Method for detecting and identifying tea seed oil by extracting DNA (Deoxyribose Nucleic Acid) based on membrane technology

Abstract

According to the method for detecting and identifying the tea-seed oil by extracting DNA based on the membrane technology, the DNA of the tea-seed oil is separated, enriched and purified through a glass fiber membrane, the tea-seed oil is identified by combining a real-time fluorescent polymerase chain reaction (PCR) technology, and the method specifically comprises the steps of n-hexane fat dissolution, water phase separation and extraction, thermal cracking, purification, membrane method DNA extraction and real-time fluorescent PCR detection. According to the pretreatment method for extracting the DNA in the tea-seed oil based on the membrane technology, the real attribute of the tea-seed oil is rapidly identified through qualitative detection of endogenous genes by PCR (Polymerase Chain Reaction) detection; according to the method, trace DNA can be extracted from large-volume tea-seed oil, so that the requirements of a PCR detection method are met, and the problem that DNA in the tea-seed oil is seriously degraded, is damaged into small molecules and cannot be efficiently extracted can be well solved; nucleic acid molecules are effectively purified and separated through the glass fiber membrane, the detection cost is reduced, and the detection period is shortened.

Description

Technical Field

[0001] The invention relates to a method for extracting DNA, detecting and identifying tea seed oil based on membrane technology, and particularly belongs to the technical field of oil and fat detection. Background Art

[0002] Camellia oil contains unsaturated fatty acids, fat-soluble vitamins, flavonoids, and other ingredients beneficial to human health. It also contains specific physiologically active substances not found in olive oil, such as tea polyphenols and camellia glycosides. Due to its multiple therapeutic benefits, including dietary benefits, it has become highly sought after and is known as the "Oriental Olive Oil." GB / T 11765-2018, "Camellia Oil," specifies physical and chemical indicators and testing methods for different grades of camellia oil, including acid value, peroxide value, and insoluble impurity content. However, a lack of identification methods for specific plant oil varieties has led to the frequent adulteration of tea seed oil with relatively low-priced oils.

[0003] Currently, commonly used methods for identifying and testing edible vegetable oils include physical and chemical analysis and molecular biology. Physical and chemical analysis primarily utilizes chromatography and mass spectrometry to measure differences in the composition and content of characteristic indicators such as fatty acids, triglycerides, and phytosterols in edible oils, thereby verifying the oil's authenticity. However, characteristic indicators vary among edible vegetable oils based on origin, variety, and degree of processing. Therefore, physical and chemical analysis has limitations and cannot accurately identify the oil. Molecular biology, based on the ubiquitous presence and stability of deoxyribonucleic acid (DNA) within cells, can directly reflect the authenticity of the vegetable oil raw material. Therefore, edible oil testing techniques targeting DNA are more stable, sensitive, and accurate than physical and chemical analysis techniques, making them suitable for detecting vegetable oil adulteration and tracing the origin of varieties.

[0004] Since Pauli U et al. successfully detected DNA from crude soybean oil in 1998, a number of studies have been published on the extraction and amplification of DNA from virgin vegetable oils. Molecular biological detection methods rely heavily on the quality, purity, and amount of isolated DNA, making DNA extraction technology crucial. Mainstream extraction methods include kit-based methods, the CTAB method, and the SDS method.

[0005] Among them, COSTA J et al. used a kit method to amplify soybean DNA from fully refined vegetable oil. The commercial kit method has been widely used due to its advantages such as simple operation and high product purity. The commonly used kit methods mainly include the magnetic bead method, the centrifugal column method and the resin method. The corresponding kits with better edible vegetable oil DNA extraction effects include WizardMagnetic DPSF, Nucleospin Plant and QIAamp DNA stool. Although the kit method has advantages in extraction efficiency and product purity, the amount of starting sample used for edible vegetable oil DNA extraction is dozens or hundreds of times that of general plant samples, resulting in a large consumption of reagents such as DNA lysis buffer, a sharp increase in extraction costs, and it is only suitable for small-volume extractions, which to a certain extent restricts the application of this method.

[0006] In the CTAB method, cetyltrimethylammonium bromide (CTAB) forms multiplexes with DNA. At high salt concentrations, the multiplexes dissolve. At low salt concentrations, the solubility decreases, resulting in precipitation. Polysaccharides and proteins remain dissolved in the solution. Centrifugation is then used to separate the polysaccharides, proteins, and DNA multiplexes, and the DNA is then purified to remove the CTAB. CTAB, as the primary component of the lysis buffer, lyses cells and removes impurities. Optimizing the composition of the lysis buffer can improve the efficiency of DNA extraction from vegetable oils. Due to its low cost and high extraction efficiency, it has gradually become one of the most commonly used nucleic acid extraction techniques. However, most edible vegetable oils are highly processed products, especially high-grade refined vegetable oils, which undergo complex production and processing. DNA is severely damaged and its DNA content is extremely low. The CTAB method is extremely complex and time-consuming, making it difficult to extract even trace amounts of DNA from edible vegetable oils.

[0007] The SDS method uses sodium dodecyl sulfate (SDS), an anionic detergent, to lyse plant cells at elevated temperatures of 55-65°C using a high-concentration SDS extraction buffer, causing chromosome segregation, protein denaturation, and nucleic acid release. Proteins and polysaccharides are then precipitated by increasing the potassium acetate concentration and cooling the sample in an ice bath (potassium acetate binds to proteins and polysaccharides, forming an insoluble product at low temperatures). After centrifugation to remove the precipitate, DNA in the supernatant is extracted with phenol / chloroform. After repeated extractions, the DNA in the aqueous phase is precipitated with ethanol. The SDS method is a commonly used method for DNA extraction, offering low cost and high DNA integrity. It also minimizes the fragmentation of nucleic acids in edible oils during the extraction process. However, its DNA yield and purity are inferior to those of the kit-based and CTAB methods, and it is incapable of extracting trace amounts of DNA from edible plant oils. Consequently, its application has been limited in recent studies.

[0008] However, existing technologies have not effectively solved the problem of DNA isolation and specific detection in high-fat oils. Moreover, residual phenolic compounds in the extract can inhibit the activity of DNA polymerase, leading to unstable amplification and causing great difficulties in subsequent detection. To this end, the present invention establishes a new method for extracting trace DNA from tea seed oil based on membrane technology, which is used for tea seed oil authenticity identification research. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the present invention proposes a pretreatment technology for extracting DNA from tea seed oil based on membrane method, and combines it with PCR detection means to establish a qualitative detection method for quickly identifying the true attributes of tea seed oil.

[0010] The present invention discloses a method for extracting DNA and detecting and identifying tea seed oil based on membrane technology. The method uses a glass fiber membrane to separate, enrich, and purify the DNA of tea seed oil, and combines it with real-time fluorescence polymerase chain reaction (PCR) technology to identify the tea seed oil. The specific steps are as follows: Step 1: Dissolve fat in hexane Weigh 5-10g of tea seed oil sample into a 50mL centrifuge tube, add 20mL of n-hexane, and extract at 40°C with shaking for 30min. Dissolve the tea seed oil lipids by liquid-liquid extraction, and then centrifuge at 8000rpm for 5min. Repeat the above steps for 6 parallel experiments. The centrifuge tubes are labeled Y1, Y2, Y3, Y4, Y5, and Y6 respectively. Step 2: Aqueous phase separation and extraction Add 10 mL of 0.1% DEPC (diethyl pyrocarbonate) water to Y1, vortex extract on a vortex mixer for 1 minute, then shake extract for 10 minutes, and then centrifuge at 8000 rpm for 5 minutes. Discard the upper oil phase, return to room temperature, vortex mix, and transfer all the remaining solution in Y1 to Y2. Then add 1 mL of 0.1% DEPC water to Y1 and vortex for 1 minute to extract residual DNA on the tube wall. The aqueous phase solution in Y1 is once again combined with Y2. Mix the solution in Y2 and shake extract for 10 minutes to enrich the extracted target. Centrifuge at 8000 rpm for 5 minutes, discard the upper oil phase, return to room temperature, and transfer all the lower aqueous phase in Y2 to Y3. Repeat the above steps until Y6. After centrifugation, discard the upper oil phase and wait for thermal lysis of the lower aqueous phase. Step 3: Thermal Cracking Transfer 4 mL of the aqueous phase from Y6 to a 50 mL centrifuge tube Y7, add 2 mL of lysis buffer, and heat-cleave the tube in a 65°C air bath shaker for 2 h. After lysis, centrifuge at 9000 rpm for 5 min. Transfer 1 mL of the supernatant to a 2 mL centrifuge tube Y8, add 7 μL of 20 mg / L proteinase K solution and 3 μL of 10 mg / L RNase A solution, oscillate to mix, and then place in a metal bath and incubate at 37°C for 2 h. Step 4: Purification Add 300 μL of protein precipitation solution to centrifuge tube Y8, centrifuge at 12000 rpm for 5 min, take 940 μL of the upper layer solution and add an equal volume of DNA extract solution, vortex on a vortex mixer for 1 min to mix thoroughly, centrifuge at 12000 rpm for 5 min, take 940 μL of the upper layer solution to a 2 mL centrifuge tube Y9, and add an equal volume of DNA extract solution again, vortex for 1 min to mix thoroughly, and centrifuge at 12000 rpm for 5 min; Step 5: DNA extraction by membrane method Activation: Take 1 mL of sterile water to activate the syringe filter with a built-in 0.7 μm glass fiber membrane; Sample loading: Take 700 μL of the upper layer solution in centrifuge tube Y9 and pass it through the membrane, control the flow rate to 3 seconds / drop, and discard the filtrate; Washing: Add 1 mL of 70% ethanol solution to wash the glass fiber membrane, control the flow rate, discard the filtrate, repeat the washing once, and press the glass fiber membrane dry; Elution: Add 100-150 μL of Tris-EDTA solution to elute the squeezed glass fiber membrane. Collect the eluate; control the flow rate and perform secondary elution on the glass fiber membrane with the collected eluate to obtain a DNA solution purified by the membrane method; Step 6: Real-time fluorescence PCR detection The DNA solution purified by membrane method is detected by real-time fluorescence PCR to amplify specific screening gene fragments. The genetic components in the tea seed oil sample are determined based on the real-time fluorescence amplification curve, thereby achieving the purpose of identifying whether the tea seed oil is adulterated.

[0011] The DNA extracting solution is a mixture of phenol, chloroform and isoamyl alcohol, with a volume ratio of 25:24:1.

[0012] The lysis buffer is a mixture of 55 mmol / L hexadecyltrimethylammonium bromide, 1.40 mol / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate and 100 mmol / L tris(hydroxymethyl)aminomethane, and has a pH of 8.0.

[0013] The protein precipitation solution is a mixture of 0.3 mol / L guanidine hydrochloride, 1.5 mol / L ammonium sulfate, 50 mmol / L sodium citrate, acetic acid and ethanol, with a final ethanol concentration of 75%. Acetic acid is used to adjust the pH value, and the pH is 8.0.

[0014] The membrane technology is used to extract DNA. The membrane is first activated by passing it through an aqueous solution, and then the sample is loaded. The DNA is adsorbed by the glass fiber membrane to remove salts, proteins, polysaccharides and phenolic compounds. The DNA is then rinsed with a 70% ethanol solution to further purify the DNA. Finally, a Tris-EDTA solution is used for elution to complete the membrane technology DNA extraction.

[0015] Beneficial effects of the present invention: 1. The present invention establishes a pre-treatment method for extracting DNA from tea seed oil based on membrane technology, and detects endogenous genes through PCR to qualitatively identify the true properties of tea seed oil.

[0016] 2. The present invention can extract trace amounts of DNA from large volumes of tea seed oil to meet the requirements of the PCR detection method, and can effectively solve the problem that DNA in tea seed oil is severely degraded and broken into small molecules that cannot be efficiently extracted.

[0017] 3. The present invention effectively purifies and separates nucleic acid molecules through glass fiber membranes, reducing detection costs and shortening detection cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0019] Figure 1 : Flow chart of the experimental steps for extracting DNA from vegetable oil using the membrane method of the present invention; Figure 1 In: (1) n-hexane dissolution process; (2) aqueous phase separation and extraction process; (3) thermal cracking process; (4) DNA purification process; (5) membrane DNA extraction process; (6) real-time fluorescence PCR detection process.

[0020] 1. 50ml centrifuge tube; 2. tea seed oil; 3. n-hexane; 4. defatted tea seed oil; 5 and 21. DEPC water; 6. centrifuge; 7. aqueous phase after tea seed oil extraction; 8. aqueous phase after circulating tea seed oil extraction; 9. lysis buffer; 10. temperature-controlled incubator; 11. 2ml centrifuge tube; 12. lysis solution; 13. proteinase K; 14. RNase A; 15. protein precipitation solution; 16. metal bath; 17. deproteinization solution; 18 and 20. DNA extraction solution; 19 and 24. DNA purification solution; 22. syringe; 23. glass fiber membrane filter; 25. activated glass fiber membrane; 26. 70% ethanol; 27. glass fiber membrane for DNA enrichment; 28. TE buffer; 29. ​​washed glass fiber membrane; 30. 1.5ml centrifuge tube; 31. DNA elution solution; 32. real-time fluorescence PCR instrument.

[0021] Figure 2 : Camellia oil DNA amplification curve of Example 1 of the present invention; Figure 3 : Rapeseed oil DNA amplification curve diagram of Example 2 of the present invention; Figure 4 : Amplification curve of rapeseed DNA in tea seed oil according to Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the embodiments.

[0023] Example 1

[0024] The sample in this example is tea seed oil. The DNA of tea seed oil is separated, enriched and purified by glass fiber membrane, and combined with PCR detection to quickly identify tea seed oil. The specific steps are as follows: Step 1: Dissolve fat in hexane

[0025] Weigh 10 g of tea seed oil sample into a 50 mL centrifuge tube and add 20 mL of n-hexane. Extract at 40°C for 30 min. Dissolve tea seed oil lipids by liquid-liquid extraction. Centrifuge at 8000 rpm for 5 min. Repeat the above steps for 6 parallel experiments. The centrifuge tube samples in the parallel experiments are marked as Y1, Y2, Y3, Y4, Y5, and Y6 respectively. Step 2: Aqueous phase separation and extraction Add 10 mL of 0.1% DEPC (diethyl pyrocarbonate) water to Y1, vortex extract on a vortex mixer for 1 minute, then shake extract for 10 minutes, and then centrifuge at 8000 rpm for 5 minutes. Discard the upper oil phase, return to room temperature, vortex mix, and transfer all the remaining solution in Y1 to Y2. Add 1 mL of 0.1% DEPC water to Y1 and vortex for 1 minute to extract residual DNA on the tube wall. Combine all the aqueous phase solutions in Y1 into Y2 again. Mix the solution in Y2 and shake extract for 10 minutes to enrich the extracted target. Centrifuge at 8000 rpm for 5 minutes, discard the upper oil phase, return to room temperature, and transfer all the lower aqueous phase in Y2 to Y3. Repeat the above steps until Y6. After centrifugation, discard the upper oil phase and wait for thermal lysis of the lower aqueous phase. Step 3: Thermal Cracking 4 mL of the aqueous phase from Y6 was placed in a 50 mL centrifuge tube Y7, and 2 mL of lysis buffer (a mixture of 55 mmol / L hexadecyltrimethylammonium bromide, 1.40 mol / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetic acid, and 100 mmol / L tris(hydroxymethyl)aminomethane, with a pH of 8.0) was added. The mixture was placed in a 65°C air bath shaker for thermal lysis for 2 h. After lysis, the mixture was centrifuged at 9000 rpm for 5 min. 1 mL of the supernatant was placed in a 2 mL centrifuge tube Y8, and 7 μL of proteinase K solution (20 mg / L) and 3 μL of RNase A (10 mg / L) solution were added. The mixture was shaken and placed in a metal bath and incubated at 37°C for 2 h. Step 4: Purification To centrifuge tube Y8, add 300 μL of protein precipitation solution (a mixture of 0.3 mol / L guanidine hydrochloride, 1.5 mol / L ammonium sulfate, 50 mmol / L sodium citrate, acetic acid, and ethanol, with a final ethanol concentration of 75%. Acetic acid is used to adjust the pH to 8.0). After centrifugation at 12,000 rpm for 5 min, remove 940 μL of the upper layer and add an equal volume of DNA extraction solution (phenol: chloroform: isoamyl alcohol, 25:24:1, v / v / v). Vortex and mix thoroughly on a vortex mixer for 1 min. After centrifugation at 12,000 rpm for 5 min, remove 940 μL of the upper layer and transfer it to a 2-mL centrifuge tube Y9. Again, add an equal volume of DNA extraction solution and vortex and mix thoroughly. Centrifuge at 12,000 rpm for 5 min. Step 5: DNA extraction by membrane method Activation: Take 1 mL of sterile water to activate the syringe filter with a built-in 0.7 μm glass fiber membrane; Sample loading: Take 700 μL of the upper layer solution in centrifuge tube Y9 and pass it through the membrane, control the flow rate to 3 seconds / drop, and discard the filtrate; Washing: Add 1 mL of 70% ethanol solution to wash the glass fiber membrane, control the flow rate, discard the filtrate, repeat the washing once, and press the glass fiber membrane dry; Elution: Add 150 μL of Tris-EDTA solution (a mixture of 10 mmol / L tris(hydroxymethyl)aminomethane-hydrochloric acid and 1 mmol / L disodium ethylenediaminetetraacetic acid, pH 8.0) to elute the dried glass fiber membrane and collect the eluate. Control the flow rate and use the collected eluate to elute the glass fiber membrane a second time to obtain the membrane-purified DNA solution. Step 6: Real-time fluorescence PCR detection The membrane-purified DNA solution was tested using real-time fluorescence PCR to amplify specific screening gene fragments. Based on the real-time fluorescence amplification curve, the genetic components of the tea seed oil sample were determined, thereby achieving the qualitative identification of the tea seed oil. Primers and probes were designed for endogenous genes in tea seeds, and their nucleic acid sequences are shown in Table 1. The tea seed qPCR amplification reaction system is shown in Table 2. The real-time fluorescence PCR amplification reaction program was 95°C for 30 seconds, followed by 60 cycles of 95°C for 10 seconds and 60 seconds at 60°C.

[0026] ; ; Due to the hydrophilic nature of DNA, the DNA content in fat-soluble samples is already extremely low, and it remains to be seen whether DNA from other oils can be extracted on this basis. To this end, in order to increase the amount of tea oil sample extracted, this embodiment repeatedly extracts multiple oil phases through the aqueous phase, increases the number of extractions, achieves the purpose of large-volume extraction, and reduces dependence on large-volume refrigerated high-speed centrifuges. In addition, by utilizing the hydrophilicity of DNA, n-hexane is first added to dissolve the oil to release the DNA, and then the aqueous phase is introduced for extraction to transfer the DNA from the oil phase to the aqueous phase. After centrifugation, the upper organic phase is removed to achieve the purpose of removing oil and fat, thereby improving the efficiency of liquid-liquid extraction. This embodiment purifies DNA through a glass fiber membrane to reduce the interference of high salt, protein, polysaccharide and phenolic compounds on DNA detection and improve detection sensitivity. The test results are as follows Figure 2 shown.

[0027] The "S"-shaped amplification curve of the real-time fluorescence PCR results indicates that the endogenous gene of the sample was successfully amplified and detected. Figure 2 The real-time fluorescence PCR amplification curve showed successful amplification at cycle 30, detecting the tea seed 1214 gene. This result demonstrates that the glass fiber membrane pretreatment method for extracting DNA from tea seed oil can overcome the difficulty of extracting trace amounts of DNA from fat-soluble samples. Furthermore, the presence of tea seed oil in this sample was accurately confirmed.

[0028] Example 2 This example differs from Example 1 in that the samples to be tested are rapeseed oil and commercially available tea seed oil. This study investigates whether the pretreatment method for DNA extraction using the glass fiber membrane method is applicable to DNA extraction from other vegetable oils, and verifies the feasibility of this method by testing actual samples. The specific steps are as follows: The pretreatment steps for extracting DNA from pure rapeseed oil and commercially available tea seed oil were carried out according to the steps described in Example 1. Primers and probes were designed for the endogenous gene of rapeseed. The nucleic acid sequences are shown in Table 3. The rapeseed qPCR amplification reaction system is shown in Table 4. The total reaction volume was 25 μL. The real-time fluorescence PCR amplification reaction program was 95°C for 30 s, and the cycle program was 95°C for 10 s; 60°C for 60 s, for a total of 60 cycles. The detection results are shown in Table 3. Figure 3-4 shown.

[0029] ; ' To identify tea seed oil, it is necessary to extract not only tea seed DNA but also DNA from other cheap oil crops. To this end, the present embodiment adopts the method described in Example 1 to first extract rapeseed oil, and then Figure 3 It can be seen that the real-time fluorescence PCR amplification curve successfully amplified at the 38th cycle on average, and the rapeseed PEP gene was detected, proving that this method can be applied to the extraction of DNA from rapeseed oil. The method described in Example 1 was then used to detect whether the actual samples of commercially available tea seed oil contained the rapeseed gene, and to identify whether the tea seed oil was adulterated with rapeseed oil. The results are as follows: Figure 4 shown.

[0030] Depend on Figure 4 It can be seen that two real-time fluorescence PCR amplification curves are "S"-shaped, indicating that the rapeseed PEP gene was detected in two wells, and only two out of six wells were detected, which also indicates that the extracted rapeseed DNA content is low. Through testing actual samples of commercially available tea seed oil, a case study of tea seed oil adulterated with rapeseed oil was successfully identified, demonstrating that the membrane-based DNA extraction method of the present invention can be used for qualitative detection of the true properties of tea seed oil, and provides a detection basis for the identification of tea seed oil adulterated with multiple edible vegetable oils and the screening of unknown edible vegetable oils.

Claims

1. A method for extracting DNA and detecting and identifying tea seed oil based on membrane technology, characterized in that: The method described herein uses a glass fiber membrane to separate, enrich, and purify tea seed oil DNA, and combines it with real-time fluorescence polymerase chain reaction (PCR) technology to identify tea seed oil. The specific steps are as follows: Step 1: Dissolve fat in hexane Weigh 5-10g of tea seed oil sample into a 50mL centrifuge tube, add 20mL of n-hexane, and extract at 40°C with shaking for 30min. Dissolve the tea seed oil lipids by liquid-liquid extraction, and then centrifuge at 8000rpm for 5min. Repeat the above steps for 6 parallel experiments. The centrifuge tubes are labeled Y1, Y2, Y3, Y4, Y5, and Y6 respectively. Step 2: Aqueous phase separation and extraction Add 10 mL of 0.1% DEPC water by volume to Y1, vortex extract on a vortex mixer for 1 minute, then shake extract for 10 minutes, and then centrifuge at 8000 rpm for 5 minutes. Discard the upper oil phase, return to room temperature, vortex mix, and transfer all the remaining solution in Y1 to Y2; then add 1 mL of 0.1% DEPC water by volume to Y1, vortex for 1 minute to extract residual DNA on the tube wall, and once again combine all the aqueous phase solutions in Y1 into Y2; mix the solution in Y2, shake extract for 10 minutes, enrich the extracted target, centrifuge at 8000 rpm for 5 minutes, discard the upper oil phase, return to room temperature, and transfer all the lower aqueous phase in Y2 to Y3; repeat the above steps to Y6, discard the upper oil phase after centrifugation, and wait for thermal cracking of the lower aqueous phase; Step 3: Thermal Cracking Transfer 4 mL of the aqueous phase from Y6 to a 50 mL centrifuge tube Y7, add 2 mL of lysis buffer, and heat-cleave the tube in a 65°C air bath shaker for 2 h. After lysis, centrifuge at 9000 rpm for 5 min. Transfer 1 mL of the supernatant to a 2 mL centrifuge tube Y8, add 7 μL of 20 mg / L proteinase K solution and 3 μL of 10 mg / L RNase A solution, oscillate to mix, and then place in a metal bath and incubate at 37°C for 2 h. Step 4: Purification Add 300 μL of protein precipitation solution to centrifuge tube Y8, centrifuge at 12000 rpm for 5 min, take 940 μL of the upper layer solution and add an equal volume of DNA extract solution, vortex on a vortex mixer for 1 min to mix thoroughly, centrifuge at 12000 rpm for 5 min, take 940 μL of the upper layer solution to a 2 mL centrifuge tube Y9, and add an equal volume of DNA extract solution again, vortex for 1 min to mix thoroughly, and centrifuge at 12000 rpm for 5 min; Step 5: DNA extraction by membrane method Activation: Take 1 mL of sterile water to activate the syringe filter with a built-in 0.7 μm glass fiber membrane; Sample loading: Take 700 μL of the upper layer solution in centrifuge tube Y9 and pass it through the membrane, control the flow rate to 3 seconds / drop, and discard the filtrate; Washing: Add 1 mL of 70% ethanol solution to wash the glass fiber membrane, control the flow rate, discard the filtrate, repeat the washing once, and press the glass fiber membrane dry; Elution: Add 100-150 μL of Tris-EDTA solution to elute the squeezed glass fiber membrane; Collect the eluate; control the flow rate and perform secondary elution of the collected eluate on the glass fiber membrane to obtain a DNA solution purified by the membrane method; Step 6: Real-time fluorescence PCR detection The DNA solution purified by membrane method is detected by real-time fluorescence PCR to amplify specific screening gene fragments. The genetic components in the tea seed oil sample are determined based on the real-time fluorescence amplification curve, thereby achieving the purpose of identifying whether the tea seed oil is adulterated.

2. The method for extracting DNA and detecting and identifying squeezed camellia oil based on membrane technology according to claim 1, characterized in that: The DNA extracting solution is a mixture of phenol, chloroform and isoamyl alcohol, with the volume ratio of the mixture being 25:24:

1.

3. The method for extracting DNA and detecting and identifying tea seed oil based on membrane technology according to claim 1, characterized in that: The lysis buffer is a mixture of 55 mmol / L hexadecyltrimethylammonium bromide, 1.40 mol / L sodium chloride, 20 mmol / L disodium ethylenediaminetetraacetate and 100 mmol / L tris(hydroxymethyl)aminomethane, and has a pH of 8.

0.

4. The method for extracting DNA and detecting and identifying squeezed camellia oil based on membrane technology according to claim 1, characterized in that: The protein precipitation solution is a mixture of 0.3 mol / L guanidine hydrochloride, 1.5 mol / L ammonium sulfate, 50 mmol / L sodium citrate, acetic acid and ethanol, with a final ethanol concentration of 75%. Acetic acid is used to adjust the pH value, and the pH is 8.

0.

5. The method for extracting DNA and detecting and identifying squeezed camellia oil based on membrane technology according to claim 1, characterized in that: The membrane technology is used to extract DNA. The membrane is first activated by passing it through an aqueous solution, and then the sample is loaded. The DNA is adsorbed by the glass fiber membrane to remove salts, proteins, polysaccharides and phenolic compounds. The DNA is then rinsed with a 70% ethanol solution to further purify the DNA. Finally, a Tris-EDTA solution is used for elution to complete the membrane technology DNA extraction.

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