final concentration 2 mg / mL) and proteinase K (final concentration 0.8 mg / mL), and vortex thoroughly.
[0046] Step 3: Incubate in a 65°C water bath for 60 minutes, gently inverting and mixing every 15 minutes during this period.
[0047] Step 4: Add an equal volume of chloroform:isoamyl alcohol (24:1), gently invert and mix for 10 min, centrifuge at 4℃ and 12000 rpm for 10 min, and collect the supernatant.
[0048] Step 5: Add 1 / 10 volume of CTAB precipitation solution (0.5% CTAB, 0.04 mol / L NaCl) to the supernatant, mix well, let stand at room temperature for 30 min, centrifuge and discard the supernatant.
[0049] Step 6: Wash the precipitate once with 1.2 mol / L NaCl solution, twice with 70% ethanol, air dry, and then dissolve in 50 μL TE buffer.
[0050] Scoring criteria: DNA peak patterns, purity, concentration, and agarose gel electrophoresis were performed on 15 groups of DNA solutions. The lysis effect of various methods was evaluated according to a 4:3:2:1 ratio, with a maximum score of 100 points. For purity, A260 / 280 = 1.9 was the maximum score; 1 point was deducted for every 0.01 ng / μL decrease compared to 1.9. For concentration, 860 ng / μL was the maximum score; 1 point was deducted for every 8.6 ng / μL decrease compared to 860 ng / μL. The scores for each group are shown in Table 2.
[0051] Table 2 Results of Orthogonal Experiments
[0052] Level / Factor Lysis buffer [surfactant concentration (%), NaCl concentration (mol / L)] Effect rating 1 1% DTAB, NaCl (1.4 mol / L) 80.800±0.10349 2 2% DTAB, NaCl (1.4 mol / L) 74.497±0.16773 3 3% DTAB, NaCl (1.4 mol / L) 78.277±0.19009 4 4% DTAB, NaCl (1.4 mol / L) 80.410±0.3 5 5% DTAB, NaCl (1.4 mol / L) 78.123±0.46264 6 0.2% OTAB, NaCl (0.75 mol / L) 84.200±0.02 7 0.4% OTAB, NaCl (0.75 mol / L) 77.677±0.15948 8 0.6% OTAB, NaCl (0.75 mol / L) 88.053±0.16743 9 0.8% OTAB, NaCl (0.75 mol / L) 82.307±0.02887 10 1% OTAB, NaCl (0.75 mol / L) 78.590±0.17349 11 1% CTAB, NaCl (1.4 mol / L) 77.313±0.00577 12 1.5% CTAB, NaCl (1.4 mol / L) 78.090±0.17321 13 2% CTAB, NaCl (1.4 mol / L) 78.150±0.17321 14 2.5% CTAB, NaCl (1.4 mol / L) 79.457±0.01528 15 3% CTAB, NaCl (1.4 mol / L) 78.510±0.34641
[0053] Three parallel experiments were conducted using the optimal extraction scheme (0.6% OTAB, 0.75 mol / L NaCl). The electrophoresis results are as follows: Figure 1 As shown in the figure. Using SPSS Statistics 25 software, the coefficient of variation analysis revealed that the optimal treatment mean was 89.637, with a coefficient of variation of 0.6%. This indicates that the parallel experimental results fluctuated around 89.637, and the degree of fluctuation was relatively small.
[0054] Therefore, compared with CTAB or DTAB, the OTB lysis buffer system offers higher DNA yield (i.e., concentration) and purity, achieving a DNA extraction score of 89.64, with an A260 / A280 ratio close to 1.9, and less protein and phenolic contamination. Furthermore, the optimized salt concentration in the lysis system (0.75 mol / L NaCl) avoids repeated freeze-thaw cycles, and the combination with proteinase K effectively removes histones, thereby simplifying the operation and shortening the extraction time by approximately 20%.
[0055] Comparative Example 1
[0056] Based on Example 1, this comparative example adjusted the OTB concentration in the OTB lysis buffer system to 0.05%, 0.10%, 2.0%, and 3.0%, respectively, to investigate the effect of OTB concentration on DNA extraction efficiency.
[0057] The same extraction steps and evaluation indicators as in Example 1 were used, with the only variable being the OTAB concentration.
[0058] The results showed that when the OTAB concentration was below 0.2%, lysis was incomplete, and the DNA yield was only 380–520 ng / μL, with the A260 / A280 ratio as low as 1.42–1.58. When the OTAB concentration was above 1.0%, excessive surfactant caused DNA emulsification and loss during the chloroform extraction step, and the yield dropped to 550–640 ng / μL. Therefore, only when the OTAB concentration is within the range of 0.2%–1.0% (especially 0.6%) can both DNA yield and purity reach optimal levels.
[0059] Example 2
[0060] This embodiment compares the ectobiological / endobiological DNA of different types of black tea samples based on the optimal lysis buffer system of Example 1.
[0061] Test samples: Three types of black tea were selected: Keemun black tea, Darjeeling black tea and Ceylon black tea. Each type was repeated and numbered: Keemun black tea (G6~G8), Darjeeling black tea (G2,G4), and Ceylon black tea (G1,G3,G5).
[0062] Test method:
[0063] 1. The extraction method of Example 1 was used. Endophytic bacteria were surface sterilized with 7.5% sodium hypochlorite + Tween 80 before extraction; ectophytic bacteria were directly ground.
[0064] 2. Perform amplification of the 16S V3-V4 region and the 18S V4 region.
[0065] Quality control points: ① The pH of the lysis buffer must be strictly controlled at 8.0; ② The incubation time is 60±5 min; ③ Chloroform extraction must be performed gently to avoid DNA breakage; ④ The final DNA solution should be stored at -20℃.
[0066] Experimental results: Endophytic and ectophytic DNA extracted from all 8 samples using the 0.6% OTAB method was successfully amplified (see...). Figure 2-5 Both methods can obtain high-quality DNA, meeting the requirements for high-throughput sequencing library construction, thus demonstrating the universality of the method.
Claims
1. A lysis buffer system for extracting total DNA from black tea, comprising surfactants, salts, chelating agents, a buffer system, and enzymes, characterized in that, The surfactant is 0.2%~1.0% (w / v) OTAB, and the concentration of the salt is 0.50~1.00 mol / L.
2. The lysis buffer system according to claim 1, characterized in that, The surfactant is 0.6%~0.8% (w / v) OTAB.
3. The lysis buffer system according to claim 1, characterized in that, The salt is NaCl, the chelating agent is EDTA, the buffer system is Tris-HCl, and the enzymes are lysozyme and protease.
4. The application of the lysis buffer system according to any one of claims 1 to 3 in the extraction of total DNA from black tea.
5. The application according to claim 4, characterized in that, The total DNA of the black tea includes black tea DNA and black tea microbial DNA.
6. The application according to claim 5, characterized in that, The black tea microorganisms include endophytic and ectophytic bacteria.
7. The application according to claim 4, characterized in that, The extraction steps for total DNA from the black tea are as follows: (1) Grinding tea samples with liquid nitrogen; (2) Add the lysis buffer system; (3) Water bath incubation; (4) Add extraction buffer, centrifuge, and collect the supernatant; (5) Add the precipitate to the supernatant, let stand, centrifuge, and discard the supernatant; (6) Wash the precipitate, dry it and then redissolve it.
8. The application according to claim 7, characterized in that, In step (3), the incubation time is 60±5 min and the temperature is 60~70℃.
9. The application according to claim 7, characterized in that, In step (6), the washing is performed sequentially with 1.0~1.5 mol / L NaCl solution and 65%~75% ethanol aqueous solution.
10. The application according to claim 7, characterized in that, In step (5), the precipitate is an aqueous solution containing 0.4%~0.6% CTAB and 0.03~0.05 mol / L NaCl.