CN1438328A - Quantitative detection method for transgenic farm product and food mixture - Google Patents
Quantitative detection method for transgenic farm product and food mixture Download PDFInfo
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Abstract
The invention refers to the ration checking method for transferring gene farm produce and food compound. The method includes folloiwng steps: the extraction of gene group DNA from transferring gene farm produce and food compound; checks the design of sign gene, special gene added ingredient and probe; ascertains the standard curve of ration checking sample; real-time fluorescence ratio polymerase chain reaction (PCR) checking, ration analysis.
Description
Technical field:
What the present invention relates to is the quantitative detection method of transgene agricultural product and food mixture.
Background technology
Fast development along with biotechnology, by foreign gene being imported the natural crops gene, realization is transformed its original gene, with expectation obtain having additional nutrients component content, improve improvement such as resistance and optimize the farm crop product innovation of proterties, and and then improve quality and the function of its processed food aspect corresponding, carrying out extensive studies and application at present.Meanwhile, the plant that this class is obtained by transgenic technology and the safety in utilization problem of its converted products have also caused people's attention day by day.Therefore carry out fluorescence quantitative PCR detection by genetic material to plant genetic material or its processed food, also promptly transgenic plant or food are carried out the quantitative PCR detection of transgenosis rate and the problem of analysis, being an urgent demand of current social development, also is the inexorable trend of biotech development.
The detection method that has occurred some relevant transgenic plant or food in the market, Sichuan a company develops and is used for transgenic plant genes identified detection chip, determine detected DNA sample by known oligonucleotide sequence, come test sample by making nucleic acid molecular hybridization, the discriminatory analysis of evaluation can only be made to sample, quantitative analysis can not be accomplished; Dalian TAKARA company also releases the qualitative PCR detection kit of transgenic product, needs by agarose gel electrophoresis, and whether be transgenic product, can not carry out quantitative analysis if also can only identify plant.
Summary of the invention
The quantitative detection method that the purpose of this invention is to provide transgene agricultural product and food mixture.
The quantitative detection method of transgene agricultural product of the present invention and food mixture comprised for five steps: the extraction of transgene agricultural product and food mixture genomic dna; The design of certification mark gene and house-keeping gene primer and probe; Determine the typical curve of detection by quantitative sample; Real time fluorescent quantitative poly chain reaction (PCR) detects; Quantitative analysis.
Concrete steps are as follows:
The extraction of the first step, transgene agricultural product and food mixture genomic dna
For liquid testing sample, adopt the freeze-drying drying and be milled to Powdered; For non-pulverous solid testing sample, adopt milled processed powdered, extract the genomic dna of sample then;
Second step, at transgenosis certification mark gene and house-keeping gene design primer and probe
1) obtain the house-keeping gene of transgenosis certification mark gene and plant or agricultural-food from ncbi genbank database, wherein the certification mark gene mainly comprises CAMV 35S promoter, NOS terminator, Km
r(anti-kanamycin gene), Neo
r(anti-neomycin gene), Kyg
r(the mould plain gene of moisture resistance), Bar
r(anti-careless fourth phosphino-because of), NPT-II (neomycin phosphotransferase gene), manually insert the special foreign gene of Plant Genome change agricultural-food and Food Quality and function;
2) at the conservative region design primer and the Taqman probe of the house-keeping gene that obtains transgenosis certification mark gene and plant or agricultural-food from ncbi genbank database, make the primer of design satisfy following condition with probe: primer and probe that the report fluorescence of (1) marker gene and house-keeping gene mark has different emission wavelengths (2) marker gene and house-keeping gene can react in same pipe;
3) primer and the probe of synthetic above-mentioned design on the dna synthesizer device;
The 3rd goes on foot, determines the standard substance of detection by quantitative sample
The standard substance of the transgenosis detection by quantitative that the selected Chinese government is admitted perhaps obtain standard substance according to following steps:
1) primer of standard product, the fragment that this primer amplification is come out comprise the marker gene or the house-keeping gene fragment of detection by quantitative, and length is between 100bp～400bp;
2) with standard substance primer 100～400nm Forward Primer of above-mentioned design, 100～400nmReverse Primer and 1 * PCR Buffer, 1.5～6mM MgCl2,100～400 μ MDntp (dATP, dCTP, dGTP, dTTP), 1～100ng contains the genomic dna of transgenosis certification mark gene or house-keeping gene, and 0.5U～5U AmpliTaq DNA polymerase places the pcr reaction tubes, mends to 50 microlitres with redistilled water, carry out the polymerase chain reaction, polymerase chain reaction (PCR) reaction conditions: 94 ℃ of pre-sex change 2 minutes, react 25～35 circulations again, each circulation comprises: 94 ℃ of sex change 30 seconds～1 minute, annealed 30 seconds～1 minute and 30 seconds for 45 ℃～67 ℃, 72 ℃ were extended 30 seconds～1 minute and 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes;
3) above-mentioned PCR product is carried out gel electrophoresis, the rubber tapping of purpose fragment is received together, the fragment that reclaims is connected in the plasmid vector, then with this recombinant plasmid transformed in intestinal bacteria, allow recombinant plasmid great expression in intestinal bacteria, again recombinant plasmid dna is extracted and purifying, obtain a large amount of recombinant plasmids;
4) utilize on the plasmid vector restriction endonuclease sites that recombinant plasmid is carried out enzyme and cut, utilize the order-checking of dna sequencing instrument to identify again to cut and identify and contain the segmental recombinant plasmid of purpose, determine to contain right-on purpose fragment through the plasmid after the reorganization through enzyme;
5) calculating contains the segmental recombinant plasmid concentration of entirely true purpose, the concentration that will contain marker gene and contain the house-keeping gene recombinant plasmid is mixed according to 0～100% ratio, gets wherein at least four standard substance that ratio detects as the real-time fluorescence quantitative PCR transgenosis;
The 4th step, real time fluorescent quantitative poly chain reaction (PCR) detect
With 1 * PCR Buffer, 1.5～6mM MgCl2,100～400 μ M dNTP (dATP, dCTP, dGTP), 400 μ M dUTP, 100～900nm, second step gained marker gene Forward Primer and ReversePrimer, 50～300nM, second step gained marker gene Taqman probe, 100～900nm, second step gained house-keeping gene Forward Primer and Reverse Primer, 50～300nM, second step gained house-keeping gene Taqman probe, 10～100ng sample gene group DNA, 0.01～0.05U Amp Erase UNG enzyme, 0.5～5U AmpliTaqGod DNA polymerase, place in the same pcr reaction tubes, mend to 50 microlitres, utilize hyperchannel real-time fluorescence quantitative PCR instrument that the marker gene and the house-keeping gene of sample and standard substance are carried out real time fluorescent quantitative poly chain reaction (PCR) and detection with redistilled water;
Polymerase chain reaction (PCR) reaction conditions: 37 ℃ were reacted 5～15 minutes, 95 ℃ of pre-sex change 5～15 minutes, react 25～45 circulations again, each circulation comprises: 94 ℃ of sex change 30 seconds～1 minute, annealed 30 seconds～1 minute and 30 seconds for 45 ℃～67 ℃, 72 ℃ were extended 30 seconds～1 minute and 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes;
The 5th step, quantitative calculation and analysis
1) the quantitative and typical curve of standard substance determines
From real-time fluorescence quantitative PCR instrument detection software, obtain the certification mark gene of standard substance and the Ct numerical value of house-keeping gene, with the ratio of certification mark gene and house-keeping gene in the standard substance as X-coordinate, the absolute value of the Ct value difference value of certification mark gene and house-keeping gene is an ordinate zou in the standard substance, with in the 3rd step 5) absolute value of the certification mark gene of the marker gene recombinant plasmid of the standard substance got and four ratios of house-keeping gene recombinant plasmid concentration and each ratio correspondence and the Ct value difference value of house-keeping gene makes straight line, utilize the linear regression statistical study that this fitting of a straight line is become y=-Klog (x)+B, in the formula-K is this collinear slope, B is a constant, with this straight line as transgenosis examination criteria curve, y is the absolute value of certification mark gene and house-keeping gene Ct value difference value in the formula, and x is the ratio of certification mark gene and house-keeping gene;
2) calculating of sample and quantitative
Detect the Ct value that obtains the certification mark gene and the house-keeping gene of sample the software from the real-time fluorescence quantitative PCR instrument, get the absolute value of the Ct value difference value of the certification mark gene of same sample and house-keeping gene, typical curve formula y=-Klog (the x)+B that detects to transgenosis as y numerical value generation, the x value that obtains is exactly the ratio of certification mark gene and house-keeping gene, just the transgenosis rate of this transgene agricultural product or food.
The extraction of sample gene group DNA can be adopted CTAB extracting genome DNA method in the above-mentioned steps the first step, or the test kit extraction method, or the magnetic bead extraction method.
Designed Taqman probe can be conventional TaqMan fluorescent probe or TaqManMGB probe during above-mentioned steps second went on foot, preferably select the TaqManMGB probe for use, since the TaqManMGB probe be on the basis of oligonucleotide probe (ODN) in original design its 5 ' or 3 ' connection on a group---dihydro ring annulated indole porphyrin-tripeptides (DPI3), no matter be 3 '-MGB oligonucleotide probe or 5 '-the MGB oligonucleotide probe, all shown stronger sequence-specific than the oligonucleotide probe that does not have MGB, compare with conventional TaqMan fluorescent probe, MGB can significantly increase the stability of heteroduplex, MGB probe background fluorescence is low, because the distance of MGB probes report group and quenching group is short and structurally have the factor of other more effective cancellation fluorescence, so background value is low; The MGB probe has good susceptibility and quantitative effect, does not have the problem with the DNA nonspecific action of high complexity, guarantees the accuracy and the susceptibility of experimental result.
Said carrier can be the commercially available carrier that does not have homologous sequence with marker gene and house-keeping gene the transgenosis detection among the present invention.
Said Ct value is meant in the real-time fluorescence quantitative PCR amplification procedure among the present invention, and fluorescent signal begins to be entered by background the pairing cycle index of flex point of exponential growth phase.
The making of typical curve and sample quantitative Analysis can utilize computer software to realize among the present invention.
The marker gene recombinant plasmid of the standard substance of being got the 3rd step 5 of the present invention) and four ratios of house-keeping gene recombinant plasmid concentration generally are respectively 0.1%, 0.5%, 1%, 2%.
Advantage of the present invention:
Method of the present invention uses the real-time fluorescence quantitative PCR instrument can monitor in real time the process that detects, adopt the MGB probe, it is more accurate to guarantee to detect data, because the detection data are that the making of typical curve and the computer software that quantitatively all adopts of sample are finished automatically, therefore can guarantee the unalterable feature and the exactness of data simultaneously.
Description of drawings
Fig. 1 is a plasmid enzyme restriction site collection of illustrative plates;
Fig. 2 is the typical curve that transgenosis detects;
Embodiment
The invention will be further described below by specific embodiment:
Embodiment 1: the corn (suppose contain antiweed BAR gene) of the food that is detected for buying from market, and concrete working method is as follows:
The extraction of the first step, corn gene group
Earlier corn is clayed into power with abrasive method, adopt TAKARA company corn gene group to extract kit method then and extract corn gene group DNA, the concrete operations step has detailed explanation in the specification sheets of test kit.
Second step, design certification mark gene and house-keeping gene primer and probe
From the house-keeping gene invertase gene order U16123 of ncbi genbank acquisition antiweed BAR gene order X17220 and corn, design primer and MGB probe mark are as follows on the house-keeping gene invertase of detection by quantitative marker gene antiweed BAR gene and corn gene:
Design antiweed BAR gene primer and probe, below product that primer increased be 86bp～149bp in the X17220 scope
BAR-FOR：5’-CGGCGGTCTGCACCAT-3’
BAR-REV：5’-GGCTCGGTACGGAAGTTGAC-3’
BAR-PROBE：5’-FAM--TCAACCACTACATCGAGAC-MGB-3’
Design house-keeping gene invertase gene primer probe, below the scope of the product that primer increased be 2303bp～2364bp
INVERTASE-FOR：5’-CGTGCACCACGTGAGAATTT-3’
INVERTASE-REV：5’-TCCTCTCGTTTTCCCGTCTAAC-3’
INVERTASE-PROBE：5’-VIC-CGTCTACTCGAGCCTAG-MGB-3
On the dna synthesizer device, synthesize antiweed BAR gene and house-keeping gene invertase gene primer and probe then.
The 3rd goes on foot, determines the standard substance of detection by quantitative sample
1. determine the standard substance of antiweed bar gene:
1) design antiweed BAR gene (gene order X17220) primer, below product that primer increased be the fragment of 28bp～380bp in the nucleotide sequence of antiweed BAR gene order X17220, comprised detection by quantitative and got the product that primer obtains.
BAR-FOR：5’-ACCATGAGCCCAGAACGAC-3’
BAR-REV：5’-ACTTCAGGGACCTCCGTGT-3’
2) reaction system: 50 microlitre reaction systems
With 300nm Bar Forward Primer and 300nm Bar Reverse Primer, 1 * PCRBuffer, 1.5mM MgCl2,200 μ M dNTP (dATP, dCTP, dGTP, dTTP), 10ng contains the genomic dna of bar gene, 2U AmpliTaq DNA polymerase, place the pcr reaction tubes, mend to 50 microlitres with redistilled water, carry out the polymerase chain reaction, the polymerase chain reaction condition: 94 ℃ of pre-sex change 2 minutes, react 30 circulations, each circulation comprises: 94 ℃ of sex change 30 seconds, and 58 ℃ of annealing 30 seconds, 72 ℃ were extended 30 seconds, after the loop ends, proceed 72 ℃ of extensions 5 minutes
3) the standard substance product of the bar that above-mentioned PCR is obtained carries out electrophoresis on 1% sepharose (ethidium bromide that has added 0.5ng/ul), if PCR success, contrast DNA ladder can see that with bar purpose clip size be 352bp under ultraviolet lamp, with QIAGEN company glue purification test kit purifying, concrete operations are carried out according to the specification sheets of test kit, the purpose fragment that reclaims being got trace, to carry out electrophoresis quantitative again, the intensity of middle per sample ethidium bromide emitted fluorescence is estimated the content of nucleic acid, PGEM-T Easy VECTOR system I test kit with promega company is connected to the purpose fragment that reclaims on the PGEM-T Easy, connect product and be transformed in the intestinal bacteria DH-5 α bacterium, concrete steps are as follows:
A), after 45 minutes competence is placed on ice according to the PGEM-T Easy VECTOR system I test kit ligation of promega company.(the competence preparation has concrete method on " molecular cloning experiment guide ")
B) respectively add 200 microlitre LB (no penbritin) with 1.5 milliliters of centrifuge tubes and put into 0 ℃ of freeze thawing
C) product with above-mentioned ligation all changes in the competence, and mixing is placed on 0 ℃ and left standstill 15 minutes gently, builds the lid of ep pipe with ice bag, and the temperature of Water Tank with Temp.-controlled ℃ is adjusted to 42 ℃.
D) competence is put into 42 ℃ of tanks 45 seconds (time is accurate), be placed at once 0 ℃ 5 minutes.
E) above-mentioned competence is joined among the 200 microlitre LB (no penbritin) of previous preparation, in 37 ℃, placed 30 minutes.
F) 50mg/mlX-GAL of adding 100mMIPTG and 20ul in above-mentioned EP pipe, rifle head mixing is put upside down mixing, changes over to contain on the 50ug/ml penbritin LB flat board evenly to smear overnight incubation in 37 ℃ of incubators.
G) on the LB of overnight incubation flat board, choose a white mono-clonal and select several mono-clonals at the LB of 7ML50ug/ml penbritin substratum and cultivate, extract and plasmid purification.
4) the restriction enzyme site collection of illustrative plates of plasmid vector is seen accompanying drawing 1, utilize the ECORI enzyme to cut (promega company) the above-mentioned recombinant plasmid that obtains, the product that enzyme is cut carries out electrophoresis on 1% sepharose (ethidium bromide that has added 0.5ng/ul), contrast DNA ladder under ultraviolet lamp, can judge the size of recombinant plasmid, the picking enzyme cuts that the standard substance of bar have the mono-clonal of 352bp band to carry out the dna sequencing analysis in the product, utilize the order-checking of dna sequencing instrument to identify whether the 28bp～380bp that inserts fragment sequence and X17220 in this recombinant plasmid is identical, if identical, then this recombinant plasmid can be used as bar gene standard substance.Two bacterium liquid of the bar gene standard substance that obtain are cultivated in the LB of 50ug/ml penbritin substratum in a large number, extracted plasmid and purifying.The recombinant plasmid of the standard substance of bar gene is calculated recombinant plasmid concentration (concrete seeing " molecular cloning experiment guide " go up DNA and RNA quantitatively) according to following method of calculation, AMERSHAM3000 spectrophotometry base absorbs the amount of uv-radiation, the recombinant plasmid of bar standard substance, dilute 100 times, OD260=0.225, OD280=0.126, the OD260/OD280 value is 1.78, equal the concentration of recombinant plasmid according to OD260 * extension rate * 50, can know the recombinant plasmid 1.125mg/ml of bar standard substance, again according to the molecular weight of recombinant plasmid (molecular weight 3015 * 324.5g/moL of PGEM-Teasv add insert segmental molecular weight 352 * 324.5g/moL add insert segmental molecular weight) contain 6.02 * 10 in 3367 * 324.5g/mol and 1 mole of sample
23Individual copy number, the copy number that obtains 1 microlitre bar recombinant plasmid equals 6.2 * 10
11Individual.
2. determine the standard substance of corn house-keeping gene INVERTASE:
1) design corn house-keeping gene INVERTASE (gene order U16123) primer, the product that following primer increased is the fragment of 2147bp～2420bp in the nucleotide sequence of corn house-keeping gene INVERTASE gene order U16123, has comprised detection by quantitative and has got the product that primer obtains.
INVERTASE-FOR：5’-TACCACCGTCCAAACTGAA-3’
INVERTASE-REV：5’-ACTGAACAGAGGCCAACCG-3’
2) reaction system: 50 microlitre reaction systems, with 300nm INVERTASE Forward Primer and 300nm INVERTASEReverse Primer, 1 * PCR Buffer, 1.5mM MgCl2,200 μ MdNTP (dATP, dCTP, dGTP, dTTP), 10ng contains the genomic dna of corn house-keeping gene INVERTASE, 2U AmpliTaq DNA polymerase places the pcr reaction tubes, mends to 50 microlitres with redistilled water, carry out the polymerase chain reaction, the polymerase chain reaction condition: 94 ℃ of pre-sex change 2 minutes, react 30 circulations, each circulation comprises: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 56 ℃, 72 ℃ were extended 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes
3) the standard substance product of the invertase that above-mentioned PCR is obtained carries out electrophoresis on 1% sepharose (ethidium bromide that has added 0.5ng/ul), if PCR success, contrast DNAladder can see that invertase purpose fragment is 273bp under ultraviolet lamp, with QIAGEN company glue purification test kit purifying, concrete operations are carried out according to the specification sheets of test kit, to carry out electrophoresis quantitative the purpose fragment that reclaims being got trace, the intensity of middle per sample ethidium bromide emitted fluorescence is estimated the content of nucleic acid, PGEM-T Easy VECTOR system I test kit with promega company is connected to the purpose fragment that reclaims on the PGEM-TEasy, connect product and be transformed in the intestinal bacteria DH-5 α bacterium, concrete steps are as follows:
A) when after 45 minutes, competence being placed on ice according to the PGEM-T Easy VECTOR system I test kit ligation of promega company.(the competence preparation has concrete method on " molecular cloning experiment guide ")
B) respectively add 200 microlitre LB (no penbritin) with 1.5 milliliters of centrifuge tubes and put into 0 ℃ of freeze thawing
C) product with above-mentioned ligation all changes in the competence, and mixing is placed on 0 ℃ and left standstill 15 minutes gently, builds the lid of ep pipe with ice bag, and the temperature of Water Tank with Temp.-controlled ℃ is adjusted to 42 ℃.
D) competence is put into 42 ℃ of tanks 45 seconds (time is accurate), be placed at once 0 ℃ 5 minutes.
E) above-mentioned competence is joined among the 200 microlitre LB (no penbritin) of previous preparation, in 37 ℃, placed 30 minutes
F) 50mg/mlX-GAL of adding 100mMIPTG and 20ul in above-mentioned EP pipe, rifle head mixing is put upside down mixing, changes over to contain on the 50ug/ml penbritin LB flat board evenly to smear overnight incubation in 37 ℃ of incubators.
G) on the LB of overnight incubation flat board, choose a white mono-clonal and select several mono-clonals at the LB of 7ml50ug/ml penbritin substratum and cultivate, extract and plasmid purification.
4) utilize the ECORI enzyme to cut (promega company) the above-mentioned recombinant plasmid that obtains, the product that enzyme is cut carries out electrophoresis on 1% sepharose (ethidium bromide that has added 0.5ng/ul), under ultraviolet lamp, contrast DNAladder, can judge the size of recombinant plasmid, the picking enzyme cuts that the standard substance of invertase have the mono-clonal of 273bp band to carry out the dna sequencing analysis in the product, if it is identical to insert 2147bp～2420bp of fragment sequence and U16123 in the recombinant plasmid of invertase, then this recombinant plasmid can be used as invertase gene standard substance.Two bacterium liquid of the invertase gene standard substance that obtain are cultivated in the LB of 50ug/ml penbritin substratum in a large number, extracted plasmid and purifying.The recombinant plasmid of the standard substance of invertase gene is calculated recombinant plasmid concentration (concrete seeing " molecular cloning experiment guide " go up DNA and RNA quantitatively) according to following method of calculation, utilize AMERSHAM3000 spectrophotometry base to absorb the amount of uv-radiation, the recombinant plasmid of invertase standard substance, dilute 100 times, OD260=0.218, OD280=0.119, the OD260/OD280 value is 1.83, equal the concentration of recombinant plasmid according to OD260 * extension rate * 50, can know the recombinant plasmid 1.09mg/ml of invertase standard substance, again according to the molecular weight of recombinant plasmid (molecular weight 3015 * 324.5g/moL of PGEM Teasy add insert segmental molecular weight 273 * 324.5g/moL add insert segmental molecular weight) contain 6.02 * 10 in 3288 * 324.5g/mol and 1 mole of sample
23Individual copy number, the copy number that obtains 1 microlitre invertase recombinant plasmid equals 6.13 * 10
11Individual.
With bar marker gene and the resulting plasmid original copy number of invertase house-keeping gene according to 50%, 25%, 15%, 6.25% ratio mixes the standard substance as real-time fluorescence quantitative PCR transgenosis sample detection.
The 4th step, real time fluorescent quantitative poly chain reaction (PCR) detect
With 1 * PCR Buffer, 4mM MgCl2,200 μ M dNTP (dATP, dCTP, dGTP,), 400 μ M dUTP, 900nm second step gained Bar Forward Primer and Bar Reverse Primer, 250nM Bar-probe probe, 900nm second step gained Invertase Forward Primer and Invertase Reverse Primer, 250nM Invertase-probe probe, the corn gene group sample DNA of buying from market of 100ng said extracted, 2U AmpliTaqGod DNA polymerase places same pcr reaction tubes, carries out the polymerase chain reaction with redistilled water benefit to 50 microlitres, the polymerase chain reaction condition: 37 ℃ were reacted 10 minutes, 40 circulations are reacted in 95 ℃ of pre-sex change 10 minutes again, and each circulation comprises: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 60 ℃, 72 ℃ were extended 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes
The 5th step, quantitative calculation and analysis
Standard substance quantitatively and typical curve really normal root according to the data adjustment of testing in this example, play between the 15th circulation from the 3rd circulation and to determine the baseline scope, pairing PCR cycle index when above baseline, producing on can detected statistics significant fluorescent emission, in real-time fluorescence quantitative PCR instrument detection software, obtain the certification mark gene Bar of standard substance and the Ct numerical value of house-keeping gene invertase, with the ratio of certification mark gene Bar in the standard substance and house-keeping gene invertase as X-coordinate, the absolute value of the Ct value difference value of the invertase of certification mark gene Bar and house-keeping gene is an ordinate zou in the standard substance; get marker gene Bar and house-keeping gene invertase ratio 50%; 25%; 15%; 6.25% and the absolute value of the Ct value difference value of the certification mark gene Bar of each ratio correspondence and house-keeping gene invertase make straight line and see Fig. 2; utilize the linear regression statistical study that this fitting of a straight line is become y=-3.32log (x)+6.64; relation conefficient=1; with this straight line as transgenosis examination criteria curve; y is the absolute value of certification mark gene Bar (FAM) and house-keeping gene invertase (VIC) Ct value difference value in the formula; x is the ratio of certification mark gene Bar (FAM) and house-keeping gene invertase (VIC), just the transgenosis rate of this corn.
2. the calculating of sample and quantitative
From real-time fluorescence quantitative PCR instrument detection software, obtain the certification mark gene Bar of sample and the Ct numerical value of house-keeping gene invertase, the typical curve formula y=-3.32log (x)+6.64 that absolute value generation of the difference of Ct numerical value is detected to transgenosis, obtain containing the ratio 25% of certification mark antiweed BAR gene and house-keeping gene invertase, just the transgenosis rate 25% of this corn.
Embodiment 2: the soybean of the food that is detected for buying from market, and concrete working method is as follows:
The extraction of the first step, soybean gene group
Earlier soybean is clayed into power with abrasive method, adopt TAKARA company soybean gene group to extract kit method then and extract soybean gene group DNA, the concrete operations step has detailed explanation in the specification sheets of test kit.
Second step, design certification mark gene and house-keeping gene primer and probe
Obtain the house-keeping gene LECTIN gene order K00821 of CAMV35S gene order number and soybean from ncbi genbank, design primer and MGB probe mark are as follows on the house-keeping gene LECTIN of detection by quantitative marker gene CAMV35S gene and soybean gene:
Design CAMV35S gene primer and probe, below product that primer increased be 2343bp～2404bp in the AJ251014 scope
CAMV35S-FOR：5’-GACCAAAGGGCAATTGAGACTT-3’
CAMV35S-REV：5’-TGGAATCCGAGGAGGTTTCC-3’
CAMV3?5S-PROBE：5’-FAM--TCAACAAAGGGTAATATC-MGB-3’
Design house-keeping gene LECTIN gene primer probe, below product that primer increased be 1274bp～1332bp in the K00821 scope
LECTIN-FOR：5’-CGCCGCTTCCTTCAACTTC-3’
LECTIN-REV：5’-GCCCATCTGCAAGCCTTTT-3’
LECT?IN-PROBE：5’-VIC-CCTTATGCCCCTGACA-MGB-3
On the dna synthesizer device, synthesize CAMV35S gene and house-keeping gene LECTIN gene primer and probe then.
The 3rd goes on foot, determines the standard substance of detection by quantitative sample
Buy the DNA of genetically engineered soybean standard substance 0.1%, 0.5%, 1%, 2% from department of the Chinese government.
The 4th step, real time fluorescent quantitative poly chain reaction (PCR) detect
With 1 * PCR Buffer, 4mM MgCl2,200 μ M dNTP (dATP, dCTP, dGTP,), 400 μ M dUTP, 900nm second step gained CAMV35S Forward Primer and CAMV35S ReversePrimer, 250nM CAMV35S-probe probe, 900nm second step gained LECTIN ForwardPrimer and LECTIN Reverse Primer, 250nM LECTIN-probe probe, the soybean gene group sample DNA of buying from market of 100ng said extracted, 2U AmpliTaqGod DNA polymerase places same pcr reaction tubes, carries out the polymerase chain reaction with redistilled water benefit to 50 microlitres, the polymerase chain reaction condition: 37 ℃ were reacted 10 minutes, 40 circulations are reacted in 95 ℃ of pre-sex change 10 minutes again, and each circulation comprises: 94 ℃ of sex change 30 seconds, annealed 30 seconds for 60 ℃, 72 ℃ were extended 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes
The 5th step, quantitative calculation and analysis
1. the quantitative and typical curve of standard substance determines
According to the data adjustment in this example, play between the 15th circulation from the 3rd circulation and to determine the baseline scope, pairing PCR cycle index when above baseline, producing on can detected statistics significant fluorescent emission, in real-time fluorescence quantitative PCR instrument detection software, obtain the certification mark gene C AMV35S of standard substance and the Ct numerical value of house-keeping gene LECTIN, with the ratio of certification mark gene C AMV35S in the standard substance and house-keeping gene LECTIN as X-coordinate, the absolute value of the Ct value difference value of the LECTIN of certification mark gene C AMV35S and house-keeping gene is an ordinate zou in the standard substance; get marker gene CAMV35S and house-keeping gene LECTIN ratio 0.1%; 0.5%; 1%; 2% and the absolute value of the Ct value difference value of the LECTIN of the certification mark gene C AMV35S of each ratio correspondence and house-keeping gene be ordinate zou; make straight line; utilize the linear regression statistical study that this fitting of a straight line is become y=-3.29log (x)+8.9; relation conefficient=0.997; with this straight line as transgenosis examination criteria curve; y is the absolute value of certification mark gene C AMV35S (FAM) and house-keeping gene LECITN (VIC) Ct value difference value in the formula; x is certification mark gene C AMV35S and house-keeping gene LECTIN ratio, just the transgenosis rate of this soybean.
2. the calculating of sample and quantitative
Detect certification mark gene C AMV35S and Ct numerical value house-keeping gene LECTIN that obtains sample the software from the real-time fluorescence quantitative PCR instrument, the typical curve formula y=-3.29log (x)+8.9 that absolute value generation of the difference of Ct numerical value is detected to transgenosis, obtain containing marker gene CAMV35S and ratio 1.5% house-keeping gene LECTIN, just the transgenosis rate 1.5% of this corn.
Claims (4)
1. the quantitative detection method of transgene agricultural product and food mixture is characterized in that may further comprise the steps:
The extraction of the first step, transgene agricultural product and food mixture genomic dna
For liquid testing sample, adopt the freeze-drying drying and be milled to Powdered; For non-pulverous solid testing sample, adopt milled processed powdered, extract the genomic dna of sample then;
Second step, at transgenosis certification mark gene and house-keeping gene design primer and probe
1) obtain the house-keeping gene of transgenosis certification mark gene and plant or agricultural-food from ncbi genbank database, wherein the certification mark gene mainly comprises CAMV 35S promoter, NOS terminator, Km
r(anti-kanamycin gene), Neo
r(anti-neomycin gene), Kyg
r(the mould plain gene of moisture resistance), Bar
r(anti-careless fourth phosphino-because of), NPT-II (neomycin phosphotransferase gene), manually insert the special foreign gene of Plant Genome change agricultural-food and Food Quality and function;
2) at the conservative region design primer and the Taqman probe of the house-keeping gene that obtains transgenosis certification mark gene and plant or agricultural-food from ncbi genbank database, make the primer of design satisfy following condition with probe: primer and probe that the report fluorescence of (1) marker gene and house-keeping gene mark has different emission wavelengths (2) marker gene and house-keeping gene can react in same pipe;
3) primer and the probe of synthetic above-mentioned design on the dna synthesizer device;
The 3rd goes on foot, determines the standard substance of detection by quantitative sample
The standard substance of the transgenosis detection by quantitative that the selected Chinese government is admitted perhaps obtain standard substance according to following steps:
1) primer of standard product, the fragment that this primer amplification is come out comprise the marker gene or the house-keeping gene fragment of detection by quantitative, and length is between 100bp～400bp;
2) with standard substance primer 100～400nm Forward Primer of above-mentioned design, 100～400nmReverse Primer and 1 * PCRBuffer, 1.5～6mM MgCl2,100～400 μ MDntp (dATP, dCTP, dGTP, dTTP), 1～100ng contains the genomic dna of transgenosis certification mark gene or house-keeping gene, and 0.5U～5U AmpliTaq DNA polymerase places the pcr reaction tubes, mends to 50 microlitres with redistilled water, carry out the polymerase chain reaction, polymerase chain reaction (PCR) reaction conditions: 94 ℃ of pre-sex change 2 minutes, react 25～35 circulations again, each circulation comprises: 94 ℃ of sex change 30 seconds～1 minute, annealed 30 seconds～1 minute and 30 seconds for 45 ℃～67 ℃, 72 ℃ were extended 30 seconds～1 minute and 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes;
3) above-mentioned PCR product is carried out gel electrophoresis, the rubber tapping of purpose fragment is reclaimed, the fragment that reclaims is connected in the plasmid vector, then with this recombinant plasmid transformed in intestinal bacteria, allow recombinant plasmid great expression in intestinal bacteria, again recombinant plasmid dna is extracted and purifying, obtain a large amount of recombinant plasmids;
4) utilize on the plasmid vector restriction endonuclease sites that recombinant plasmid is carried out enzyme and cut, utilize the order-checking of dna sequencing instrument to identify again to cut and identify and contain the segmental recombinant plasmid of purpose, determine to contain right-on purpose fragment through the plasmid after the reorganization through enzyme;
5) calculating contains the segmental recombinant plasmid concentration of entirely true purpose, the concentration that will contain marker gene and contain the house-keeping gene recombinant plasmid is mixed according to 0～100% ratio, gets wherein at least four standard substance that ratio detects as the real-time fluorescence quantitative PCR transgenosis;
The 4th step, real time fluorescent quantitative poly chain reaction (PCR) detect
With 1 * PCR Buffer, 1.5～6mM MgCl2,100～400 μ M dNTP (dATP, dCTP, dGTP), 400 μ M dUTP, 100～900nm, second step gained marker gene Forward Primer and ReversePrimer, 50～300nM, second step gained marker gene Taqman probe, 100～900nm, second step gained house-keeping gene Forward Primer and Reverse Primer, 50～300nM, second step gained house-keeping gene Taqman probe, 10～100ng sample gene group DNA, 0.01～0.05U Amp Erase UNG enzyme, 0.5～5U AmpliTaqGod DNA polymerase, place in the same pcr reaction tubes, mend to 50 microlitres, utilize hyperchannel real-time fluorescence quantitative PCR instrument that the marker gene and the house-keeping gene of sample and standard substance are carried out real time fluorescent quantitative poly chain reaction (PCR) and detection with redistilled water;
Polymerase chain reaction (PCR) reaction conditions: 37 ℃ were reacted 5～15 minutes, 95 ℃ of pre-sex change 5～15 minutes, react 25～45 circulations again, each circulation comprises: 94 ℃ of sex change 30 seconds～1 minute, annealed 30 seconds～1 minute and 30 seconds for 45 ℃～67 ℃, 72 ℃ were extended 30 seconds～1 minute and 30 seconds, and after the loop ends, proceeded 72 ℃ of extensions 5 minutes;
The 5th step, quantitative calculation and analysis
1) the quantitative and typical curve of standard substance determines
From real-time fluorescence quantitative PCR instrument detection software, obtain the certification mark gene of standard substance and the Ct numerical value of house-keeping gene, with the ratio of certification mark gene and house-keeping gene in the standard substance as X-coordinate, the absolute value of the Ct value difference value of certification mark gene and house-keeping gene is an ordinate zou in the standard substance, with in the 3rd step 5) absolute value of the certification mark gene of the marker gene recombinant plasmid of the standard substance got and four ratios of house-keeping gene recombinant plasmid concentration and each ratio correspondence and the Ct value difference value of house-keeping gene makes straight line, utilize the linear regression statistical study that this fitting of a straight line is become y=-Klog (x)+B, in the formula-K is this collinear slope, B is a constant, with this straight line as transgenosis examination criteria curve, y is the absolute value of certification mark gene and house-keeping gene Ct value difference value in the formula, and x is the ratio of certification mark gene and house-keeping gene;
2) calculating of sample and quantitative
Detect the Ct value that obtains the certification mark gene and the house-keeping gene of sample the software from the real-time fluorescence quantitative PCR instrument, get the absolute value of the Ct value difference value of the certification mark gene of same sample and house-keeping gene, typical curve formula y=-Klog (the x)+B that detects to transgenosis as y numerical value generation, the x value that obtains is exactly the ratio of certification mark gene and house-keeping gene, just the transgenosis rate of this transgene agricultural product or food.
2. the quantitative detection method of transgene agricultural product according to claim 1 and food mixture is characterized in that sample gene group DNA extraction in the first step, adopts CTAB extracting genome DNA method, or the test kit extraction method, or the magnetic bead extraction method.
3. the quantitative detection method of transgene agricultural product according to claim 1 and food mixture is characterized in that the designed Taqman probe of marker gene and house-keeping gene mark is the TaqManMGB probe.
4. the quantitative detection method of transgene agricultural product according to claim 1 and food mixture, it is characterized in that in the 3rd step 5) the marker gene recombinant plasmid of the standard substance got and four ratios of house-keeping gene recombinant plasmid concentration are respectively 0.1%, 0.5%, 1%, 2%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297668C (en) * | 2004-05-20 | 2007-01-31 | 厦门大学 | Transgenic product low-density gene chip detecting method |
| CN100340675C (en) * | 2005-07-04 | 2007-10-03 | 黑龙江省烟草科学研究所 | Transgene tobacco detecting method and reagent box |
| CN1715912B (en) * | 2005-06-30 | 2010-04-28 | 曹际娟 | Transgenetic soybean detection power identifying sample and preparing method |
| CN1858220B (en) * | 2005-04-30 | 2011-08-10 | 徐定邦 | Double inverse transcription polymerase chain reaction method containing house keeping gene |
| CN107488732A (en) * | 2017-10-09 | 2017-12-19 | 贵州省产品质量监督检验院 | Detect triple fluorescent PCR primer group, probe groups, kit and the method for capsicum transgene component |
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2003
- 2003-03-08 CN CN 03115716 patent/CN1438328A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297668C (en) * | 2004-05-20 | 2007-01-31 | 厦门大学 | Transgenic product low-density gene chip detecting method |
| CN1858220B (en) * | 2005-04-30 | 2011-08-10 | 徐定邦 | Double inverse transcription polymerase chain reaction method containing house keeping gene |
| CN1715912B (en) * | 2005-06-30 | 2010-04-28 | 曹际娟 | Transgenetic soybean detection power identifying sample and preparing method |
| CN100340675C (en) * | 2005-07-04 | 2007-10-03 | 黑龙江省烟草科学研究所 | Transgene tobacco detecting method and reagent box |
| CN107488732A (en) * | 2017-10-09 | 2017-12-19 | 贵州省产品质量监督检验院 | Detect triple fluorescent PCR primer group, probe groups, kit and the method for capsicum transgene component |
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