A method for DNA extraction from edible oils

Abstract

This invention discloses a method for DNA extraction from edible oils. The method includes: weighing 10g of oil or phospholipid into a first centrifuge tube, adding 15ml of n-hexane and 2ml of EFG buffer, vortexing and centrifuging, and collecting 1ml of the bottom liquid after separation into a second centrifuge tube; adding 500μl of chloroform to the second centrifuge tube, inverting to mix, and centrifuging again, collecting the supernatant into a third centrifuge tube; adding 0.8 times the volume of isopropanol and 4μl of glycogen to the third centrifuge tube, mixing, and allowing to stand at room temperature for at least 30 minutes, centrifuging again, discarding the supernatant, adding 500μl of 75% ethanol, centrifuging again, discarding the supernatant, drying at 65°C for 5 minutes, adding 60μl of TE buffer to dissolve the precipitate, placing at 65°C for 2 minutes, and storing the DNA at -20°C for later use. This invention saves time and costs, yields highly stable DNA, has relatively low environmental toxicity, is safe and reliable, uses fewer consumables, and is inexpensive.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for extracting DNA from edible oils. Background Technology

[0002] For the identification and detection of edible oil varieties, traditional methods include physicochemical detection, primarily using mass spectrometry and spectroscopy (Dmam L et al., 2008). However, these two techniques are easily affected by production conditions, season, and origin, and have relatively high detection limits (≥5%) (Qi Lingqian et al., 2016). Compared to traditional methods, molecular biology techniques for the detection of edible oils offer advantages such as higher detection sensitivity, greater operability, and higher efficiency (Zhang Hailiang et al., 2010). DNA extraction from edible oils is a key and challenging aspect of molecular biological detection (He Jing, 2012).

[0003] Extensive comparative studies of DNA extracted from vegetable oils revealed that more research has been conducted on olive oil abroad (Vietina M et al., 2013). Due to the excellent properties of olive oil, it does not require refining before consumption, thus minimizing DNA damage and facilitating extraction (Scollo F et al., 2016). In my country, soybean oil is the most common edible oil, followed by tea seed oil, cottonseed oil, etc. (Fu Xiaohua et al., 2014). For DNA extraction from vegetable oils, international methods primarily utilize commercial kits and hexane emulsification (Scollo F et al., 2016). Z et al., 2014; Raieta K et al., 2015). Domestically, most methods utilize the modified CTAB method and commercially available kits (Yao Fei et al., 2012; Wu Xingquan et al., 2012).

[0004] From seeds to edible oils, multiple processes are required, such as deacidification, decolorization, and deodorization. These processes severely damage DNA, preserving only small amounts of DNA fragments. Furthermore, the presence of impurities and PCR inhibitors increases the difficulty of DNA isolation and purification (Costa J et al., 2012). Therefore, the success of DNA extraction from oils is crucial for detection results. Current research indicates that some edible oils still suffer from unsuccessful DNA extraction and low extraction concentrations, affecting subsequent detection. Phospholipids are mostly highly processed products; after extraction with alcohols and ethers, acetone, etc., the integrity of their genome is also damaged, and they are difficult to dissolve, all of which restrict the extraction and detection of their DNA (Han Jianxun et al., 2011).

[0005] References:

[0006] 1. Dmam L, Smvan R. An overview of analytical methods for determining the geographical origin of food products. Food Chemistry 2008,107:897-911.

[0007] 2. Qi Lingqian, Liu Xiu, Ding Mengxuan, Liu Yuanyuan, Ke Runhui, Yin Jianjun. Research progress on DNA extraction and gene detection of vegetable oils [J]. Food Research and Development, 2016, 37(01):220-224.

[0008] 3. Zhang Hailiang, Wu Yajun, Chen Yinji, et al. Research progress on DNA extraction methods from edible oils. Food and Fermentation Industries, 2010, 11: 128-132.

[0009] 4. He Jing, Xu Wentao, Huang Kunlun. Research progress on DNA extraction and detection technology of edible oil. Food Industry Technology, 2012, 12: 382-387.

[0010] 5. Vietina M, Agrimonti C, Marmiroli N. Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration. Food Chemistry 2013,141:3820-3826.

[0011] 6. Scollo F, Egea LA, Gentile A, et al. Absolute quantification of olive oil DNA by droplet digital-PCR (dd PCR): Comparison of isolation and amplification methodologies. Food Chemistry 2016, 213: 388-394.

[0012] 7. Fu Xiaohua, Zhang Yan, Zhang Wei, et al. Comparative study on different methods of DNA extraction from cottonseed oil. Journal of Chinese Cereals and Oils, 2014, 29:42-46.

[0013] 8. Z, I, Zdjelar G, et al. Detection of genetically modified soybean in crude soybean oil. Food Chemistry 2014,145:1072-1075.

[0014] 9. Raieta K, Muccillo L, Colantuoni VA novel reliable method of DNA extraction from olive oil suitable for molecular traceability. Food Chemistry2015,172:596-602.

[0015] 10. Yao Fei, Zhou Hui, Wu Suxi. Comparison of DNA extraction methods for camellia seed oil. Grain and Oil Food Science and Technology, 2012, 20: 17-20.

[0016] 11. Wu Xingquan, Zhang Haiyan, Hou Dongdong, et al. Research on DNA analysis technology of vegetable oils. 21st Annual Academic Conference of the Oil Branch of the Chinese Cereals and Oils Association and Forum on the Development of China's Edible Oil Industry, 2012, 227-229.

[0017] 12. Costa J, Mafra I, Mbpp O. Advances in vegetable oil authentication by DNA-based markers. Trends in Food Science & Technology 2012, 26: 43-55.

[0018] 13. Han Jianxun, Wu Yajun, Wang Bin, Yang Hairong, Chen Ying. Extraction method and application of DNA from soybean lecithin [J]. Food and Fermentation Industries, 2011, 37(09):185-190. Summary of the Invention

[0019] The purpose of this invention is to provide a method for DNA extraction from edible oils to solve the problems of unsuccessful DNA extraction and low extraction concentration in the existing technology.

[0020] This invention provides a method for DNA extraction from edible oils, comprising the following steps:

[0021] Step 1: Weigh 10g of oil or phospholipid into the first centrifuge tube;

[0022] Step 2: Add 15 ml of n-hexane and 2 ml of EFG buffer to the first centrifuge tube;

[0023] Step 3: Vortex the first centrifuge tube to thoroughly mix the oil, n-hexane, and EFG buffer in the first centrifuge tube, and then centrifuge the first centrifuge tube.

[0024] Step 4: Take 1 ml of the bottom liquid after separation in the first centrifuge tube and transfer it to the second centrifuge tube;

[0025] Step 5: Add 500 μl of chloroform to the second centrifuge tube, invert and mix the bottom liquid and chloroform, and then centrifuge the second centrifuge tube.

[0026] Step 6: Transfer the supernatant from the second centrifuge tube to the third centrifuge tube, and add 0.8 times the volume of isopropanol and 4 μl of glycogen to the third centrifuge tube;

[0027] Step 7: Mix the supernatant, isopropanol and glycogen in the third centrifuge tube, let it stand at room temperature for at least 30 minutes, then perform the first centrifugation operation on the third centrifuge tube, discard the supernatant in the third centrifuge tube, add 500 μl of 75% ethanol to the third centrifuge tube, and then perform the second centrifugation operation on the third centrifuge tube.

[0028] Step 8: Discard the supernatant in the third centrifuge tube, dry the third centrifuge tube at 65°C for 5 min, add 60 μl of TE buffer to the third centrifuge tube to dissolve the precipitate, place at 65°C for 2 min, and store the DNA at -20°C for later use.

[0029] Furthermore, in step one, the first centrifuge tube is a 50ml centrifuge tube.

[0030] Furthermore, in step two, if the oil is a type of oil that is difficult to melt, add 15 ml of n-hexane and 2 ml of EFG buffer to the first centrifuge tube, and then place the first centrifuge tube in a 60°C water bath until the oil melts.

[0031] Furthermore, in step three, the first centrifuge tube is centrifuged at 7500 rpm for 20 minutes.

[0032] Furthermore, in step four, the second centrifuge tube is a 2ml centrifuge tube.

[0033] Furthermore, in step four, if the liquid in the first centrifuge tube does not separate into layers, add another 2 ml of EFG to the first centrifuge tube and centrifuge for 20 minutes, repeating this process until the liquid in the first centrifuge tube separates into layers.

[0034] Furthermore, in step five, the second centrifuge tube is centrifuged at 14,000 rpm for 10 minutes.

[0035] Furthermore, in step six, the third centrifuge tube is a 2ml centrifuge tube.

[0036] Furthermore, in step seven, the third centrifuge tube is subjected to a first centrifugation operation at 14,000 rpm for 20 minutes.

[0037] Furthermore, in step seven, the third centrifuge tube is subjected to a second centrifugation operation at 14,000 rpm for 10 minutes.

[0038] The present invention has the following beneficial effects:

[0039] This invention provides a method for DNA extraction from edible oils that is more efficient than traditional methods, significantly saving time and costs compared to emulsification methods, and yielding highly stable DNA. A series of comparative experiments have shown that the DNA extracted using this method exhibits greater stability in subsequent experiments. It also has relatively low environmental toxicity, is safe and reliable to operate, and uses relatively few organic reagents, posing less risk to laboratory personnel and the environment. Furthermore, it is more cost-effective. Compared to methods using adsorption columns or magnetic bead reagent kits, this invention uses fewer consumables and is less expensive; compared to emulsification methods, it has lower time and labor costs. This invention provides a better foundation for DNA extraction from edible oils and phospholipids and for the biological detection of nucleic acids. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 A flowchart of the method for DNA extraction from edible oils provided by the present invention;

[0042] Figure 2 RT-qPCR amplification curves for four different types of phospholipids;

[0043] Figure 3 RT-qPCR amplification curves for four different soybean oils. Detailed Implementation

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be pointed out that the following detailed description is illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] This invention references various oil DNA extraction methods and, after extensive experimentation, has finalized an optimized DNA extraction scheme for edible oils. Please refer to [link to relevant documentation]. Figure 1 The present invention provides a method for DNA extraction from edible oils, which specifically includes the following steps:

[0046] Step 1: Weigh 10g of oil or phospholipid into the first centrifuge tube.

[0047] Specifically, in step one, the first centrifuge tube is a 50ml centrifuge tube.

[0048] Step 2: Add 15 ml of n-hexane and 2 ml of EFG buffer to the first centrifuge tube.

[0049] Leaching is a DNA extraction method that utilizes the principle of extraction. The oils in oilseeds can dissolve in n-hexane. Through contact (soaking) with the oil, the oils are extracted. EFG buffer denatures proteins, effectively removing proteins from DNA and releasing the DNA.

[0050] Specifically, the EFG buffer was purchased from Eurofins GeneScan. In addition, in step two, if the oil is a type of oil that is difficult to melt, 15 ml of n-hexane and 2 ml of EFG buffer are added to the first centrifuge tube, and then the first centrifuge tube is placed in a 60°C water bath until the oil melts.

[0051] Step 3: Vortex the first centrifuge tube to thoroughly mix the oil, n-hexane, and EFG buffer in the first centrifuge tube, and then centrifuge the first centrifuge tube.

[0052] Specifically, in step three, the first centrifuge tube is centrifuged at 7500 rpm for 20 minutes.

[0053] Step 4: Take 1 ml of the bottom liquid after separation in the first centrifuge tube and transfer it to the second centrifuge tube.

[0054] After organic extraction, the solution separates into three layers, with DNA present in the bottom layer.

[0055] Specifically, in step four, the second centrifuge tube is a 2ml centrifuge tube. Further, in step four, if the liquid in the first centrifuge tube does not separate into layers, then add another 2ml of EFG to the first centrifuge tube and centrifuge for 20 minutes, repeating this process until the liquid in the first centrifuge tube separates into layers.

[0056] Step 5: Add 500 μl of chloroform to the second centrifuge tube, invert and mix the bottom liquid and chloroform, and then centrifuge the second centrifuge tube.

[0057] Adding chloroform can accelerate the separation of the organic phase and the liquid phase, and remove trace amounts of residual phenols in the DNA aqueous solution.

[0058] Specifically, in step five, the second centrifuge tube is centrifuged at 14,000 rpm for 10 minutes.

[0059] Step 6: Transfer the supernatant from the second centrifuge tube to the third centrifuge tube, and add 0.8 times the volume of isopropanol and 4 μl of glycogen to the third centrifuge tube.

[0060] Specifically, after centrifugation, the lower layer is the organic phase, and the upper layer is the aqueous solution of the desired DNA. Isopropanol is used to selectively precipitate DNA. Nucleic acids precipitated using glycogen as a co-precipitant are more suitable for subsequent PCR, RT-PCR, and nuclease reactions such as restriction enzymes.

[0061] Specifically, in step six, the third centrifuge tube is a 2ml centrifuge tube.

[0062] Step 7: Mix the supernatant, isopropanol and glycogen in the third centrifuge tube, let it stand at room temperature for at least 30 minutes, then perform the first centrifugation operation on the third centrifuge tube, discard the supernatant in the third centrifuge tube, add 500 μl of 75% ethanol to the third centrifuge tube, and then perform the second centrifugation operation on the third centrifuge tube.

[0063] Specifically, discard the supernatant in the third centrifuge tube and collect the DNA precipitate at the bottom of the flask. 75% ethanol is more cost-effective than anhydrous ethanol. The DNA solution is stable in a hydrated state; upon adding ethanol, the ethanol removes water molecules surrounding the DNA, causing the DNA to dehydrate, polymerize, and precipitate.

[0064] Specifically, in step seven, the third centrifuge tube is centrifuged at 14,000 rpm for 20 minutes. Further, in step seven, the third centrifuge tube is centrifuged at 14,000 rpm for 10 minutes.

[0065] Step 8: Discard the supernatant in the third centrifuge tube, dry the third centrifuge tube at 65°C for 5 min, add 60 μl of TE buffer to the third centrifuge tube to dissolve the precipitate, place at 65°C for 2 min, and store the DNA at -20°C for later use.

[0066] Carefully clean the waste liquid; the precipitate at the bottom of the bottle is the required DNA. Specifically, the amount of TE buffer used can be adjusted according to the actual concentration results.

[0067] It should be noted that this invention requires strict control over the sample amount of the extracted matrix; too much or too little will affect the extraction results. Compared to the smaller amount of oil used in adsorption column methods, the weight used in this invention makes it easier to extract the corresponding DNA. Furthermore, for poorly soluble oils, careful control of temperature and time is necessary during heating to prevent DNA damage; the temperature used in this invention is the optimal temperature after testing. This description is not found in existing methods for extracting DNA from edible oils and phospholipids. Finally, if no stratification occurs after the first centrifugation with EFG buffer, the volume of EFG buffer added again must be carefully controlled to avoid affecting subsequent extractions; the volume added in this invention is the result of multiple experiments. Figure 2 The graphs show the RT-qPCR amplification curves for four different types of phospholipids. Figure 3 The RT-qPCR amplification curves of four different soybean oils show that the DNA extraction results were good and the amplification curves were normal.

[0068] As can be seen from the above embodiments, the edible oil DNA extraction method of the present invention has the following advantages:

[0069] (1) The DNA extraction method for oils and phospholipids in this invention is more efficient. It saves a lot of time and cost compared to methods such as emulsification.

[0070] (2) The DNA extracted by this invention has high stability. Through a series of comparative experiments, it was found that the DNA extracted by this method is more stable in subsequent experiments.

[0071] (3) The present invention has relatively low toxicity to the environment and is safe and reliable to operate. The method uses relatively few organic reagents and has relatively low harm to experimental personnel and the environment.

[0072] (4) The present invention is more cost-effective. Compared with methods such as adsorption column method and magnetic bead reagent kit, the present invention uses fewer consumables and has lower costs; compared with emulsification method, the present invention has lower time and labor costs.

[0073] Therefore, this invention provides a better foundation for DNA extraction and nucleic acid bioassay of edible oils and phospholipids.

[0074] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0075] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented, for example, in sequences other than those illustrated or described herein.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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 method for extracting DNA from edible oils, characterized in that, Includes the following steps: Step 1: Weigh 10g of oil or phospholipid into the first centrifuge tube; Step 2: Add 15 ml of n-hexane and 2 ml of EFG buffer to the first centrifuge tube; Step 3: Vortex the first centrifuge tube to thoroughly mix the oil, n-hexane, and EFG buffer in the first centrifuge tube, and then centrifuge the first centrifuge tube. Step 4: Take 1 ml of the bottom liquid after separation in the first centrifuge tube and transfer it to the second centrifuge tube; Step 5: Add 500 μl of chloroform to the second centrifuge tube, invert and mix the bottom liquid and chloroform, and then centrifuge the second centrifuge tube. Step 6: Transfer the supernatant from the second centrifuge tube to the third centrifuge tube, and add 0.8 times the volume of isopropanol and 4 μl of glycogen to the third centrifuge tube; Step 7: Mix the supernatant, isopropanol and glycogen in the third centrifuge tube, let it stand at room temperature for at least 30 minutes, then perform the first centrifugation operation on the third centrifuge tube, discard the supernatant in the third centrifuge tube, add 500 μl of 75% ethanol to the third centrifuge tube, and then perform the second centrifugation operation on the third centrifuge tube. Step 8: Discard the supernatant in the third centrifuge tube, dry the third centrifuge tube at 65°C for 5 min, add 60 μl of TE buffer to the third centrifuge tube to dissolve the precipitate, place at 65°C for 2 min, and store the DNA at -20°C for later use.

2. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step one, the first centrifuge tube is a 50ml centrifuge tube.

3. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step two, if the oil is a type of oil that is difficult to melt, add 15 ml of n-hexane and 2 ml of EFG buffer to the first centrifuge tube, and then place the first centrifuge tube in a 60°C water bath until the oil melts.

4. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step three, the first centrifuge tube is centrifuged at 7500 rpm for 20 minutes.

5. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step four, the second centrifuge tube is a 2ml centrifuge tube.

6. The method for DNA extraction from edible oils according to claim 5, characterized in that, In step four, if the liquid in the first centrifuge tube does not separate into layers, add another 2 ml of EFG to the first centrifuge tube and centrifuge for 20 min. Repeat this process until the liquid in the first centrifuge tube separates into layers.

7. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step five, the second centrifuge tube is centrifuged at 14,000 rpm for 10 minutes.

8. The method for DNA extraction from edible oils according to claim 1, characterized in that, In step six, the third centrifuge tube is a 2ml centrifuge tube.

9. The method for extracting DNA from edible oils according to claim 8, characterized in that, In step seven, the third centrifuge tube is subjected to a first centrifugation operation at 14,000 rpm for 20 minutes.

10. A method for DNA extraction from edible oils according to claim 9, characterized in that, In step seven, the third centrifuge tube is subjected to a second centrifugation operation at 14,000 rpm for 10 minutes.

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