CN107904288A - A kind of detection method for identifying olive oil doping - Google Patents
A kind of detection method for identifying olive oil doping Download PDFInfo
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- CN107904288A CN107904288A CN201810015118.XA CN201810015118A CN107904288A CN 107904288 A CN107904288 A CN 107904288A CN 201810015118 A CN201810015118 A CN 201810015118A CN 107904288 A CN107904288 A CN 107904288A
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- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses a kind of detection method for identifying olive oil doping, comprise the following steps：Step 1: design of primers：The gene synthesized for PALM FATTY ACID in NCBI storehouses is designed and screened；Step 2: the DNA extractions of olive oil to be detected；Step 3: real-time fluorescence quantitative PCR reacts：Primer pair GL1, GL2, EO1, EO2 and TY2 are added in the DNA oil samples to be detected to be expanded, and whether to read Ct values as standard in 40 circulations, are judged whether to obtain positive amplification, are judged whether olive oil adulterates accordingly.The present invention can effectively identify other oil products of incorporation from olive oil.
Description
Technical field
The present invention relates to the technical field of biological detection, more particularly to a kind of detection method for identifying olive oil doping.
Background technology
Olive oil is increasingly favored with its unique physicochemical property and nutritive value be subject to people, its unrighted acid
Up to 82%-87%, oleic acid, linoleic acid, the ratio of linolenic acid content are to be most suitable for the ratio that human body needs.Olive oil has
Anti-oxidant, pre- anti-cancer, beauty and other effects, price is more much higher than other plant oil.But some illegal businessmans are sudden and violent to try to gain
Profit, just mixes the vegetable oil or other virgin oils and olive oil pomace etc. of low price in the olive oil of high price.It is this to mix
False oil list is difficult to tell truth from falsehood in appearance from color, smell etc., seriously compromises the health and rights and interests of consumer.Italy
The high-end olive oil of multi-brand is by the quick-fried cheap olive oil of incorporation；Taiwan " taste is complete " brand is found incorporation Marc oil and Taiwan
Regional " gutter oil " event, the edible vegetable oil adulteration incident constantly produced cause people to the extensive of vegetable oil safe mass
Concern.Therefore, to ensure the rights and interests and health of consumer, a kind of identification method of the olive oil true and false is developed, to controlling food matter
Amount is safe and prevents olive oil adulteration incident important in inhibiting.
Each state all very pays close attention to olive oil adulteration incident, and substantial amounts of analysis method is established to this.In recent years, olive oil is mixed
Miscellaneous identification technology mainly has atom conversion mass spectrum, nuclear magnetic resonance, spectrum analysis, high performance liquid chromatography and other methods.However,
Since the chemical composition of olive oil changes with the change of the season of growth and environment, can not accurately be reflected by physico-chemical method
The other olive oil true and false.Due to process of the refining of edible vegetable oil by the complexity such as degumming, depickling, decoloration, its nucleic acid by
Serious destruction, content is very low, and along with the main component of vegetable oil is lipid material, water miscible DNA content is quite few
It is and of poor quality.It is frequently encountered and is carried less than DNA or the various situations mentioned a small amount of DNA but can not expanded Chu Lai during extraction.Cause
This, DAN's focuses on DNA in how effective, convenient enrichment and extraction olive oil in extraction olive oil.Remained in olive oil
Trace dna illustrate that the gene for representing species specificity may be retained, its hereditary property of different plant species has different
Specificity, can be by examining its specific gene to differentiate species.Therefore the molecular biology method based on DNA analysis is identification
The detection of olive oil doping provides powerful guarantee.
In view of this, the present inventor studies and devises a kind of detection method for identifying olive oil doping, thus this case is produced
It is raw.
The content of the invention
It is an object of the invention to provide a kind of detection method for identifying olive oil doping.
To achieve these goals, the technical scheme adopted by the invention to solve the technical problem is that：
A kind of detection method for identifying olive oil doping, comprises the following steps：
Step 1: design of primers：The gene synthesized for PALM FATTY ACID in NCBI storehouses is designed and screened：
For olive oil, filter out the first primer pair GL1 and the second primer pair GL2, the first primer pair GL1 draw including upstream
Thing GL1F and anti-sense primer GL1R, the second primer pair GL2 include sense primer GL2F and anti-sense primer GL2R, described
The nucleotide base sequence of GL1F, GL1R, GL2F, GL2R are respectively such as sequence table SEQ NO:1 to SEQ NO:Shown in 4；
For target detection thing, the first primer pair EO1 and the second primer pair EO2, the first primer pair EO1 and second is filtered out
Primer pair EO2 is designed according to the gene that PALM FATTY ACID in the NCBI storehouses of realistic objective detectable substance synthesizes and is screened to obtain；
As the internal reference of real-time fluorescence quantitative PCR, using primer pair TY2, the primer pair TY2 include sense primer TY2F and under
The nucleotide base sequence of primer TY2R, the TY2F and the TY2R are swum respectively such as sequence table SEQ NO:5 and SEQ NO:6 institutes
Show；
Step 2: the DNA extractions of olive oil to be detected：Extracted using plasmid extraction kit, obtain DNA oil samples to be detected；
As the preferred embodiment of embodiment, the plasmid extraction kit is using the green skies Bioisystech Co., Ltd production in Shanghai
The plasmid extraction kit D0026 of sale.
Step 3: real-time fluorescence quantitative PCR reacts：Added in the DNA oil samples to be detected primer pair GL1, GL2,
EO1, EO2 and TY2 are expanded, and whether to read Ct values as standard in 40 circulations, judge whether to obtain positive amplification,
Judge whether olive oil adulterates accordingly.
As the preferred embodiment of embodiment, in the step 1, the target detection thing is palm oil, first primer
Including sense primer EO1F and anti-sense primer EO1R, the second primer pair EO2 to EO1 includes sense primer EO2F and EO2R,
The EO1F, the EO1R, the nucleotide base sequence of the EO2F and the EO2R are respectively such as sequence table SEQ NO:7 to SEQ
NO:Shown in 10.
As the preferred embodiment of embodiment, the step 2 concretely comprises the following steps, and takes the test tube of two 50ml, often manages and respectively adds
Enter suspension 6.5ml and lysate 6.5ml, and add detected sample to 50ml, overturn and mix concussion 10min, 10000r/
Min centrifuges 2min, removes oil phase, and olive oil is added in water phase to 50ml, the above-mentioned action of repetition 4 times；The 4th in two test tubes
The combination liquid added in the water phase retained after centrifugation in 13ml kits turns upside down 6 times immediately, 10000r/min centrifugations
20min, then illustrates to carry out, merges the DNA oil samples of two test tubes, DNA oil samples to be detected are made according to kit subsequent operation,
Finally DNA oil samples to be detected are stored in -20 DEG C.
As the preferred embodiment of embodiment, the Amplification of the real-time fluorescence quantitative PCR is as follows：Amplified reaction volume is
20ul, ROX I 0.25ul, 2X Syker Mix 10ul, concentration are the primer 0.4ul, ddH of 10umol/L2O 7.35ul,
DNA profiling 2.0ul；Amplification reaction condition：95 DEG C of 5min, 95 DEG C of 5s, 60 DEG C of 31s, period 40.
As the preferred embodiment of embodiment, the drafting of real-time fluorescence quantitative PCR standard curve is further included：Copied known to using
The olive oil and target detection thing of shellfish number are as standard sample；After amplification, the Ct values of fluorescence associated label in each oil sample are measured,
The Ct values and the natural logrithm of copy number are in a linear relationship, and it is bent to draw SYBR Green I real-time fluorescence quantitative PCRs standard accordingly
Line.
The present invention is used for a series of problems, such as vegetable fat Nucleic Acid is low, destruction is serious and DNA is difficult to extraction
A kind of nucleic acid enriching method and DNA extraction kit method, are effectively extracted available for PCR amplification from olive oil and palm oil
The DNA profiling of reaction, in addition, designing and the primer that can effectively expand DAN templates being filtered out by quantitative fluorescent PCR.This hair
Bright other oil products that incorporation can be effectively identified from olive oil.
Embodiment
The detection of 1 olive oil of embodiment
Material：Olive oil
Reagent：The a large amount of extraction agent box (models of plasmid：) and small amount plasmid extraction agent box (model D0026：D0003), purchase
From the green skies Bioisystech Co., Ltd in Shanghai.
The extraction step of olive oil DNA：The test tube of two 50ml is taken, often manages each addition suspension 6.5ml and lysate
6.5ml, and olive oil is added to 50ml, overturn and mix concussion 10min, 10000r/min centrifugation 2min, oil phase is removed, in water phase
Olive oil is added to 50ml, the above-mentioned action of repetition 4 times.13ml is added in the water phase retained in two test tubes after the 4th centrifugation
Combination liquid in kit turns upside down 6 times immediately, and 10000r/min centrifugation 20min, then say according to kit subsequent operation
Bright progress, merges the DNA oil samples of two test tubes, and DNA oil samples to be detected are made, DNA oil samples to be detected finally are stored in -20 DEG C
In.
Primer GL1, GL2, TY2 are added in DNA extracting solutions（Internal reference）Carry out real-time fluorescence quantitative PCR amplification：
The parameter of real-time fluorescence quantitative PCR amplification is as follows：Amplified reaction volume is 20ul, ROX I 0.25ul, 2X Syker
Mix 10ul, concentration are primer 0.4ul, the ddH2O 7.35ul, DNA profiling 2.0ul of 10umol/L；
Real-time fluorescence quantitative PCR amplification reaction condition：95 DEG C of 5min, 95 DEG C of 5s, 60 DEG C of 31s, period 40, sky is done with water
White control, judges whether to extract DNA and primer specificity from oil sample according to standard curve and Ct values.
As a result：Whether to read Ct values as standard in 40 circulations, judge whether to obtain positive amplification.GL1、GL2、
TY2 obtains 100% positive amplification rate.But Ct values are larger, possible cause is because olive oil nucleic acid damage is serious, is extracted
DNA concentration is relatively low, and the amplification efficiency of primer is different, and comprehensive analysis can show that all samples all extract DNA and primer
With specificity.
The detection of 2 palm oil of embodiment
Material：Palm oil
Reagent：The a large amount of extraction agent box (models of plasmid：) and small amount plasmid extraction agent box (model D0026：D0003), purchase
From the green skies Bioisystech Co., Ltd in Shanghai.
The extraction step of palm oil DNA：The test tube of two 50ml is taken, often manages each addition suspension 6.5ml and lysate
6.5ml, and palm oil is added to 50ml, overturn and mix concussion 10min, 10000r/min centrifugation 2min, oil phase is removed, in water phase
Olive oil is added to 50ml, the above-mentioned action of repetition 4 times.13ml is added in the water phase retained in two test tubes after the 4th centrifugation
Combination liquid in kit turns upside down 6 times immediately, and 10000r/min centrifugation 20min, then say according to kit subsequent operation
DNA oil samples, are finally stored in -20 DEG C by bright progress.
Primer EO1, EO2, TY2 are added in DNA extracting solutions（Internal reference）Carry out real-time fluorescence quantitative PCR amplification；
The parameter of real-time fluorescence quantitative PCR amplification is as follows：Amplified reaction volume is 20ul, ROX I 0.25ul, 2X Syker
Mix 10ul, concentration are primer 0.4ul, the ddH2O 7.35ul, DNA profiling 2.0ul of 10umol/L；
Real-time fluorescence quantitative PCR amplification reaction condition：95 DEG C of 5min, 95 DEG C of 5s, 60 DEG C of 31s, period 40, sky is done with water
White control, judges whether to extract DNA and primer specificity from oil sample according to standard curve and Ct values.
As a result：Whether to read Ct values as standard in 40 circulations, judge whether to obtain positive amplification.EO1、EO2、
TY2 obtains 100% positive amplification rate.But Ct values are larger, possible cause is because olive oil nucleic acid damage is serious, is extracted
DNA concentration is relatively low, and the amplification efficiency of primer is different, and comprehensive analysis can show that all samples all extract DNA and primer
With specificity.
Embodiment 3 is mixed with the olive oil detection of palm oil
Material：It is mixed with the olive oil of palm oil
Reagent：The a large amount of extraction agent box (models of plasmid：) and small amount plasmid extraction agent box (model D0026：D0003), purchase
From the green skies Bioisystech Co., Ltd in Shanghai.
The extraction step of miscella DNA：The test tube of two 50ml is taken, often manages each addition suspension 6.5ml and lysate
6.5ml, and miscella is added to 50ml, overturn and mix concussion 10min, 10000r/min centrifugation 2min, oil phase is removed, in water phase
Olive oil is added to 50ml, the above-mentioned action of repetition 4 times.13ml is added in the water phase retained in two test tubes after the 4th centrifugation
Combination liquid in kit turns upside down 6 times immediately, and 10000r/min centrifugation 20min, then say according to kit subsequent operation
DNA oil samples, are finally stored in -20 DEG C by bright progress.
Primer GL1, GL2, EO1, EO2, TY2 are added in DNA extracting solutions（Internal reference）Carry out real-time fluorescence quantitative PCR expansion
Increase；
The parameter of real-time fluorescence quantitative PCR amplification is as follows：Amplified reaction volume is 20ul, ROX I 0.25ul, 2X Syker
Mix 10ul, concentration are primer 0.4ul, the ddH2O 7.35ul, DNA profiling 2.0ul of 10umol/L；
Real-time fluorescence quantitative PCR amplification reaction condition：95 DEG C of 5min, 95 DEG C of 5s, 60 DEG C of 31s, period 40, sky is done with water
White control, judges whether to extract DNA from oil sample according to standard curve and Ct values and whether amplifies palm fibre in olive miscella
Palmitic acid oil specific gene fragment.
As a result：Whether to read Ct values as standard in 40 circulations, judge whether to obtain positive amplification.EO1、EO2、
GL1, GL2, TY2 obtain 100% positive amplification rate.But Ct values are larger, possible cause be because olive oil nucleic acid damage it is serious,
Extract that DNA concentration is relatively low, the amplification efficiency of primer is different, and comprehensive analysis can show that all samples all extract DNA
And palm oil specific gene is detected in olive miscella.
Although olive oil by the complicated process such as multiple degumming, depickling, decoloration, the present invention be enriched with by DNA and
Extracting method, finds to go back residual minim nucleic acid in the vegetable oil such as olive oil, and the present invention can detect olive with the method
Olive oil doping other plant oil product kind, the detection method of the palm oil of the similar present invention, except that the primer of design is different,
Need to design primer according to specific target detection thing.
The above, is only present pre-ferred embodiments, therefore cannot limit the scope implemented of the present invention according to this, i.e., according to
The equivalent changes and modifications that the scope of the claims of the present invention and description are made, all should still belong in the range of the present invention covers.
Claims (6)
- A kind of 1. detection method for identifying olive oil doping, it is characterised in that：Comprise the following steps：Step 1: design of primers：The gene synthesized for PALM FATTY ACID in NCBI storehouses is designed and screened：For olive oil, filter out the first primer pair GL1 and the second primer pair GL2, the first primer pair GL1 draw including upstream Thing GL1F and anti-sense primer GL1R, the second primer pair GL2 include sense primer GL2F and anti-sense primer GL2R, described The nucleotide base sequence of GL1F, GL1R, GL2F, GL2R are respectively such as sequence table SEQ NO:1 to SEQ NO:Shown in 4；For target detection thing, the first primer pair EO1 and the second primer pair EO2, the first primer pair EO1 and second is filtered out Primer pair EO2 is designed according to the gene that PALM FATTY ACID in the NCBI storehouses of realistic objective detectable substance synthesizes and is screened to obtain；As the internal reference of real-time fluorescence quantitative PCR, using primer pair TY2, the primer pair TY2 include sense primer TY2F and under The nucleotide base sequence of primer TY2R, the TY2F and the TY2R are swum respectively such as sequence table SEQ NO:5 and SEQ NO:6 institutes Show；Step 2: the DNA extractions of olive oil to be detected：Extracted using plasmid extraction kit, obtain DNA oil samples to be detected；Step 3: real-time fluorescence quantitative PCR reacts：Primer pair GL1, GL2, EO1, EO2 are added in the DNA oil samples to be detected And TY2 is expanded, whether to read Ct values as standard in 40 circulations, judge whether to obtain positive amplification, sentence accordingly Whether disconnected olive oil adulterates.
- A kind of 2. detection method for identifying olive oil doping as claimed in claim 1, it is characterised in that：In the step 1, The target detection thing is palm oil, and the first primer pair EO1 includes sense primer EO1F and anti-sense primer EO1R, described the Two primer pair EO2 include sense primer EO2F and EO2R, the EO1F, the EO1R, the nucleosides of the EO2F and the EO2R Base sequence is respectively such as sequence table SEQ NO:7 to SEQ NO:Shown in 10.
- A kind of 3. detection method for identifying olive oil doping as claimed in claim 1, it is characterised in that：The tool of the step 2 Body step is to take the test tube of two 50ml, often manages each addition suspension 6.5ml and lysate 6.5ml, and add detected sample To 50ml, overturn and mix concussion 10min, 10000r/min centrifugation 2min, remove oil phase, olive oil is added in water phase to 50ml, The above-mentioned action of repetition 4 times；Added in two test tubes after the 4th centrifugation in the water phase that retains combination liquid in 13ml kits with Turn upside down 6 times, 10000r/min centrifugation 20min, then illustrate to carry out, merge two test tubes according to kit subsequent operation DNA oil samples, be made DNA oil samples to be detected, finally DNA oil samples to be detected are stored in -20 DEG C.
- A kind of 4. detection method for identifying olive oil doping as claimed in claim 1, it is characterised in that：The matter of the step 2 The plasmid extraction kit D0026 that grain extraction agent box is produced using the green skies Bioisystech Co., Ltd in Shanghai.
- A kind of 5. detection method for identifying olive oil doping as claimed in claim 1, it is characterised in that：The real-time fluorescence is determined The Amplification for measuring PCR is as follows：Amplified reaction volume is 20ul, ROX I 0.25ul, 2X Syker Mix 10ul, and concentration is The primer 0.4ul, ddH of 10umol/L2O 7.35ul, DNA profiling 2.0ul；Amplification reaction condition：95 DEG C of 5min, 95 DEG C 5s, 60 DEG C of 31s, period 40.
- A kind of 6. detection method for identifying olive oil doping as claimed in claim 1, it is characterised in that：Further include real-time fluorescence The drafting of quantitative PCR standard curve：I.e. using the olive oil and target detection thing of known copy number as standard sample；Amplification Afterwards, the Ct values of fluorescence associated label in each oil sample are measured, the Ct values and the natural logrithm of copy number are in a linear relationship, paint accordingly SYBR Green I real-time fluorescence quantitative PCR standard curves processed.
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Cited By (1)
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| CN113667724A (en) * | 2021-07-02 | 2021-11-19 | 集美大学 | Detection method for identifying olive oil doping |
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Application publication date: 20180413 |