CN107904288A - A kind of detection method for identifying olive oil doping - Google Patents

Abstract

The invention discloses a detection method for identifying adulterated olive oil, which comprises the following steps: step 1, primer design: designing and screening genes for palm fatty acid synthesis in NCBI library; step 2, DNA extraction of olive oil to be detected; step 3. Real-time fluorescence quantitative PCR reaction: add primers to GL1, GL2, EO1, EO2 and TY2 in the DNA oil sample to be detected to amplify, to read the Ct value as a standard within 40 cycles, and judge whether Positive amplification was obtained, based on which it was judged whether the olive oil was adulterated. The invention can effectively identify other mixed oil products from olive oil.

Description

A detection method for identifying adulterated olive oil

technical field

The invention relates to the technical field of biological detection, in particular to a detection method for identifying olive oil adulteration.

Background technique

Olive oil is more and more popular for its unique physical and chemical properties and nutritional value. Its unsaturated fatty acids are as high as 82%-87%, and the ratio of oleic acid, linoleic acid, and linoleic acid is the most suitable for human needs. . Olive oil has anti-oxidation, cancer prevention, beauty and other effects, and its price is much higher than other vegetable oils. However, in order to make huge profits, some unscrupulous merchants mix low-priced vegetable oil or other virgin olive oil and olive oil pomace into high-priced olive oil. It is difficult to distinguish the authenticity of this kind of adulterated oil from the appearance of color and smell, which seriously damages the health and rights of consumers. Italian multi-brand high-end olive oil was exposed to be mixed with cheap olive oil; Taiwan's "Weiquan" brand was found to be mixed with pomace oil and Taiwan's "waste oil" incident. There is widespread concern about the safety and quality of vegetable oils. Therefore, in order to protect the rights and health of consumers, developing a method for identifying the authenticity of olive oil is of great significance for controlling food quality and safety and eliminating adulteration incidents of olive oil.

All countries are very concerned about olive oil adulteration incidents, and have established a large number of analytical methods. In recent years, the identification techniques of olive oil adulteration mainly include atomic conversion mass spectrometry, nuclear magnetic resonance, spectral analysis, high performance liquid chromatography and other methods. However, since the chemical composition of olive oil changes with the growing season and the environment, it is impossible to accurately identify the authenticity of olive oil through physical and chemical methods. Because the refined edible vegetable oil has undergone complex processing processes such as degumming, deacidification, and decolorization, its nucleic acid has been severely damaged, and its content is very low. In addition, the main component of vegetable oil is lipid substances, and the content of water-soluble DNA is quite small and the quality is low. Difference. When extracting, we often encounter various situations where no DNA is mentioned or a small amount of DNA is mentioned but cannot be amplified. Therefore, the focus of extracting DNA in olive oil is how to effectively and conveniently enrich and extract DNA in olive oil. The trace amount of nucleic acid remaining in olive oil indicates that the genes representing species-specificity may be preserved. Different species have different specificities in their genetic properties, and species can be identified by testing their specific genes. Therefore, molecular biology methods based on DNA analysis provide a strong guarantee for the detection of olive oil adulteration.

In view of this, the inventor researched and designed a detection method for identifying olive oil adulteration, and this case arose from it.

Contents of the invention

The object of the present invention is to provide a kind of detection method of identification olive oil adulteration.

In order to achieve the above object, the technical solution taken by the present invention to solve the technical problems is:

A detection method for identifying adulterated olive oil, comprising the steps of:

Step 1. Primer design: Design and screen for the gene synthesis of palm fatty acids in the NCBI library:

For olive oil, the first primer pair GL1 and the second primer pair GL2 are screened out, the first primer pair GL1 includes the upstream primer GL1F and the downstream primer GL1R, and the second primer pair GL2 includes the upstream primer GL2F and the downstream primer GL2R, The nucleotide base sequences of the GL1F, GL1R, GL2F, and GL2R are respectively shown in the sequence table SEQ NO:1 to SEQ NO:4;

For the target detection substance, the first primer pair EO1 and the second primer pair EO2 are screened out, and the first primer pair EO1 and the second primer pair EO2 are designed and screened according to the gene synthesis of palm fatty acid in the NCBI library of the actual target detection substance ;

As an internal reference for real-time fluorescence quantitative PCR, a primer pair TY2 is used, and the primer pair TY2 includes an upstream primer TY2F and a downstream primer TY2R, and the nucleotide base sequences of the TY2F and the TY2R are respectively as shown in the sequence table SEQ NO: 5 and Shown in SEQ NO:6;

Step 2, DNA extraction of the olive oil to be tested: extracting with a plasmid extraction kit to obtain a DNA oil sample to be tested;

As a preferred mode of the embodiment, the plasmid extraction kit D0026 produced and sold by Shanghai Beyontian Biotechnology Co., Ltd. was used as the plasmid extraction kit.

Step 3, real-time fluorescent quantitative PCR reaction: add primer pairs GL1, GL2, EO1, EO2 and TY2 to the DNA oil sample to be detected to amplify, and determine whether the Ct value is read as a standard within 40 cycles Whether positive amplification is obtained, based on which it is judged whether the olive oil is adulterated.

As a preferred mode of the embodiment, in the first step, the target detection substance is palm oil, the first primer pair EO1 includes upstream primer EO1F and downstream primer EO1R, and the second primer pair EO2 includes upstream primer EO2F and The nucleotide base sequences of EO2R, EO1F, EO1R, EO2F and EO2R are respectively shown in SEQ NO: 7 to SEQ NO: 10 in the sequence table.

As a preferred method of the embodiment, the specific steps of the second step are: take two 50ml test tubes, add 6.5ml of suspension and 6.5ml of lysate to each tube, add the sample to be tested to 50ml, mix and shake for 10min , centrifuge at 10000r/min for 2min, remove the oil phase, add olive oil to the water phase to 50ml, repeat the above action 4 times; add 13ml of the binding solution in the kit to the water phase retained after the fourth centrifugation in the two test tubes Then turn it upside down 6 times, centrifuge at 10,000r/min for 20min, and follow the kit’s follow-up instructions to combine the DNA oil samples from the two test tubes to obtain the DNA oil sample to be tested, and finally store the DNA oil sample to be tested at -20°C middle.

As a preferred mode of the embodiment, the amplification parameters of the real-time fluorescent quantitative PCR are as follows: the amplification reaction volume is 20ul, ROX I 0.25ul, 2X Syker Mix 10ul, the concentration is 0.4ul of primers of 10umol/L, ddH ₂ O 7.35 ul, DNA template 2.0ul; amplification reaction conditions: 95°C for 5min, 95°C for 5s, 60°C for 31s, cycle number 40.

As a preferred mode of the embodiment, it also includes the drawing of real-time fluorescent quantitative PCR standard curve: promptly adopt olive oil and target detection substance of known copy number as standard sample; After amplification, measure the Ct of relevant fluorescent markers in each oil sample Value, the Ct value has a linear relationship with the natural logarithm of the copy number, and the SYBR Green I real-time fluorescent quantitative PCR standard curve is drawn accordingly.

Aiming at a series of problems such as low nucleic acid content, serious damage, and difficult DNA extraction in vegetable oils, the present invention adopts a nucleic acid enrichment method and a DNA extraction kit method to effectively extract DNA from olive oil and palm oil that can be used for PCR amplification. In addition, primers that can effectively amplify the DNA template were designed and screened by fluorescent quantitative PCR. The invention can effectively identify other mixed oil products from olive oil.

Detailed ways

The detection of embodiment 1 olive oil

Material: olive oil

Reagents: Plasmid Large Scale Extraction Kit (Model: D0026) and Plasmid Small Scale Extraction Kit (Model: D0003), both were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

Extraction steps of olive oil DNA: Take two 50ml test tubes, add 6.5ml of suspension and 6.5ml of lysate to each tube, and add olive oil to 50ml, invert and shake for 10min, centrifuge at 10000r/min for 2min, remove the oil phase , add olive oil to the water phase to 50ml, repeat the above action 4 times. Add 13ml of the binding solution in the kit to the water phase retained after the fourth centrifugation in the two test tubes, then turn it upside down 6 times, centrifuge at 10,000r/min for 20min, and then follow the subsequent operation instructions of the kit to combine the DNA of the two test tubes For the oil sample, prepare the DNA oil sample to be detected, and finally store the DNA oil sample to be detected at -20°C.

Add primers GL1, GL2, TY2 (internal reference) to the DNA extraction solution for real-time fluorescent quantitative PCR amplification:

The parameters of the real-time fluorescent quantitative PCR amplification are as follows: the amplification reaction volume is 20ul, ROX I 0.25ul, 2X SykerMix 10ul, the primer concentration is 10umol/L 0.4ul, ddH2O 7.35ul, DNA template 2.0ul;

Real-time fluorescent quantitative PCR amplification reaction conditions: 95°C for 5min, 95°C for 5s, 60°C for 31s, cycle number 40, use water as blank control, judge whether DNA is extracted from oil samples and primer specificity according to standard curve and Ct value .

Results: Based on whether the Ct value was read within 40 cycles or not, it was judged whether positive amplification was obtained. GL1, GL2, TY2 all obtained 100% positive amplification rate. However, the Ct value is relatively large, which may be due to the serious damage to the olive oil nucleic acid, the low concentration of extracted DNA, and the different amplification efficiencies of the primers. Comprehensive analysis shows that DNA was extracted from all samples and the primers were specific.

The detection of embodiment 2 palm oil

Material: palm oil

Reagents: Plasmid Large Scale Extraction Kit (Model: D0026) and Plasmid Small Scale Extraction Kit (Model: D0003), both were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

Extraction steps of palm oil DNA: take two 50ml test tubes, add 6.5ml of suspension and 6.5ml of lysate to each tube, and add palm oil to 50ml, mix and shake upside down for 10min, centrifuge at 10000r/min for 2min, remove the oil phase , add olive oil to the water phase to 50ml, repeat the above action 4 times. Add 13ml of the binding solution in the kit to the water phase retained after the fourth centrifugation in the two test tubes, then turn it upside down 6 times, centrifuge at 10000r/min for 20min, then follow the subsequent operation instructions of the kit, and finally store the DNA oil sample. at -20°C.

Add primers EO1, EO2, TY2 (internal reference) to the DNA extraction solution for real-time fluorescent quantitative PCR amplification;

The parameters of the real-time fluorescent quantitative PCR amplification are as follows: the amplification reaction volume is 20ul, ROX I 0.25ul, 2X SykerMix 10ul, the primer concentration is 10umol/L 0.4ul, ddH2O 7.35ul, DNA template 2.0ul;

Real-time fluorescent quantitative PCR amplification reaction conditions: 95°C for 5min, 95°C for 5s, 60°C for 31s, cycle number 40, use water as blank control, judge whether DNA is extracted from oil samples and primer specificity according to standard curve and Ct value .

Results: Based on whether the Ct value was read within 40 cycles or not, it was judged whether positive amplification was obtained. EO1, EO2, TY2 all obtained 100% positive amplification rate. However, the Ct value is relatively large, which may be due to the serious damage to the olive oil nucleic acid, the low concentration of extracted DNA, and the different amplification efficiencies of the primers. Comprehensive analysis shows that DNA was extracted from all samples and the primers were specific.

Embodiment 3 is mixed with the olive oil detection of palm oil

Ingredients: olive oil mixed with palm oil

Reagents: Plasmid Large Scale Extraction Kit (Model: D0026) and Plasmid Small Scale Extraction Kit (Model: D0003), both were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

Extraction steps of mixed oil DNA: Take two 50ml test tubes, add 6.5ml of suspension and 6.5ml of lysate to each tube, add mixed oil to 50ml, invert and shake for 10min, centrifuge at 10000r/min for 2min, remove the oil phase , add olive oil to the water phase to 50ml, repeat the above action 4 times. Add 13ml of the binding solution in the kit to the water phase retained after the fourth centrifugation in the two test tubes, then turn it upside down 6 times, centrifuge at 10000r/min for 20min, then follow the subsequent operation instructions of the kit, and finally store the DNA oil sample. at -20°C.

Add primers GL1, GL2, EO1, EO2, TY2 (internal reference) to the DNA extraction solution for real-time fluorescent quantitative PCR amplification;

The parameters of the real-time fluorescent quantitative PCR amplification are as follows: the amplification reaction volume is 20ul, ROX I 0.25ul, 2X SykerMix 10ul, the primer concentration is 10umol/L 0.4ul, ddH2O 7.35ul, DNA template 2.0ul;

Real-time fluorescent quantitative PCR amplification reaction conditions: 95°C for 5min, 95°C for 5s, 60°C for 31s, cycle number 40, use water as blank control, judge whether DNA is extracted from oil samples and whether olives are mixed according to the standard curve and Ct value A palm oil-specific gene fragment was amplified from the oil.

Results: Based on whether the Ct value was read within 40 cycles or not, it was judged whether positive amplification was obtained. EO1, EO2, GL1, GL2, TY2 all obtained 100% positive amplification rate. However, the Ct value is relatively large, which may be due to the severe damage to the nucleic acid in olive oil, the low concentration of extracted DNA, and the different amplification efficiencies of primers. Comprehensive analysis can conclude that DNA was extracted from all samples and detected in olive oil mixture Palm oil-specific genes.

Although olive oil has undergone complex processing processes such as degumming, deacidification, and decolorization for many times, the present invention finds trace amounts of nucleic acid in olive oil and other vegetable oils through DNA enrichment and extraction methods, and the present invention can use this method to detect olive oil Doping with other vegetable oil varieties is similar to the palm oil detection method of the present invention, except that the designed primers are different, that is, primers need to be designed according to specific target detection substances.

The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, the equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention within range.

Claims (6)

1. a detection method for identifying adulterated olive oil, characterized in that: comprises the following steps: Step 1. Primer design: Design and screen for the gene synthesis of palm fatty acids in the NCBI library: For olive oil, the first primer pair GL1 and the second primer pair GL2 are screened out, the first primer pair GL1 includes the upstream primer GL1F and the downstream primer GL1R, and the second primer pair GL2 includes the upstream primer GL2F and the downstream primer GL2R, The nucleotide base sequences of the GL1F, GL1R, GL2F, and GL2R are respectively shown in the sequence table SEQ NO:1 to SEQ NO:4; For the target detection substance, the first primer pair EO1 and the second primer pair EO2 are screened out, and the first primer pair EO1 and the second primer pair EO2 are designed and screened according to the gene synthesis of palm fatty acid in the NCBI library of the actual target detection substance ; As an internal reference for real-time fluorescence quantitative PCR, a primer pair TY2 is used, and the primer pair TY2 includes an upstream primer TY2F and a downstream primer TY2R, and the nucleotide base sequences of the TY2F and the TY2R are respectively as shown in the sequence table SEQ NO: 5 and Shown in SEQ NO:6; Step 2, DNA extraction of the olive oil to be tested: extracting with a plasmid extraction kit to obtain a DNA oil sample to be tested; Step 3, real-time fluorescent quantitative PCR reaction: add primer pairs GL1, GL2, EO1, EO2 and TY2 to the DNA oil sample to be detected to amplify, and determine whether the Ct value is read as a standard within 40 cycles Whether positive amplification is obtained, based on which it is judged whether the olive oil is adulterated. 2. a kind of detection method of identification olive oil adulteration as claimed in claim 1, is characterized in that: in described step 1, described target detection substance is palm oil, and described first primer pair EO1 comprises upstream primer EO1F And the downstream primer EO1R, the second primer pair EO2 includes upstream primers EO2F and EO2R, the nucleotide base sequences of the EO1F, the EO1R, the EO2F and the EO2R are as shown in the sequence table SEQ NO:7 to Shown in SEQ NO:10. 3. a kind of detection method of identification olive oil adulteration as claimed in claim 1, it is characterized in that: the concrete step of described step 2 is, get two 50ml test tubes, each tube respectively adds suspension 6.5ml and cracks Add 6.5ml of the sample to be tested to 50ml, invert and shake for 10min, centrifuge at 10000r/min for 2min, remove the oil phase, add olive oil to the water phase to 50ml, repeat the above action 4 times; Add 13ml of the binding solution in the kit to the water phase retained after 4 times of centrifugation, then turn it upside down 6 times, centrifuge at 10,000r/min for 20min, then follow the subsequent operation instructions of the kit, combine the DNA oil samples of the two test tubes, and prepare the Detect the DNA oil sample, and finally store the DNA oil sample to be tested at -20°C. 4. a kind of detection method for identifying adulterated olive oil as claimed in claim 1, is characterized in that: the plasmid extraction kit of described step 2 adopts the plasmid extraction kit D0026 that Shanghai Biyuntian Biotechnology Co., Ltd. produces . 5. a kind of detection method of identification olive oil adulteration as claimed in claim 1, is characterized in that: the amplification parameter of described real-time fluorescence quantitative PCR is as follows: amplification reaction volume is 20ul, ROX 1 0.25ul, 2X Syker Mix 10ul, primer 0.4ul with a concentration of 10umol/L, ddH ₂ O 7.35ul, DNA template 2.0ul; amplification reaction conditions: 95°C for 5min, 95°C for 5s, 60°C for 31s, cycle number 40. 6. a kind of detection method of identifying olive oil adulteration as claimed in claim 1, is characterized in that: also comprise the drawing of real-time fluorescence quantitative PCR standard curve: promptly adopt the olive oil of known copy number and target detection substance as standard Sample; after amplification, measure the Ct value of the relevant fluorescent marker in each oil sample, the Ct value is linear with the natural logarithm of the copy number, and draw the SYBR Green I real-time fluorescent quantitative PCR standard curve accordingly.