A plant oil nucleic acid extraction kit and extraction method

Abstract

The present invention belongs to the field of nucleic acid extraction technology, and in particular to a plant oil nucleic acid extraction kit and extraction method. The present invention provides a kit and extraction method for extracting DNA in plant oils by optimizing the composition of extraction reagents such as enrichment composition, lysis composition and purification solution. Wherein, the enrichment composition includes 5-10 parts of sodium lauryl sarcosine, 5-8 parts of disodium ethylenediaminetetraacetic acid, 6-12 parts of sodium chloride, 2-12 parts of trishydroxymethylaminomethane, and 0.02-0.04 parts of polysaccharide nucleic acid precipitation aids. The present invention has the advantages of mild extraction conditions, small amount of organic reagents, and is suitable for extraction of a large number of samples. It has a good application prospect in DNA extraction of plant oils, especially in DNA extraction of deep-processed plant oils.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid extraction, and in particular relates to a plant oil nucleic acid extraction kit and an extraction method. Background Art

[0002] Edible vegetable oil is edible oil made from edible vegetable oil or crude vegetable oil. Common ones in our daily life include soybean oil, rapeseed oil, peanut oil, sesame oil, edible vegetable blended oil, etc. In recent years, the consumption of edible oil has increased year by year, and the global production of edible vegetable oil has exceeded 200 million tons. There is increasing concern about the quality and safety of edible vegetable oil. The adulteration of low-cost vegetable oils, the blending and adulteration of edible oils that replace high-cost oils, and the entry of genetically modified vegetable oil products into the market have attracted widespread attention. There is an increasing demand for the qualitative detection of edible vegetable oils using molecular biotechnology. DNA qualitative detection is the main means of authenticity detection and genetically modified detection of edible vegetable oils.

[0003] Plant oils and fats are deeply processed products of plants. After multiple stages of extreme processing, plant genomic DNA is greatly lost and degraded into fragments of varying sizes. At the same time, the solubility of DNA in oils and fats is very low, resulting in extremely low genomic DNA content in plant oils and fats. Currently, commonly used commercial nucleic acid extraction kits are mainly used for nucleic acid extraction from a small number of samples. They do not have the process of enriching nucleic acids in oils and fats, and are not suitable for large-scale enrichment and extraction of plant oils and fats. In practical applications, they are mainly used for DNA extraction from primary pressed oils and fats such as olive oil (small DNA loss), and are not suitable for DNA extraction from refined oils and fats. The research results of Costa J (European Food Research and Technology, 2010, 230 (6): 915-923.), Raieta K (Food Chemistry, 2015, 172: 596-602.), You Shangqi (Optimization of DNA Extraction Methods for Edible Vegetable Oils [J]. Journal of Food Safety and Quality, 2021.) and many other scholars on the DNA extraction methods of plant oils and fats have proved that compared with the general plant genomic DNA extraction, the extraction of plant oil and fat DNA is more difficult, and the extraction process is often accompanied by long-term high-speed centrifugation (14000-20000g, 30min-3h). With the improvement of the processing grade of plant oils and fats, the difficulty of extracting plant oil and fat DNA continues to increase, and the quality of the extracted plant oil and fat DNA is poor (many impurities, DNA fragment degradation, etc.), and subsequent inspection experiments are often impossible.

[0004] At present, the emulsification method is one of the main methods for extracting nucleic acids from plant oils. The emulsification method mainly uses the system of "organic reagent + aqueous phase lysis solution + plant oil" in the stage of large-scale oil enrichment, and uses the principle that nucleic acids are dissolved in the aqueous phase and oils are dissolved in the organic phase to achieve full emulsification of plant oils and nucleic acid enrichment. Organic solvents such as n-hexane and chloroform commonly used in the enrichment process of the emulsification method are volatile, toxic and hazardous chemicals. The enrichment of a large amount of plant oils produces a large amount of organic solvent waste liquid. In addition, the method of n-hexane + CTAB / SDS solution + plant oil is commonly used in the domestic emulsification method to enrich plant oil DNA. The water phase recovery efficiency of this enrichment method is not high, and high-temperature heating is required during the lysis process after enrichment. Long-term high-temperature heating will cause a certain degree of damage to the nucleic acid, which is not conducive to subsequent amplification detection.

[0005] In summary, there is an urgent need in the art for a method for extracting nucleic acids from plant oils and fats that has mild extraction conditions, uses a small amount of organic reagents, and is suitable for extracting a large number of samples. Summary of the invention

[0006] In view of the problems of the prior art, the present invention provides a plant oil nucleic acid extraction kit and an extraction method, with the aim of providing new enrichment liquid (enrichment composition) and other reagents to achieve a plant oil nucleic acid extraction method with mild extraction conditions, low organic reagent usage, and applicability to the extraction of a large number of samples.

[0007] An enrichment composition comprising the following components in parts by weight:

[0008] 5-10 parts of sodium lauryl sarcosinate,

[0009] 5-8 parts of ethylenediaminetetraacetic acid disodium salt,

[0010] 6-12 parts of sodium chloride,

[0011] 2-12 parts of tris(hydroxymethyl)aminomethane,

[0012] 0.02-0.04 parts of polysaccharide nucleic acid sedimentation aid.

[0013] Preferably, the following components are included in parts by weight:

[0014] 10 parts of sodium lauryl sarcosinate,

[0015] 8 parts of ethylenediaminetetraacetic acid disodium salt,

[0016] 6 parts of sodium chloride,

[0017] 12 parts of tris(hydroxymethyl)aminomethane,

[0018] 0.03 parts of polysaccharide nucleic acid sedimentation aid.

[0019] Preferably, it is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent:

[0020] Sodium lauryl sarcosinate 0.5%-1%,

[0021] Ethylenediaminetetraacetic acid disodium salt 0.5%-0.8%,

[0022] Sodium chloride 0.6-1.2%,

[0023] Tris(hydroxymethyl)aminomethane 0.2-1.2%,

[0024] Polysaccharide nucleic acid sedimentation aid 0.002-0.004%.

[0025] Preferably, it is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent:

[0026] Sodium lauryl sarcosinate 1%,

[0027] Ethylenediaminetetraacetic acid disodium salt 0.8%,

[0028] Sodium chloride 0.6%,

[0029] Tris(hydroxymethyl)aminomethane 1.2%,

[0030] Polysaccharide nucleic acid precipitation aid 0.003%.

[0031] Preferably, the polysaccharide nucleic acid sedimentation aid is molecular biology grade Glycogen (glycogen) that does not contain DNase and RNase.

[0032] The present invention also provides a plant oil nucleic acid extraction kit, which comprises a lysis composition and the above-mentioned enrichment composition, wherein the lysis composition comprises the following components in parts by weight:

[0033] 10-20 parts of sodium lauryl sarcosinate,

[0034] 7-19 parts of ethylenediaminetetraacetic acid disodium salt,

[0035] Sodium chloride 12-30 parts,

[0036] 12-24 parts of tris(hydroxymethyl)aminomethane.

[0037] Preferably, the pyrolysis composition comprises the following components in parts by weight:

[0038] 15 parts of sodium lauryl sarcosinate,

[0039] 8 parts of ethylenediaminetetraacetic acid disodium salt,

[0040] 12 parts of sodium chloride,

[0041] 12 parts of tris(hydroxymethyl)aminomethane.

[0042] Preferably, the lysis composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent:

[0043] Sodium lauryl sarcosinate 1%-2%,

[0044] Ethylenediaminetetraacetic acid disodium salt 0.7%-1.9%,

[0045] Sodium chloride 1.2%-3%,

[0046] Tris(hydroxymethyl)aminomethane 1.2-2.4%.

[0047] Preferably, the lysis composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent:

[0048] Sodium lauryl sarcosinate 1.5%,

[0049] Ethylenediaminetetraacetic acid disodium salt 0.8%,

[0050] Sodium chloride 1.2%,

[0051] Tris(hydroxymethyl)aminomethane 1.2%.

[0052] Preferably, the method further comprises a purified solution, wherein the purified solution is an ethanol aqueous solution containing 1.2M-2M salt with a volume fraction of 20%-30%, and the salt is a sodium salt or a potassium salt.

[0053] Preferably, the salt is selected from at least one of sodium acetate, potassium acetate, sodium chloride or potassium chloride.

[0054] Preferably, it also includes a sedimentation agent, and the sedimentation agent is a polysaccharide nucleic acid sedimentation aid.

[0055] Preferably, the polysaccharide nucleic acid sedimentation aid is molecular biology grade Glycogen (glycogen) that does not contain DNase and RNase.

[0056] The present invention also provides a method for extracting nucleic acid from plant oil using the above kit, comprising the following steps:

[0057] Step 1, using the enrichment composition and vegetable oil to repeat the operations of mixing, emulsifying, centrifuging and collecting the aqueous phase for several times to obtain an aqueous phase enrichment solution;

[0058] Step 2, adding a cleavage composition to the aqueous phase enriched solution obtained in step 1, incubating, and obtaining a cleavage product;

[0059] Step 3, adding a purification solution and chloroform to the cleavage product obtained in step 2, and separating the supernatant after purification;

[0060] Step 4, adding isopropanol and a sedimentation agent to the supernatant obtained in step 3, freezing and sedimenting, separating, washing, and drying to obtain.

[0061] Preferably, in step 1, the enrichment composition is an aqueous solution, and each ml of the enrichment composition is enriched with at least 30 ml of vegetable oil;

[0062] And / or, in step 2, the lysis composition is an aqueous solution, and the volume ratio of the lysis composition to the aqueous phase enrichment solution is (1:1)-(2:1);

[0063] And / or, in step 2, the incubation temperature is 35-40°C;

[0064] And / or, in step 3, the volume ratio of the purified solution, chloroform and the cleavage product is (0.3-0.5):(0.7-1):1.

[0065] In the kit of the present invention, various reagents can be used in the form of aqueous solutions, and steps such as enrichment and cleavage can be achieved without using organic reagents, thereby reducing the generation of organic reagent waste liquid. At the same time, reagents such as enrichment compositions, cleavage compositions, and purification agents achieve good enrichment, cleavage, purification, and other effects by optimizing the components and their dosage ratios.

[0066] Among them, sodium lauryl sarcosine in the enrichment composition has a good emulsifying and solubilizing effect on plant oils and fats, which not only promotes the full mixing of the aqueous phase and the plant oils and fats, but also can achieve the separation of the aqueous phase and the oils and fats through simple operations such as centrifugation without the use of organic reagents, thereby reducing the generation of organic reagent waste liquid. At the same time, sodium lauryl sarcosine has the effect of denaturing and cracking proteins, denaturing proteins in plant oils and fats, releasing nucleic acid DNA, and increasing the enrichment of trace free DNA in the aqueous phase solution through glycogen, thereby improving the plant oil DNA enrichment effect, and realizing the simple and efficient enrichment of nucleic acid DNA in oils and fats.

[0067] The surfactant used in the lysis composition is sodium lauryl sarcosinate. Compared with CTAB, SDS and the like commonly used in plant oil extraction, sodium lauryl sarcosinate is relatively stable to heat, acid, alkali and the like under normal conditions. It does not require high-temperature heating and can achieve efficient lysis by incubation at room temperature, thereby reducing the damage of high-temperature heating to nucleic acid DNA in plant oils.

[0068] In addition, unlike the previous single chloroform extraction of protein impurities, the present invention uses a purification solution (high concentration salt + alcohol) to remove polysaccharide impurities, and then chloroform extraction is used to remove protein, pigment and other impurities, thereby reducing the inhibition of impurities on downstream PCR amplification.

[0069] Therefore, the present invention realizes a plant oil nucleic acid extraction method with mild extraction conditions, small amount of organic reagents, and applicability to a large number of sample extractions, and has a good application prospect.

[0070] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0071] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The results of the conventional PCR test for plant oil DNA in Experimental Example 1 (commercially available plant nucleic acid extraction kit);

[0073] Figure 2 The results of corn oil DNA fluorescence PCR detection in Experimental Example 1;

[0074] Figure 3 The results of rapeseed oil DNA fluorescence PCR test in Experimental Example 1;

[0075] Figure 4 The results of soybean oil DNA fluorescence PCR detection in Experimental Example 1;

[0076] Figure 5 The liquid phase separation after enrichment in Experimental Example 2, where from left to right are TE+vegetable oil group, CTAB+vegetable oil group, SDS+vegetable oil group and enriched liquid+vegetable oil group;

[0077] Figure 6 The liquid phase separation after enrichment in Experimental Example 2, where from left to right are TE+vegetable oil+n-hexane group, CTAB+vegetable oil+n-hexane group, SDS+vegetable oil+n-hexane group and enriched liquid+vegetable oil+n-hexane group;

[0078] Figure 7 The fluorescence PCR test results of the TE enrichment + CTAB extraction solution group and the TE enrichment + SDS extraction solution group in Experimental Example 3;

[0079] Figure 8 The fluorescence PCR test results of the TE enrichment + SDS extraction solution group, TE enrichment + lysis solution group and enrichment solution enrichment + lysis solution group in Experimental Example 3;

[0080] Fig. 9These are the fluorescence PCR test results of the chloroform extraction purification group and the purification solution + chloroform extraction purification group in Experimental Example 4. DETAILED DESCRIPTION

[0081] In the following examples and experimental examples, all reagents and raw materials used are commercially available. Since the enrichment composition and the lysis composition in the examples are used in the form of aqueous solutions, the enrichment composition is referred to as "enrichment solution" and the lysis composition is referred to as "lysis solution" in the examples and experimental examples.

[0082] Example 1 Plant oil nucleic acid extraction kit

[0083] The kit of this embodiment includes an enrichment solution, a lysis solution, a purification solution and a sedimentation agent.

[0084] The enriched solution is an aqueous solution with a pH of 8.0 containing the following components:

[0085] Sodium lauryl sarcosine 1% (m / v), disodium ethylenediaminetetraacetic acid 0.8% (m / v), sodium chloride 0.6% (m / v), tris(hydroxymethyl)aminomethane 1.2% (m / v), polysaccharide nucleic acid precipitation aid 0.003% (m / v).

[0086] Sodium lauryl sarcosine and polysaccharide nucleic acid precipitation aids in the enrichment solution promote full contact between the aqueous phase enrichment solution and the plant oil, thereby achieving efficient enrichment of trace DNA in the plant oil in the aqueous phase enrichment solution.

[0087] The lysate is an aqueous solution at pH 8.0 containing the following components:

[0088] Sodium lauryl sarcosine 1.5% (m / v), disodium ethylenediaminetetraacetic acid 0.8% (m / v), sodium chloride 1.2% (m / v), tris(hydroxymethyl)aminomethane 1.2% (m / v).

[0089] The sodium lauryl sarcosine in the lysis buffer can disintegrate and denature the protein, fully and effectively lysing and releasing the nucleic acid DNA.

[0090] The purified solution is an aqueous solution containing the following components:

[0091] Sodium acetate 1.2M, ethanol 20% (v / v).

[0092] The purified solution can effectively remove impurities such as proteins and polysaccharides.

[0093] The sedimentation agent is a polysaccharide nucleic acid sedimentation aid, which can efficiently precipitate and collect the extracted DNA fragments of different sizes without affecting the subsequent PCR detection.

[0094] The polysaccharide nucleic acid precipitation aid is preferably a molecular biology grade (glycogen) free of DNase and RNase. The polysaccharide nucleic acid precipitation aid used in the embodiment is biosharp's nucleic acid precipitation aid Glycogen (product number BL154A).

[0095] Example 2 Plant oil nucleic acid extraction kit

[0096] The kit of this embodiment includes an enrichment solution, a lysis solution, a purification solution and a sedimentation agent.

[0097] The enriched solution is an aqueous solution with a pH of 8.0 containing the following components:

[0098] Sodium lauryl sarcosine 0.5% (m / v), disodium ethylenediaminetetraacetic acid 0.5% (m / v), sodium chloride 0.6% (m / v), tris(hydroxymethylaminomethane) 1.2% (m / v), polysaccharide nucleic acid precipitation aid 0.003% (m / v).

[0099] The lysate is an aqueous solution at pH 8.0 containing the following components:

[0100] Sodium lauryl sarcosine 1% (m / v), disodium ethylenediaminetetraacetic acid 0.5% (m / v), sodium chloride 2.4% (m / v), tris(hydroxymethyl)aminomethane 1.2% (m / v).

[0101] The purified solution is an aqueous solution containing the following components:

[0102] Sodium acetate 2M, ethanol 30% (v / v).

[0103] The sedimentation agent is a polysaccharide nucleic acid sedimentation aid.

[0104] The polysaccharide nucleic acid precipitation aid is preferably molecular biology grade Glycogen (glycogen) that does not contain DNase and RNase. The polysaccharide nucleic acid precipitation aid used in the embodiment is biosharp's nucleic acid precipitation aid Glycogen (product number BL154A).

[0105] Example 3 Plant oil nucleic acid extraction method

[0106] This example uses the kit described in Example 1 to extract nucleic acid from plant oils and fats, and the specific steps are as follows:

[0107] ① Enrichment: Take 4 ml of the enrichment solution and add it to a 50 ml centrifuge tube, add 30-40 ml of vegetable oil each time (preferably 40 ml in this embodiment), fully shake in a vortex for 15-20 seconds (preferably 20 seconds in this embodiment) to fully mix and emulsify the oil and the enrichment solution, then centrifuge at 10000 rpm for 10-20 minutes (preferably 15 minutes in this embodiment), separate the water phase and the oil phase, remove the upper vegetable oil layer, and collect the water phase enrichment solution. If the oil phase and the water phase cannot be separated by centrifugation, continue to add 1 ml of enrichment solution and shake and centrifuge.

[0108] ② Enrichment: Continue to add 30-40 ml of vegetable oil (preferably 40 ml in this embodiment) to the aqueous phase enriched liquid obtained by centrifugation in step 1, continue to fully shake and mix, and then centrifuge at high speed (conditions are the same as step 1) to enrich the aqueous phase enriched liquid with not less than 120 ml of vegetable oil (the higher the refining grade of the vegetable oil, the greater the amount of enriched vegetable oil. In this embodiment, the enrichment was repeated 3 times, and a total of 120 ml of vegetable oil was enriched).

[0109] ③ Lysis: Add an equal volume of lysis solution to the aqueous phase enriched solution obtained by centrifugation in step 2, incubate at 35-40°C for 30-60 min (preferably 40°C for 60 min in this embodiment), and shake the mixture several times during the process.

[0110] ④ Purification: Add 4 ml of purification solution, mix thoroughly, add 0.7-1 times the volume of chloroform (8.4 ml in this embodiment), shake thoroughly, centrifuge at low temperature, and collect the supernatant into a 15 ml centrifuge tube.

[0111] ⑤ Sedimentation: Add 0.7-1 times the volume of pre-cooled isopropanol and glycogen solution as a sedimentation aid to the collected supernatant to make the pre-cooled isopropanol and glycogen (the concentration of the sedimentation aid glycogen reaches 20-30 μg / ml, preferably 30 μg / ml in this embodiment), freeze and precipitate in a -20°C or -80°C refrigerator for more than 2 hours, then centrifuge at low temperature and high speed for 15-20 minutes (preferably 15 minutes in this embodiment), discard the supernatant, and the white precipitate at the bottom of the tube is DNA.

[0112] ⑥ Cleaning: Use 70%-75% (preferably 70% in this embodiment) ethanol solution to clean the DNA at the bottom of the tube, centrifuge at high speed and discard the supernatant, and wash twice.

[0113] ⑦ Drying: Centrifuge again and carefully absorb the remaining ethanol solution at the bottom of the tube with a pipette.

[0114] ⑧ Drying: After opening the lid, place it at room temperature for more than 10 minutes to completely dry the residual ethanol.

[0115] ⑨ Dissolution: Dissolve the DNA at the bottom of the tube in 50-100 μL (preferably 80 μL in this embodiment) of TE buffer.

[0116] The technical solution of the present invention is further described below through experiments. The steps not specifically described in the experimental example are the same as those in Example 3.

[0117] Experimental Example 1: Effect of commercial kits on the extraction of plant oil DNA

[0118] 1. Experimental Methods

[0119] A commercially available plant genomic DNA extraction kit (DP305) was used, and the extraction method followed the method recommended by the kit.

[0120] The plant oil DNA extracted above was subjected to fluorescent PCR and conventional PCR detection (the detection primers used the soybean standard gene primers in the Ministry of Agriculture Announcement No. 2031-8-2013, the rapeseed standard gene primers in the Ministry of Agriculture Announcement No. 2031-9-2013, and the corn standard gene primers in the Ministry of Agriculture Announcement No. 1861-3-2012), and the DNA of corn, rapeseed and soybean plants extracted from the same batch was used as a detection control.

[0121] 2. Experimental Results

[0122] The commonly used plant genomic DNA extraction kit (DP305) was used to extract DNA from rapeseed oil, soybean oil, corn oil and other plant oils commonly found in the market. Compared with general plant samples, the DNA extraction effect for plant oil samples was not good. The results are shown in Table 1.

[0123] Table 1 DNA extraction concentration

[0124]

[0125]

[0126] The results of fluorescence PCR and conventional PCR are as follows Figure 1-4 shown.

[0127] It can be seen that commercial plant genomic DNA extraction kits can achieve good DNA extraction for samples of raw materials such as corn, soybeans, and rapeseed. However, after these raw materials are made into vegetable oils, the DNA extraction effect of corn oil, rapeseed oil, and soybean oil samples is poor. Due to the insufficient amount of DNA extracted from vegetable oils, it is difficult to achieve accurate PCR detection.

[0128] Experimental Example 2 Screening of Enriched Fluid Composition

[0129] 1. Experimental Methods

[0130] The experimental group design is as follows:

[0131] (1) Enriched liquid + vegetable oil group: the enriched liquid of Example 1 was used, and the extraction method was the extraction method of Example 3;

[0132] (2)TE+vegetable oil group:

[0133] Enrichment reagent TE buffer reagent composition: 10mM Tris-HCl, 1mM EDTA

[0134] Enrichment method: Take 4 ml of TE solution and add it to a 50 ml centrifuge tube, add 40 ml of vegetable oil each time, and fully shake it in a vortex for 15-20 seconds (preferably 20 seconds in this embodiment) to fully mix and emulsify the oil and TE solution, and then centrifuge it at 10000 rpm for 15 minutes, separate the water phase and the oil phase, remove the upper vegetable oil layer, and collect the water phase enrichment solution. Then continue to add 40 ml of vegetable oil to the collected water phase enrichment solution for enrichment, enrich it multiple times, enrich 120 ml of vegetable oil, and collect the water phase enrichment solution for the next step of DNA extraction and purification.

[0135] (3) CTAB+vegetable oil group:

[0136] Enrichment reagent CTAB buffer composition: 2% CTAB, 0.1M Tris-HCl, 20mM EDTA, 1.4M NaCl

[0137] Enrichment method: Take 4 ml of CTAB solution and add it to a 50 ml centrifuge tube, add 40 ml of vegetable oil each time, and fully shake it in a vortex for 15-20 seconds (preferably 20 seconds in this embodiment) to fully mix and emulsify the oil and CTAB solution, then centrifuge it at 10000 rpm for 15 minutes, separate the water phase and the oil phase, remove the upper vegetable oil layer, and collect the water phase CTAB solution. Then continue to add 40 ml of vegetable oil to the collected water phase CTAB solution for enrichment, enrich it multiple times, enrich 120 ml of vegetable oil, and collect the water phase CTAB solution for the next step of DNA extraction and purification.

[0138] (4) SDS+vegetable oil group:

[0139] Enrichment reagent SDS buffer composition: 4% SDS, 0.1M Tris-HCl, 20mM EDTA

[0140] Enrichment method: Take 4 ml of SDS solution and add it to a 50 ml centrifuge tube, add 40 ml of vegetable oil each time, and fully shake it in a vortex for 15-20 seconds (preferably 20 seconds in this embodiment) to fully mix and emulsify the oil and SDS solution, then centrifuge it at 10000 rpm for 15 minutes, separate the water phase and the oil phase, remove the upper vegetable oil layer, and collect the water phase SDS solution. Then continue to add 40 ml of vegetable oil to the collected water phase SDS solution for enrichment, enrich it multiple times, and finally enrich 120 ml of vegetable oil, and collect the water phase SDS solution for the next step of DNA extraction and purification.

[0141] (5) Enriched liquid + vegetable oil + n-hexane group:

[0142] The enrichment solution reagent composition is the same as the enrichment solution described in Example 1, and n-hexane solution.

[0143] Enrichment method: Take 4 ml of the enrichment solution and add it to a 50 ml centrifuge tube, add 20 ml of vegetable oil and 20 ml of n-hexane each time, and fully shake it in a vortex for 20 seconds to fully mix and emulsify the vegetable oil, enrichment solution and n-hexane, then centrifuge it at 10,000 rpm for 15 minutes, separate the aqueous phase and the organic phase, remove the upper organic phase where the vegetable oil and n-hexane are located, and collect the aqueous enrichment solution. Then continue to add 20 ml of vegetable oil and 20 ml of n-hexane to the collected aqueous enrichment solution for enrichment, enrich it multiple times, and finally enrich 120 ml of vegetable oil, and collect the aqueous enrichment solution for the next step of DNA extraction and purification.

[0144] (6)TE+vegetable oil+n-hexane group:

[0145] Enrichment reagent composition: TE solution (10mM Tris-HCl, 1mM EDTA) and n-hexane solution

[0146] Enrichment method: Take 4 ml of TE solution and add it to a 50 ml centrifuge tube, add 20 ml of vegetable oil and 20 ml of n-hexane each time, and fully shake it in a vortex for 20 seconds to fully mix and emulsify the vegetable oil, enrichment solution and n-hexane, then centrifuge it at 10,000 rpm for 15 minutes, separate the aqueous phase and the organic phase, remove the upper organic phase where the vegetable oil and n-hexane are located, and collect the aqueous TE solution. Then continue to add 20 ml of vegetable oil and 20 ml of n-hexane to the collected aqueous enrichment solution for enrichment, enrich it multiple times, and finally enrich 120 ml of vegetable oil, and collect the aqueous TE solution for the next step of DNA extraction and purification.

[0147] (7) CTAB+vegetable oil+n-hexane group:

[0148] Enrichment reagent composition: 2% CTAB buffer (2% CTAB, 0.1M Tris-HCl, 20mM EDTA, 1.4M NaCl) and n-hexane

[0149] Enrichment method: Take 4 ml of CTAB solution and add it to a 50 ml centrifuge tube, add 20 ml of vegetable oil and 20 ml of n-hexane each time, and fully shake it in a vortex for 20 seconds to fully mix and emulsify the vegetable oil, enrichment solution and n-hexane, then centrifuge it at 10,000 rpm for 15 minutes, separate the aqueous phase and the organic phase, remove the upper organic phase where the vegetable oil and n-hexane are located, and collect the aqueous phase CTAB solution. Then continue to add 20 ml of vegetable oil and 20 ml of n-hexane to the collected aqueous phase enrichment solution for enrichment, enrich it multiple times, and finally enrich 120 ml of vegetable oil, and collect the aqueous phase CTAB solution for the next step of DNA extraction and purification.

[0150] (8) SDS+vegetable oil+n-hexane group:

[0151] Enrichment reagent composition: SDS buffer (4% SDS, 0.1M Tris-HCl, 20mM EDTA) and n-hexane

[0152] Enrichment method: Take 4 ml of SDS solution and add it to a 50 ml centrifuge tube, add 20 ml of vegetable oil and 20 ml of n-hexane each time, and fully shake it in a vortex for 20 seconds to fully mix and emulsify the vegetable oil, enrichment solution and n-hexane, then centrifuge it at 10,000 rpm for 15 minutes, separate the aqueous phase and the organic phase, remove the upper organic phase where the vegetable oil and n-hexane are located, and collect the aqueous phase SDS solution. Then continue to add 20 ml of vegetable oil and 20 ml of n-hexane to the collected aqueous phase enrichment solution for enrichment, enrich it multiple times, and finally enrich 120 ml of vegetable oil, and collect the aqueous phase SDS solution for the next step of DNA extraction and purification.

[0153] The plant oil DNA extracted above was subjected to fluorescent PCR and conventional PCR detection, and the method was the same as that in Experimental Example 1.

[0154] 2. Experimental Results

[0155] The effect of liquid phase separation after enrichment is as follows Figure 5 , 6 As shown. It can be seen that a large amount of precipitate material appears during the CTAB method enrichment, and the organic phase appears gelatinous during the SDS method enrichment, and the aqueous phase where the enriched DNA is located cannot be collected, resulting in poor enrichment effect. When only TE buffer and the enrichment solution of the present invention are used for enrichment, the aqueous phase and the organic phase can be separated by shaking centrifugation, and the bottom aqueous phase recovery rate is high.

[0156] Experimental Example 3 Screening of Lysate Composition

[0157] 1. Experimental Methods

[0158] Based on the comparison of enrichment solutions, an experimental comparison study was conducted on the commonly used lysis solutions with CTAB and SDS as the main components.

[0159] The experimental group design is as follows:

[0160] Enrichment solution enrichment + lysis solution group: using the enrichment solution and lysis solution of Example 1, and the extraction method is the extraction method of Example 3;

[0161] TE enrichment + CTAB extraction liquid group: 120ml of plant oil was enriched by the enrichment method of 4ml TE buffer + 40ml plant oil (the detailed operation steps are the same as the enrichment operation steps in Experimental Example 2), and the lower aqueous phase solution was collected after the enrichment was completed. 4ml of 2% CTAB extraction liquid was added to the collected enriched aqueous phase solution, incubated at 65 degrees for 60min, and then 0.7-1 volume of chloroform was added to fully shake and mix, centrifuged at 10000rpm for 15min, the upper aqueous phase was collected and transferred to another clean centrifuge tube, and an equal volume of pre-cooled isopropanol and glycogen (nucleic acid precipitation aid glycogen reached 30μg / ml) was added to the aqueous phase, after inversion and mixing, low temperature precipitation at -20℃ / -80℃ for more than 2h, and then centrifuged at 12000rpm for 15min, the supernatant was discarded, and the DNA precipitate was collected. Add more than 500 μL of 70% alcohol to wash the DNA precipitate twice, place it at room temperature to dry, and after the alcohol is dried, add 50-100 μL of TE solution to dissolve the DNA.

[0162] TE enrichment + SDS extraction liquid group: 120ml of vegetable oil was enriched by the enrichment method of 4ml TE buffer + 40ml vegetable oil (see the enrichment operation steps in Experimental Example 2 for detailed operation steps), and the lower aqueous phase solution was collected after the enrichment was completed. 1ml of 20% SDS extraction liquid was added to the collected enriched aqueous phase solution, incubated at 65 degrees for 60min, and then 1.5ml of 3M sodium acetate (pH5.2) was added, after shaking and mixing, centrifuged at 10000rpm for 15min under low temperature conditions, the supernatant was transferred to a new centrifuge tube, and then 1 volume of chloroform was added to fully shake and mix, centrifuged at 10000rpm for 15 minutes, the upper aqueous phase was collected and transferred to a new centrifuge tube, and an equal volume of pre-cooled isopropanol and glycogen (nucleic acid precipitation aid glycogen reached 30μg / ml) was added to the aqueous phase solution, after inversion and mixing, -20℃ / -80℃ low temperature sedimentation for more than 2h, and then 12000rpm high-speed centrifugation for 15min, the supernatant was discarded, and the DNA precipitate was collected. Add more than 500 μL of 70% alcohol to wash the DNA precipitate twice, place it at room temperature to dry, and after the alcohol is dried, add 50-100 μL of TE solution to dissolve the DNA.

[0163] TE enrichment + lysis buffer group: 120 ml of vegetable oil was enriched by using 4 ml of TE buffer + 40 ml of vegetable oil (see the enrichment operation steps in Experimental Example 2 for detailed operation steps). After the enrichment, the lower aqueous phase solution was collected. 4 ml of lysis buffer was added to the aqueous phase solution, incubated at 40 degrees for 60 minutes, and the operation steps after lysis were the same as those in Example 3.

[0164] The plant oil DNA extracted above was subjected to fluorescent PCR and conventional PCR detection, and the method was the same as that in Experimental Example 1.

[0165] 2. Experimental Results

[0166] The extraction results of different experimental groups are shown in the following table:

[0167] Table 2 Concentration detection of enrichment solution and lysate comparison experiment

[0168] concentration A260 / A280 TE enrichment + CTAB extraction solution 1.32±0.85 1.10 TE enrichment + SDS extraction solution 34.57±9.64 1.85 TE enrichment + lysis buffer 21.2±7.09 1.54 Enrichment solution Enrichment + lysis solution 30.51±5.81 1.58

[0169] It can be seen from the data in the table that the combination of the enrichment solution and the lysis solution provided by the present invention can be used to extract DNA at room temperature, and the extraction effect is equivalent to that of the TE enrichment + SDS extraction solution group that requires high temperature incubation in the prior art.

[0170] Fluorescence PCR was used to verify the extraction effect. Figure 7 As shown in the figure, the TE enrichment + CTAB extraction and TE enrichment + SDS extraction methods were compared. From the results of fluorescent PCR, it was found that the DNA extracted by the CTAB method had poor repeatability, while the DNA extracted by SDS had better quality and good repeatability. Figure 8 As shown in the figure, the three extraction methods of TE enrichment + SDS extraction solution, TE enrichment + lysis solution, and enrichment solution enrichment + lysis solution were compared. From the fluorescence PCR results, it can be seen that the DNA extracted by these three methods has good repeatability, and the enrichment solution enrichment + lysis solution group has the best performance in fluorescence PCR.

[0171] It can be seen from the above experimental examples that the enrichment solution and lysis solution selected in the present invention are used together to extract high-concentration DNA samples to meet the requirements of PCR and other detection.

[0172] Experimental Example 4 Screening of Purified Solution Composition

[0173] 1. Experimental Methods

[0174] The experimental groups were set up as follows:

[0175] Purification solution + chloroform extraction purification group: using the enrichment solution, lysate, and purification solution of Example 1, and the extraction method is the extraction method of Example 3;

[0176] Chloroform extraction and purification group: 4 ml of TE buffer + 40 ml of vegetable oil was used to enrich 120 ml of vegetable oil. After the enrichment was completed, the aqueous phase solution was collected, 4 ml of lysate was added to the aqueous phase solution, incubated at 40 degrees for 60 minutes, and then 0.7 volumes of chloroform was directly added for extraction and purification without adding purification solution. The operation steps after purification were the same as those in Example 3.

[0177] The plant oil DNA extracted above was subjected to fluorescent PCR and conventional PCR detection, and the method was the same as that in Experimental Example 1.

[0178] 2. Experimental Results

[0179] The extraction results of different experimental groups are shown in Table 3. Fluorescence PCR detection was used to verify the extraction effect. Fig. 9 shown.

[0180] Table 3 Concentration detection of purified solution comparison experiment

[0181] concentration A260 / A280 Chloroform extraction and purification 28.07±5.05 1.51 Purification solution + chloroform extraction purification 30.51±5.81 1.58

[0182] It can be seen that DNA was also extracted from the sample using only chloroform extraction and purification, but the chloroform extraction and purification effect was poor, which had a greater impact on the fluorescent PCR amplification. Therefore, the purified solution of the present invention can effectively improve the purity of the DNA extracted from the plant oil and fat, and enhance the fluorescent PCR detection effect.

[0183] Through the above embodiments and experimental examples, it can be seen that the present invention provides a kit and extraction method for extracting DNA from plant oils and fats by optimizing the composition of extraction reagents such as enrichment solution, lysate and purification solution. The present invention has the advantages of mild extraction conditions, small amount of organic reagents, and applicability to a large number of sample extractions, and has a good application prospect in DNA extraction from plant oils and fats, especially DNA extraction from deep-processed plant oils and fats.

Claims

1. A plant oil nucleic acid extraction kit, characterized in that: The invention comprises a lysis composition, an enrichment composition, a purification solution and a sedimentation agent. The lysis composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent: Sodium lauryl sarcosinate 1%-2%, Ethylenediaminetetraacetic acid disodium salt 0.7%-1.9%, Sodium chloride 1.2%-3%, Tris(hydroxymethyl)aminomethane 1.2-2.4%; The enrichment composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent: Sodium lauryl sarcosinate 0.5%-1%, Ethylenediaminetetraacetic acid disodium salt 0.5%-0.8%, Sodium chloride 0.6-1.2%, Tris(hydroxymethyl)aminomethane 0.2-1.2%, Polysaccharide nucleic acid sedimentation aid 0.002-0.004%; The purified solution is an ethanol aqueous solution containing 1.2M-2M salt with a volume fraction of 20%-30%, and the salt in the purified solution is a sodium salt or a potassium salt; The sedimentation agent is a polysaccharide nucleic acid sedimentation aid.

2. The kit according to claim 1, characterized in that: The enrichment composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent: Sodium lauryl sarcosinate 1%, Ethylenediaminetetraacetic acid disodium salt 0.8%, Sodium chloride 0.6%, Tris(hydroxymethyl)aminomethane 1.2%, Polysaccharide nucleic acid sedimentation aid 0.003%.

3. The kit according to claim 1, characterized in that: The lysis composition is a solution containing the following components in mass volume concentrations, prepared using enzyme-free sterile water as a solvent: Sodium lauryl sarcosinate 1.5%, Ethylenediaminetetraacetic acid disodium salt 0.8%, Sodium chloride 1.2%, Tris(hydroxymethyl)aminomethane 1.2%.

4. The kit according to claim 1, characterized in that: The salt in the purified solution is selected from at least one of sodium acetate, potassium acetate, sodium chloride or potassium chloride.

5. A method for extracting nucleic acid from plant oil using the kit according to any one of claims 1 to 4, characterized in that: The steps include: Step 1, using the enrichment composition and vegetable oil to repeat the operations of mixing, emulsifying, centrifuging and collecting the aqueous phase for several times to obtain an aqueous phase enrichment solution; Step 2, adding the lysis composition to the aqueous phase enriched solution obtained in step 1, incubating, and obtaining a lysis product; Step 3, adding the purification solution and chloroform to the cleavage product obtained in step 2, and separating the supernatant after purification; Step 4, adding isopropanol and the sedimentation agent to the supernatant obtained in step 3, freezing sedimentation, separation, washing, and drying to obtain.

6. The method according to claim 5, characterized in that: In step 1, the enrichment composition is an aqueous solution, and each ml of the enrichment composition is enriched with at least 30 ml of vegetable oil; And / or, in step 2, the lysis composition is an aqueous solution, and the usage ratio of the lysis composition to the aqueous phase enrichment solution is a volume ratio of (1:1) to (2:1); And / or, in step 2, the incubation temperature is 35-40°C; And / or, in step 3, the volume ratio of the purified solution, chloroform and the cleavage product is (0.3-0.5):(0.7-1):1.

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