CN103642934A - Fluorescent quantitative PCR (Polymerase Chain Reaction) detection method of transgenic soybean edible oil - Google Patents
Fluorescent quantitative PCR (Polymerase Chain Reaction) detection method of transgenic soybean edible oil Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection and in particular relates to a fluorescent quantitative PCR (Polymerase Chain Reaction) detection method of transgenic soybean edible oil. With the adoption of the fluorescent quantitative PCR detection method, aiming to the characteristics of edible grease of low nucleic acid, severe destroy and short DNA (Deoxyribonucleic Acid) sequence fragment, by utilizing a DNA extraction kit method and a nucleic acid enrichment treatment step, a DNA template which can be used for PCR amplification reaction is effectively extracted from the edible soybean oil, primers which can be used for effectively amplifying the DNA template are designed and screened, endogenous genes and exogenous control sequences of soybeans are qualitatively detected by an ordinary fluorescent quantitative PCR technology in real time, and a stable and effective method is provided for detecting whether the edible oil raw material is transgenic. The method is also suitable for the edible oil after high-temperature treatment (cooking).
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of fluorescent quantitative PCR detection method of genetically engineered soybean vegetables oil.
Background technology
Soybean is important edible oil, food protein and feed protein raw material, in the world the demand of soybean is grown with each passing day, people's applying transgene technique carries out directional transformation to the hereditary property of soybean for this reason, makes it to have nutritive ingredient high, output is high, the pest-resistant good character of Denging.Along with the development of genetic engineering technique, the farm crop such as genetically engineered soybean, with its good resistance, high yield, relatively low production cost, have occupied world market very soon, have realized its marketization and commercialization.Yet these genetically modified crops and food thereof are to ecotope and human health safety whether, are the large problem of public's concern all the time.Accordingly, international and domestic priority has been put into effect transgene agricultural product identity management policy, to protect right to know and the preference of consumption.According to the relevant regulations of the Ministry of Agriculture's appearance in 2002, the transgenosis edible oil in China market must clearly identify.Just need to there is the detection method of the transgenosis edible oil matching with it for this reason.And grease is because through squeezing or extracting and separating, wherein seldom, and structure is imperfect for the composition of DNA, is difficult to applicable general DNA detection technology.At present, on market, the transgenic technology of soybean is the strategy that adopts 35S promoter and goal gene simultaneously to proceed to, international method is also to take round pcr as platform, by specific amplified transgenic regulation original paper cauliflower mosaic virus CaMV35S promotor, nopaline synthase NOS terminator and goal gene anti-herbicide gene CP4-EPSPS, screens detection genetically modified food.But edible oil is after the step height refinings such as high temperature, high pressure, extraction, nucleic acid havoc, content is extremely low, extracts very difficultly, and therefore how the DNA in high efficiency extraction refining oil is the bottleneck difficult point that transgenosis detects all the time.Although the existing concerned countries standard of the detection of transgene component is put into effect in grease, Zhou Hongxia etc. find by many experiments, and the method for standard can not extract the DNA in grease completely effectively.They with reference to and improve the method that forefathers introduce, by CTAB method, add nucleic acid enriching treatment step, from the oil sample of 15-20ml, extracted and can be used in the DNA that follow-up PCR detects, improved the validity of DNA extraction, but the electrophoresis detection poor effect of pcr amplification product may be due to the considerably less expanding effect that affects PCR of the nucleic acid amount of extracting.The present invention is on the basis of forefathers' research, with improved CTAB, extract test kit method and add nucleic acid enriching treatment step, extract the DNA that is applicable to fluorescent quantitative PCR in soybean edible oil stability and high efficiency, adopted extraction and the detection that sensitive SYBR Green I Real-Time Fluorescent Quantitative PCR Technique is genetically engineered soybean edible oil DNA that a kind of stable effective means is provided.And adopting actual cooking temp to process edible oil, checking present method is applicable too to the soybean oil of cooking.
Summary of the invention
A kind of method that the object of this invention is to provide genetically engineered soybean edible oil fluorescence quantitative PCR detection, and confirmation detects applicable too for the edible oil after pyroprocessing.
The molecular detecting method of genetically engineered soybean edible oil provided by the invention, with tRNA, 18S, Lec, 35S, NOS primer pair oil sample DNA, carrying out SYBR Green I real-time fluorescence quantitative PCR detects, as long as have a gene fragment to be amplified out in tRNA, 18S, Lectin gene, just can confirm to have extracted edible oil DNA, this method has greatly improved the recall rate of DNA, reduce false negative result, with 35S and NOS primer amplification, be used for judging whether transgenosis of oil sample used.Concrete steps are as follows:
(1) with CTAB plant genome DNA, extract test kit (choosing from Ai Delai bio tech ltd, Beijing) and extract the minim DNA in edible oil.Soon the nucleic acid enriching in edible oil, in CTAB lysate, operates the DNA in purifying lysate by test kit, and DNA is stored in elution buffer EB, leaves 2-8 ℃ in;
The concrete operation step extracting is: for certain soybean edible oil, get the test tube of two 50ml, every pipe adds lysate PL and the 40ml edible oil in 2ml test kit, put upside down and mix, 45 ℃, 250r/min shakes 1h, the centrifugal 5min of 10000r/min, the phase of deoiling, in water, add again oil phase 40ml, repeat aforesaid operations 4 times, the water of the centrifugal rear reservation of the 4th in two test tubes is divided and installed in 2ml EP pipe by every pipe 600ul, every pipe adds 600ul chloroform/primary isoamyl alcohol (24:1), put upside down and fully mix several minutes, the centrifugal 5min of 13000rpm, then by test kit subsequent operations explanation, undertaken.From DNA extraction, positive control and blank are all established in all experiments;
(2) for native gene tRNA, 18S, the Lectin of soybean, design the primer of a series of amplification different lengths fragments, filter out the best primer of a set of expanding effect, obtain tRNA, 18S, tri-pairs of best primers of amplification soybean edible oil DNA effect of Lectin;
Primer is:
tRNA： F：5’- CGAAATCGGTAGACGCTACG-3’ （SEQ.ID.NO1）
R：5’-TTCCATTGAGTCTCTGCACCT-3’ （SEQ.ID.NO2）
18S： F：5’-ATGATAACTCGACGGATCGC-3’ （SEQ.ID.NO3）
R：5’-CTTGGATGTGGTAGCCGTTT-3’ （SEQ.ID.NO4）
Lec： F 5’- ATAAGAATGCGGCCGCGGCTTGCCTTCTTTCTC-3’ （SEQ.ID.NO5）
R 5’-CAATGGAATCCGAGGAGTCCCTTCGATCTGTCACATTT-3’（SEQ.ID.NO6）；
(3) design soybean foreign gene 35S promoter primer, NOS terminator primer, be respectively:
35S： F：5' -CTAACAGAACTCGCCGTAAAGA -3' （SEQ.ID.NO7）
R：5' -AGATAGCTGGGCAATGGAAT -3' （SEQ.ID.NO8）
NOS： F 5’ -GTTCATTTCATTTGGAGAGGGATCGTTCAAACATTTGG-3’ （SEQ.ID.NO9）
R 5’-ACTCTTGTGGTGTTTGTGGCGATCTAGTAACATAGATGA-3’ （SEQ.ID.NO10）；
(4) according to the genomic dna of ordinary method extraction non-transgenic soybean, carry out gradient dilution, then to adding tRNA, 18S, Lec primer to increase in each diluent, (parameter of amplification is as follows: amplified reaction volume is 25 μ l, 2 * Premix Ex Taq, 12.5 μ l, concentration is the forward primer 0.5 μ l of 10 μ mol/L, concentration is the reverse primer 0.5 μ l of 10 μ mol/L, ddH
2o 9.5 μ l, DNA profiling 2.0 μ l, amplification reaction condition: 96 ℃ of 10min denaturations; 96 ℃ of 15s, the annealing temperature 30s of primer, 72 ℃ of 15s, cycle number 36);
(5) after amplification, measure the Ct value of fluorescence associated marker in each diluent, the natural logarithm of this Ct value and copy number is linear, draws accordingly the SYBR Green I real-time fluorescence quantitative PCR typical curve of tRNA, 18S, Lectin gene;
(6) bacterium containing with 35S promoter and NOS terminator sequence plasmid is carried out to gradient dilution, then to adding 35S promoter and NOS terminator primer to carry out pcr amplification in each diluent, (parameter of amplification is as follows: amplified reaction volume is 25 μ l, 2 * Premix Ex Taq, 12.5 μ l, concentration is the forward primer 0.5 μ l of 10 μ mol/L, concentration is the reverse primer 0.5 μ l of 10 μ mol/L, ddH
2o 9.5 μ l, DNA profiling 2.0 μ l, amplification reaction condition: 96 ℃ of 10min denaturations; 96 ℃ of 15s, the annealing temperature 30s of primer, 72 ℃ of 15s, cycle number 36); Measure the Ct value of fluorescence associated marker in each diluent, and draw the SYBR Green I real-time fluorescence quantitative PCR typical curve of 35S promoter and NOS terminator primer;
(7) with CTAB plant genome DNA, extract test kit (choosing from Ai Delai bio tech ltd, Beijing) and extract the minim DNA in soybean edible oil to be detected, then in DNA extraction liquid, add respectively tRNA, 18S, Lec, 35S and NOS primer, increase, each primer repeats 3 times; After amplification, measure the Ct value of fluorescence associated marker in DNA extraction liquid, then according to the quantitative PCR typical curve of each gene of above-mentioned drafting, estimate the content of DNA in extracting solution, thereby judged whether to extract the DNA that can detect for PCR from edible oil, whether the damaed cordition of each native gene and sample are genetically engineered soybean edible oil.
The present invention also simulates actual cooking operation, with above-mentioned extracting method and real-time fluorescence quantitative PCR detection method, explores the impact of heat treated on DNA content in edible oil.It is the applicable equally the inventive method of soybean edible oil after heat treated.For example:
(1) edible oil is processed respectively: 160 ℃ of 1min, 160 ℃ of 3min, 160 ℃ of 5min, microwave oven moderate heat heating 3min, high fire heating 3min in microwave oven, 121 degree autoclavings;
(2) according to aforementioned extracting method, extract the DNA through the edible oil of heat treated, and the DNA extracting is detected; Oily DNA copy number after heat treated and the DNA copy number of heating untreated oil are contrasted, thereby judge the impact of each heat treated on DNA content in oil;
(3) impact on DNA content in oil according to heating, confirms still effective to detection the present invention of the edible oil after pyroprocessing.
advantage and effect that the present invention has are as follows:
The extracting method cost of the edible oil DNA that the present invention is designed is low, simple to operate, and from edible oil, extracted can be for the DNA of fluorescence quantitative PCR detection stability and high efficiency.Extremely low for edible oil Nucleic Acid, destroy serious, the short feature of DNA sequencing fragment, designed a series of primers of the soybean edible oil DNA that is applicable to increase, obtained good expanding effect, greatly improved the recall rate of the edible oil DNA extracting.Adopt SYBR Green I Real-Time Fluorescent Quantitative PCR Technique, make to detect sensitive, accurately, simple, reliable.Simulate actual cooking operation, the impact of checking heat treated on DNA content in edible oil, verifies that the oil sample after method of the present invention is for pyroprocessing is also applicable.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The extraction of embodiment 1 soybean edible oil DNA
Material: soybean edible oil
Northeast soybean (doing negative control)
Reagent: CTAB plant genome DNA rapid extraction test kit is chosen from Ai Delai bio tech ltd, Beijing;
Step: for every kind of edible oil, get the test tube of two 50ml, every pipe adds lysate PL and the 40ml edible oil in 2ml test kit, put upside down and mix, 45 ℃, 250r/min shakes 1h, the centrifugal 5min of 10000r/min, the phase of deoiling, in water, add again oil phase 40ml, repeat aforesaid operations 4 times, the water of the centrifugal rear reservation of the 4th in two test tubes is divided and installed in 2ml EP pipe by every pipe 600ul, every pipe adds 600ul chloroform/primary isoamyl alcohol (24:1), put upside down and fully mix several minutes, the centrifugal 5min of 13000rpm, then by test kit subsequent operations explanation, undertaken.Soon the nucleic acid enriching in 320ml edible oil, in the CTAB lysate of 4ml, operates the DNA in purifying lysate by test kit, finally DNA is stored in 100ul elution buffer EB, leaves 2-8 ℃ in.
With CTAB plant genome DNA rapid extraction test kit, according to operation instructions, extract the genomic dna of northeast soybean, as negative control.
The SYBR Green I real-time fluorescence quantitative PCR detection method of embodiment 2 tRNA, 18S, Lectin, 35S, NOS gene.
Oil sample used: purchased from 30 parts of the soybean edible oils of supermarket and network shopping mall.
By 10 times, 100 times, 1000 times of the Soybean genomic DNA dilutions of having extracted, use respectively tRNA, 18S, the above-mentioned concentration gradient of Lec primer amplification, each concentration is established 3 repetitions, obtains the typical curve of each gene amplification.
The bacterium containing with 35S promoter sequence plasmid is diluted to 10 times, 100 times, 1000 times, use respectively 35S, the above-mentioned concentration gradient of NOS primer amplification, each concentration is established 3 repetitions, obtains the typical curve of 35S, the amplification of NOS promotor.
Amplified reaction volume is 25 μ l, 2 * Premix Ex Taq, 12.5 μ l, and concentration is the forward primer 0.5 μ l of 10 μ mol/L, concentration is the reverse primer 0.5 μ l of 10 μ mol/L, ddH
2o 9.5 μ l, DNA profiling 2.0 μ l, amplification reaction condition: 96 ℃ of 10min denaturations; 96 ℃ of 15s, the annealing temperature 30s of primer, 72 ℃ of 15s, cycle number 36.
30 oil sample DNA that extract with tRNA, 18S, Lectin, 35S, the amplification of NOS gene primer respectively, with water, make blank, according to typical curve, judge whether to go out above gene fragment from oil sample DNA cloning, thereby determine whether to have extracted oil sample DNA and differentiate that whether oil sample is containing transgene component.
Result: take whether to read in 36 circulations and obtain CT and refer to as standard, judge whether to obtain positive amplification.For tRNA, obtaining positive amplification rate is 60%; For 18S, obtaining positive amplification rate is 100%; For Lectin, obtaining positive amplification rate is 46.7%.In sum, can judge that the DNA of all oil samples is extracted, in the DNA extraction liquid of all samples, all contain 18S gene fragment.And other genes have part sample there is no positive amplification, may be because this gene disruption is more serious, content is very low, and the amplification efficiency of primer is different, although or the CT threshold value that has amplification to set lower than experiment.But comprehensive analysis can affirm that all samples have all extracted DNA.
For 35S promoter and NOS terminator, obtained same result.The positive amplification rate that is designated genetically modified sample is 100%, and the positive amplification rate that is designated not genetically modified sample is 86.7%, and the positive amplification rate of the sample of sign is not 100%, and the positive amplification rate of northeast soybean negative control is 0%.
Most soybean edible oils are on the market described, comprise being clearly designated not genetically modifiedly, be in fact genetically engineered soybean edible oil or be mixed with genetically engineered soybean composition.
Embodiment 3: the impact of heat treated on DNA content in edible oil
Soybean oil is carried out respectively to following processing: 160 ℃ of 1min, 160 ℃ of 3min, 160 ℃ of 5min, microwave oven moderate heat heating 3min, high fire heating 3min in microwave oven, autoclaving, untreated control.Oil sample DNA after extraction process, respectively with tRNA, 18S, Lectin, 35S, NOS gene primer above-mentioned 7 oil samples that increase, with SYBR Green I real-time fluorescence quantitative PCR, detect, and according to after typical curve contrast treatment and the variation of undressed oil sample DNA content, and the impact of different heating degree on DNA content in oil.
Result: for each gene, the gene fragment concentration that untreated control extracts is not the highest, after processing, the concentration of gene is low unlike what do not process, the difference that sample exists may be just because extraction efficiency difference causes, but it is little on the impact of gene stability and concentration also can to reflect above processing.Same temperature, treatment time difference also has no significant effect gene stability and concentration; Same time treatment of different temperature, has no significant effect gene stability and concentration; Short period of time inner high voltage is processed also having no significant effect the concentration of gene fragment.As can be seen here, under general condition, the gene order in oil can stable existence, preserves for a long time, makes the gene discrimination method of transgenic plant oil more reliable.And under general culinary art condition (temperature is not generally higher than 160 degree, and the time is no longer than 5min), the gene fragment in vegetables oil still can exist, whether applicable method of the present invention detects transgenosis.
<110> Fudan University
The method of a <120> genetically engineered soybean edible oil fluorescence quantitative PCR detection
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Claims (3)
1. a method for genetically engineered soybean edible oil fluorescence quantitative PCR detection, is characterized in that concrete steps are:
(1) with CTAB plant genome DNA rapid extraction test kit, extract the DNA in soybean edible oil;
(2) design and screen and obtain increasing the pair of primers of tRNA gene, the pair of primers of amplification 18S gene, the pair of primers of amplification Lectin gene, is respectively:
tRNA： SEQ.ID.NO1
SEQ.ID.NO2
18S： SEQ.ID.NO3
SEQ.ID.NO4
Lec： SEQ.ID.NO5
SEQ.ID.NO6
(3) design soybean foreign gene 35S promoter primer, NOS terminator primer, be respectively:
35S： SEQ.ID.NO7
SEQ.ID.NO8
NOS：SEQ.ID.NO9
SEQ.ID.NO10
(4) extract the genomic dna of non-transgenic northeast soybean, carry out gradient dilution, then in each diluent, add tRNA, 18S, Lec primer to increase;
(5) after amplification, measure the Ct value of fluorescence associated marker in each diluent, the natural logarithm of this Ct value and copy number is linear, draws accordingly the SYBR Green I real-time fluorescence quantitative PCR typical curve of tRNA, 18S, Lectin gene;
(6) bacterium containing with 35S promoter and NOS terminator sequence plasmid is carried out to gradient dilution, then in each diluent, add 35S promoter and NOS terminator primer to increase, measure the Ct value of fluorescence associated marker in each diluent, and draw the SYBR Green I real-time fluorescence quantitative PCR typical curve of 35S promoter and NOS terminator primer;
(7) in DNA extraction liquid, add respectively tRNA, 18S, Lec, 35S and NOS primer, increase, each primer repeats 3 times; After amplification, measure the Ct value of fluorescence associated marker in DNA extraction liquid, then according to the quantitative PCR typical curve of each gene of above-mentioned drafting, estimate the content of DNA in extracting solution, thereby judged whether to extract the DNA that can detect for PCR from edible oil, whether the damaed cordition of each native gene and sample are transgenosis edible oil.
2. the method for genetically engineered soybean edible oil fluorescence quantitative PCR detection according to claim 1, is characterized in that, described soybean edible oil is through heat treated.
3. the method for genetically engineered soybean edible oil fluorescence quantitative PCR detection according to claim 1, it is characterized in that, describedly by the step that CTAB plant genome DNA rapid extraction test kit extracts DNA in soybean edible oil, be: by the nucleic acid enriching in edible oil in CTAB lysate, by test kit, operate the DNA in purifying lysate, and DNA is stored in elution buffer EB, leave 2-8 ℃ in.
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Cited By (3)
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| CN107904288A (en) * | 2018-01-08 | 2018-04-13 | 集美大学 | A kind of detection method for identifying olive oil doping |
| CN110656107A (en) * | 2019-10-24 | 2020-01-07 | 九三集团哈尔滨惠康食品有限公司 | Method for extracting transgenic components in deep-processed vegetable oil |
| CN110904265A (en) * | 2019-12-25 | 2020-03-24 | 石盼盼 | Primer, probe, kit and method for real-time fluorescence PCR (polymerase chain reaction) detection of transgenic soybean |
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| CN102277426A (en) * | 2011-07-25 | 2011-12-14 | 南京农业大学 | Detection kit of transgenic component in soybean edible oil and its deeply-processed product and detection method thereof |
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| CN102277426A (en) * | 2011-07-25 | 2011-12-14 | 南京农业大学 | Detection kit of transgenic component in soybean edible oil and its deeply-processed product and detection method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904288A (en) * | 2018-01-08 | 2018-04-13 | 集美大学 | A kind of detection method for identifying olive oil doping |
| CN110656107A (en) * | 2019-10-24 | 2020-01-07 | 九三集团哈尔滨惠康食品有限公司 | Method for extracting transgenic components in deep-processed vegetable oil |
| CN110904265A (en) * | 2019-12-25 | 2020-03-24 | 石盼盼 | Primer, probe, kit and method for real-time fluorescence PCR (polymerase chain reaction) detection of transgenic soybean |
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