CN101045942A - Transgenic papaya and detection of transgenic component in transgenic papaya product - Google Patents
Transgenic papaya and detection of transgenic component in transgenic papaya product Download PDFInfo
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Abstract
The present invention discloses one kind of kit for detecting transgenic papaya and transgenic papaya product and its qualitative and quantitative detection method. The quantitative detection method includes the following steps: extracting DNA of the detected sample as the template, and performing PCR amplification with specific primer pair of CAR gene and specific primer pair of exogenous gene; hybridizing the amplified products separately with the probe and detecting the hybridization generated detectable signal; and analyzing the detected data to detect the transgenic component content in papaya and papaya product quantitatively. The said method of the present invention is accurate and simple.
Description
Technical field
The present invention relates to the product detection technique, more specifically, the present invention relates to detection kit and detection method that qualitative and quantitative detects transgene component in transgenic papaya and the converted products thereof.
Background technology
In recent years, along with development of biology, the rapid commercialization of genetically modified crops, and enter the world market by trade.Development of biology has improved the output and the quality of agricultural-food significantly, but has alleviated the population growth to a certain extent, natural resources scarcity farming land area reduces and the natural disaster that can not expect is brought influence etc.Yet the safety problem of transgenic crop causes that people pay close attention to greatly.
Countries such as many in the world countries such as European Union, Japan, Australia, Singapore, with legislation or other forms genetically modified organism and converted products thereof are carried out strict management one after another, for example: when requiring the import transgenic product to enter domestic markets, when surpassing certain limit, gm content answers labelling, reference when choosing for the human consumer, European Union's regulation transgene component is higher than 0.9% and adapts to labelling, and Japanese regulation transgene component is higher than at 5% o'clock and answers labelling.
Comprise that at present many countries of China have all carried out the research of transgenic papaya, and the circulation of transgenic papaya has been arranged on the market.Yet, at present both at home and abroad still not to the report of the qualitative and detection by quantitative of transgenic papaya and converted products thereof.
In the transgene agricultural product detection technique, what be most widely used at present is PCR correlation detection technology based on DNA, and the amplification of target gene needs internal standard gene to do contrast to avoid occurring false-negative result.The establishment of internal standard gene simultaneously also helps to determine the species in foreign gene source.Present soybean, corn, the internal standard gene of rice all has been determined, but the internal standard gene of papaya never is determined out.Also there be not transgene component content detecting method and detection kit in a kind of detection transgenic papaya and the converted products thereof at present.
This area presses for exploitation transgene component in transgenic papaya and the converted products thereof is carried out the method that qualitative and quantitative detects.
Summary of the invention
The test kit and the qualitative and quantitative analysis method thereof that the purpose of this invention is to provide transgene component in a kind of efficient, accurate, easy detection transgenic papaya or its product.
The invention provides a kind of test kit that is used to detect transgenic cotton seed and converted products thereof, it is characterized in that it contains:
(1) Auele Specific Primer of amplification CAR gene is right;
(2) with CAR specificity of gene amplification product bonded probe;
(3) Auele Specific Primer of amplification foreign gene is right;
(4) with foreign gene amplified production specificity bonded probe;
(5) standard reference sample.
The Auele Specific Primer of amplification CAR gene is to being in the test kit of the present invention:
Upstream primer 5 ' AGT GGC TCA ATA TGG TAT TCA CTA CAG A 3 '
Downstream primer 5 ' AAA ATG TAG ATA TAC CTC CCT TGA GCG 3 '.
In the test kit of the present invention be with CAR specificity of gene amplification product bonded probe:
5’ATA CTT ACC CAT ATG AGG GAG TGC AAC GTT ATT G 3’。
Foreign gene is not particularly limited in the test kit of the present invention, can be any foreign gene, can be selected from PRSVR gene, PRSVCP gene, the FMV35S gene one or more.
When foreign gene of the present invention was the PRSVR gene, its Auele Specific Primer was to being:
Upstream primer 5 ' TTG TCC CCT CTT CCG GAG TT 3 '
Downstream primer 5 ' CTT CCT TGC TTA GAA CGC TTT TCA 3 ',
Accordingly, with PRSVR specificity of gene amplification product bonded probe be:
5’CCT GGA GTG TAA TGA GGA AGC CAA GAC TTT CTT T 3’。
When foreign gene of the present invention was the PRSVCP gene, its Auele Specific Primer was to being:
Upstream primer 5 ' TCT AAC ACT CGC GCC ACT CA 3 '
Downstream primer 5 ' CCG TTT AAC ATT ACT TGC ATT TCG 3 ',
Accordingly, with PRSVCP specificity of gene amplification product bonded probe be:
5’ACG AGG GAG TGA GGA ATG ATT ACG GCC T 3’。
Test kit middle probe of the present invention is the probe with the marker mark, and itself and amplified production can produce detectable signal when hybridizing; Be used for detectable signal of the present invention without limits, it can be detection signal commonly used in any detection of nucleic acids field, the kind of detectable signal depends on marker used on the probe, and described probe mark thing can be selected from one or more in biotin-avidin, digoxin, radio isotope, the fluorescent mark; Preferably, described probe is a Taqman type fluorescent probe.
Standard reference sample product are that one group of transgenic papaya DNA and non-transgenic papaya DNA are in gradient ratio blended mixture in the test kit of the present invention; Preferably, the content of transgenic papaya DNA is respectively 0.05%, 0.1%, 0.5%, 1%, 2%, 5% in each mixture.
Can also contain in the test kit of the present invention:
10 * PCR reaction buffer, 10 * 25mM MgCl
2, components such as 10mM dNTP, 1U/ μ l UNG enzyme, 5U/ μ l Taq archaeal dna polymerase, sterilization ultrapure water.
The invention provides the method for a kind of qualitative detection transgenic papaya and converted products thereof, it may further comprise the steps:
(1) extracts the DNA of papaya or its converted products as masterplate;
(2) detect with test kit of the present invention, masterplate DNA is added amplification CAR gene and foreign gene reaction system separately respectively, hatch on the pcr amplification instrument, CAR gene and foreign gene increase respectively;
Each the component volume proportion of qualitative PCR reaction system of CAR gene and foreign gene of wherein increasing is: 10 * PCR reaction buffer, 5 parts by volume, 10 * 25mM MgCl
2Masterplate DNA 2 parts by volume of 5 parts by volume, 10mM dNTP 5 parts by volume, Taq archaeal dna polymerase 0.4 parts by volume, 5 μ M upstream primers, 1 parts by volume, 5 μ M downstream primers, 1 parts by volume, sterilization ultrapure water 30.6 parts by volume, adding;
The qualitative PCR loop parameter is:
95 ℃ of pre-sex change 3min; 94 ℃ of sex change 20s; 54 ℃ of annealing 40s; 72 ℃ are extended 40s, circulate 40 times;
Extend 10min at 72 ℃ at last;
(3) the qualitative PCR product is carried out detected through gel electrophoresis;
(4) the PCR product is reclaimed purifying, order-checking.
The present invention also provides the method for a kind of detection by quantitative transgenic papaya and converted products thereof, and it may further comprise the steps:
(1) extracts the DNA of papaya or its converted products as masterplate;
(2) detect with test kit of the present invention, masterplate DNA is added amplification CAR gene and foreign gene quantitative reaction system separately respectively, hatch on the quantitative pcr amplification instrument, CAR gene and foreign gene increase respectively;
Each the component volume proportion of quantitative reaction system of CAR gene and foreign gene of wherein increasing is: 10 * PCR reaction buffer, 5 parts by volume, 10 * 25mM MgCl
2Masterplate DNA 2 parts by volume of 10 parts by volume, 10mM dATP 1 parts by volume, 1mM dGTP 1 parts by volume, 1mM dUTP 1 parts by volume, 1mM dCTP 1 parts by volume, 1U/ μ l UNG enzyme 0.5 parts by volume, 5U/ μ l Taq archaeal dna polymerase 0.5 parts by volume, 5 μ M upstream primers, 4 parts by volume, 5 μ M downstream primers, 4 parts by volume, 20 μ M probes, 1 parts by volume, sterilization ultrapure water 19 parts by volume, adding;
The quantitative PCR loop parameter is:
50 ℃ of 10min; 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min circulate 50 times.
(3) respectively with amplified production and probe hybridization separately, detect the signal surveyed that hybridization is produced, obtain data;
(4) production standard curve:
As template, increase (2) set by step with marker; (3) are measured set by step, obtain data, make typical curve;
(5) data and the typical curve that step (3) is measured compares transgene component in detection by quantitative cottonseed or its converted products.
The CAR gene is the caricin gene among the present invention, and it is the endogenous marker gene of papaya.When carrying out the PCR detection by quantitative, need to detect the endogenous specific mark gene of the inherent of this crop own, come the template total amount of genetically modified crops and non-transgenic crop in the working sample, this is the basis of carrying out detection by quantitative.For example, the endogenous specific mark gene that is used for the genetically engineered soybean detection can be selected the Lectin gene for use; The endogenous specific mark gene that is used for the transgenic corns detection can be selected Zein gene or IVR gene for use.The inventor is through extensively and in depth research, and by the sequence-specific analysis, the method for PCR check and analysis and southern blot has determined that the CAR gene is the endogenous reference gene of preferred cotton, has finished the present invention on this basis.
Used herein, term " CAR Auele Specific Primer " refer to increase specifically stearic acid dehydrogenase gene or its segmental primer.
Used herein, term " foreign gene Auele Specific Primer " refers to amplify specifically foreign gene or its segmental primer that changes plant over to.Should be understood that the foreign gene Auele Specific Primer changes with the difference of foreign gene, also change with foreign gene amplified production specificity bonded probe accordingly with the different of foreign gene; For example, when foreign gene was the PRSVR gene, the foreign gene Auele Specific Primer of selecting for use was exactly the PRSVR gene-specific primer, and PRSVR gene or its segmental primer specifically can increase; Corresponding and foreign gene amplified production specificity bonded probe is PRSVR gene or its segmental probe just.Foreign gene is not particularly limited in the test kit of the present invention, can be any foreign gene, can be one or more PRSVR genes, PRSVCP gene, FMV35S gene etc.
Standard reference sample product are that one group of transgenic papaya DNA and non-transgenic papaya DNA are in gradient ratio blended mixture in the test kit of the present invention; They contain the DNA of a certain proportion of transgenic papaya DNA and non-transgenic papaya respectively; Preferably, marker is that the DNA with isolating transgenic papaya DNA and non-transgenic papaya all is diluted to 30ng/ul, by volume ratio is mixed two kinds of DNA, be made into the transgenic papaya dna content and account for 5%, 2%, 1%, 0.5%, 0.1%, 0.05% dna solution respectively, as the DNA of detection by quantitative with reference to sample.Correspondingly change with the change of foreign gene with reference to sample transfer gene papaya DNA.
Can be used for DNA extraction technology of the present invention and be not particularly limited, can be this area various extractive techniques commonly used.A kind of preferable methods is the magnetic bead extraction method, and for example the paramagnetic particle method DNA extraction test kit of producing with Promega company extracts the DNA in the papaya.
The principle of detection by quantitative of the present invention: by detecting total template amount of cotton DNA in the endogenous reference gene CAR of the papaya genetic testing sample, by detecting foreign gene, the template amount of working sample transfer gene DNA, then with the template amount of transgenosis DNA template amount, thereby calculate the per-cent of transgenic papaya content in the sample divided by the total DNA of papaya.The detection of foreign gene and native gene CAR gene is carried out in two reaction tubess respectively simultaneously, and reaction system is all identical with reaction conditions.Draw out the typical curve of Δ Ct value (Ct foreign gene-Ct CAR gene) and gm content per-cent by marker.By with the comparison of typical curve, just can quantitatively determine same or unknown papaya that the same terms is measured down and converted products thereof in transgene component.
The mensuration of template amount is by pcr amplification in the reaction system of the present invention, combine with specific probe, and according to detecting in conjunction with the detectable signal that is sent.Can be used for detectable signal of the present invention without limits, can be detection signal commonly used in any detection of nucleic acids field.The kind of detectable signal depends on marker used on the probe, wherein can be biotin-avidin, digoxin, radio isotope, fluorescent mark etc.Preferred probes is a Taqman type probe.
The result shows that test kit of the present invention and detection method thereof can satisfy the detection by quantitative demand of transgenic papaya, helps transgenic papaya and converted products thereof are identified.In addition, adopt and of the present inventionly unified help eliminating testing differentia between different laboratories with reference to sample.
Using test kit of the present invention can make detection research institution quickly and accurately the sampling observation sample be identified, carry out the laws and regulations of China effectively about transgenic product, protected human consumer's right to know effectively, set up the transgenic papaya and the converted products detection technique system thereof of China oneself simultaneously, accurately identify and prevent abroad to enter China, China's ecotope and people ' s health are caused disadvantageous effect without the transgenic papaya and the converted products thereof of label and safety evaluation; Also provide powerful technical support simultaneously for legitimacy protection this country's market for farm products and the outlet that utilizes Technical Barriers to Trade after the China joined WTO.
Description of drawings
Fig. 1: PRSVR gene primer and probe carry out the linear graph of quantitative PCR test to reference sample (5%, 2%, 1%, 0.5%,, transgenic papaya DNA sample).
Fig. 2: PRSVR gene primer and probe are to the quantitative measurement standard curve of transgenic papaya.
Fig. 3: CAR gene primer and probe carry out the linear graph of quantitative PCR test with reference to sample (5%, 2%, 1%, 0.5% transgenic papaya DNA sample).
Fig. 4: CAR gene primer and probe are to the quantitative measurement standard curve of transgenic papaya.
Fig. 5: PRSVCP gene primer and probe carry out the linear graph of quantitative PCR test to reference sample (5%, 2%, 1%, 0.5% transgenic papaya DNA sample).
Fig. 6: PRSVCP gene primer and probe are to the quantitative measurement standard curve to transgenic papaya.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1. material and instrument
1.1 vegetable material
Changeing the antiviral papaya standard specimen of PRSVR gene is provided by the Chinese Academy of Agricultural Sciences, is antiviral transgenic papaya, and fruit and leaf purity are 100%.Negative non-transgenic papaya is provided by the Chinese Academy of Agricultural Sciences.
1.2 reagent
1.2.1 the relevant reagent of DNA extraction
1) the special-purpose agarose of electrophoresis.
2) TAE (50 *): 242g Tris-alkali, the 57.1ml Glacial acetic acid, 100ml 0.5mol/lEDTA (pH 8.0), soluble in water, be settled to 1L.
3) liquid nitrogen.
4) 70% ethanol.
5) TE (pH 8.0): measure 10ml 1mol/L Tris.HCl (pH 8.0) and 2ml0.5mol/L EDTA (pH 8.0).Add water and be settled to 1000ml.Autoclaving is standby after the packing.
6) ethidium bromide: 10mg/ml.
7) 100-2000bp ladder DNA Marker: section's sky reaches biotech firm's product.
8) 6 * Loading Buffer:Promega company product.
9) total DNA reclaims test kit: worker biotech company product is given birth in Shanghai.
10) Promega paramagnetic particle method DNA extraction test kit.
1.2.2 the relevant reagent of PCR reaction
1) 10 * Buffer (PCR reaction buffer) (5 μ l): 500mmol/l KCl, 100mmol/l Tris.HCl pH 8.3 room temperatures, 15mmol/l MgCl
2: Promega company product.
2) 10 * Mg
2+(25mM): Promega company product;
3) 10 * dNTP (2mM): Promega company product;
4) primer: it is synthetic that worker bio-engineering corporation is given birth in Shanghai, is made into 100 μ M-20 ℃ storages earlier, dilute for final concentration 5 μ M for use;
5) probe: it is synthetic that worker bio-engineering corporation is given birth in Shanghai, is diluted to 20 μ M for using;
6) Taq archaeal dna polymerase, 5unit/ μ l:Promega company product;
7) UNG enzyme (1U/ μ l) (Products Co., Ltd of Applied Biosystems company product)
8) ultrapure water (dd H2O): Easy Pure pure water instrument makes.
9) TaqManTM PCR Core Regent Kit test kit Cat NO article No. .:N808-0228 (providing middle translated name) U.S. Applied Biosystems produces.
1.3 plant and instrument
1) pcr amplification instrument: U.S. MJ RESEARCH Minicycler PTC-150 type;
2) quantitative pcr amplification instrument: the ABI Prism SequenceDetector of U.S. biotech company 7000 types;
3) electrophoresis apparatus: U.S. Continental Lab Products75.2314 type;
4) gel imaging system: U.S. BIO-RAD Gel DOC-2000 type;
5) whizzer: German Eppendorf 5415D type;
6) micro sample adding appliance: German Eppendorf product has specifications such as 2.5,10,20,100,200,1000 μ l;
7) balance: German Sartorius;
8) high-pressure sterilizing pot: Japanese Tomy SS-325 type.
9) DNA freeze concentration instrument: U.S. CentriVap Console Labconco product.
2. test method
2.1 the extraction of the total DNA of papaya
Use the CTAB method to extract DNA, specified operational procedure is as follows.
Take by weighing the 100mg sample, transfer to the 1.5ml centrifuge tube; Add 700 μ l CTAB and extract damping fluid; 65 ℃ of temperature were bathed 30 minutes, during put upside down mixing frequently; 13000g, centrifugal 10 minutes; Shift supernatant to the 1.5ml centrifuge tube; Add 2/3 volume Virahol, put upside down mixing 10-15 time, room temperature 30 minutes; 12000g, centrifugal 10 minutes; Getting precipitation dissolves with 350 μ l TE; With twice of isopyknic chloroform/primary isoamyl alcohol (24: 1) extracting; Twice dehydrated alcohol precipitation 2 hours; 12000g, centrifugal 10 minutes; Washing with alcohol precipitation with 70%; Drain liquid with the freeze concentration instrument; Add 100 μ l distilled waters, the concussion mixing, 65 ℃ of temperature are bathed 10min.Centrifuge tube is kept 1min on centrifuge tube shelf, carefully liquid is transferred in another new pipe; Get 1 μ l and be PCR.
2.2 PCR test
2.2.1 design of primers
Primer sequence that this test is used and amplification fragment length see Table 1
Table 1: primer, probe sequence and amplification fragment length that this test is used
| The gene title | Sequence | Amplification length (bp) |
| CAR | Upstream primer 5 ' AGT GGC TCA ATA TGG TAT TCA CTA CAG A 3 ' downstream primer 5 ' AAA ATG TAG ATA TAC CTC CCT TGA GCG 3 ' probe 5 ' ATA CTT ACC CAT ATG AGG GAG TGC AAC GTT ATT G 3 ' | 91 |
| PRSVR | Upstream primer 5 ' TTG TCC CCT CTT CCG GAG TT 3 ' downstream primer 5 ' CTT CCT TGC TTA GAA CGC TTT TCA 3 ' probe 5 ' CCT GGA GTG TAA TGA GGA AGC CAA GAC TTT CTT T 3 ' | 100 |
| PRSVCP | Upstream primer 5 ' TCT AAC ACT CGC GCC ACT CA 3 ' downstream primer 5 ' CCG TTT AAC ATT ACT TGC ATT TCG 3 ' probe 5 ' ACG AGG GAG TGA GGA ATG ATT ACG GCC T 3 ' | 100 |
2.2.2 PCR reaction system
The reaction system of quantitative PCR test sees Table 2;
The reaction system of qualitative PCR test sees Table 3.
The reaction system of table 2 quantitative PCR test
| Reagent name | 50 μ l reaction systems | 25 μ l reaction systems |
| 10×Buffer | 5.0 | 2.5 |
| 10×Mg 2+(25mM) | 10.0 | 5.0 |
| dATP(10mM) | 1.0 | 0.5 |
| dGTP(mM) | 1.0 | 0.5 |
| dUTP(mM) | 1.0 | 0.5 |
| dCTP(mM) | 1.0 | 0.5 |
| UNG enzyme (1U/ μ l) | 0.5 | 0.25 |
| Taq archaeal dna polymerase (5U/ μ l) | 0.5 | 0.25 |
| Upstream primer (5 μ M) | 4.0 | 2.0 |
| Downstream primer (5 μ M) | 4.0 | 2.0 |
| Probe (20 μ M) | 1.0 | 0.5 |
| The sterilization ultrapure water | 19.0 | 9.5 |
| Template | 2.0 | (1.0 about 30ng DNA) |
The reaction system of table 3 qualitative PCR test
| Reagent name | 50 μ l reaction systems | 25 μ l reaction systems |
| 10×Buffer | 5.0 | 2.5 |
| 10×dNTP | 5.0 | 2.5 |
| 10×Mg 2+(25mM) | 5.0 | 2.5 |
| Taq archaeal dna polymerase (5U/ μ l) | 0.4 | 0.2 |
| Upstream primer (5 μ M) | 1.0 | 0.5 |
| Downstream primer (5 μ M) | 1.0 | 0.5 |
| The sterilization ultrapure water | 30.6 | 15.3 |
| Template | 2.0 | (1.0 about 30ng DNA) |
2.2.3 PCR reaction cycle parameter
Quantitative PCR reaction cycle parameter: 50 ℃ of 10min; 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min circulate 50 times.
Qualitative PCR reaction cycle parameter:
95 ℃ of pre-sex change 33min; 94 ℃ of sex change 20s; 54 ℃ of annealing 40s; 72 ℃ are extended 40s, circulate 40 times;
Extend 10min at 72 ℃ at last;
2.3 the detected through gel electrophoresis of qualitative PCR product
1) preparation sepharose
50 * TAE is diluted to 1 * TAE solution, and with 1 * TAE preparation agarose (concentration is 2%), gel melts with the microwave oven boiling, waits to be cooled to 60 ℃, and adding ethidium bromide to final concentration is 5 ‰, pours in the electrophoresis chamber gel slab and solidifies;
2) point sample
Get 20 μ l pcr amplification products, add 2 μ l sample-loading buffer point samples and carry out electrophoresis, and add dna molecular marker electrophoresis simultaneously, to judge the pulsating size of PCR product.
3) deposition condition
Voltage is according to the length of electrophoresis chamber and decide 3-5V/cm, and electrophoresis time is determined according to the shift position of tetrabromophenol sulfonphthalein.
4) result
Electrophoresis detection result gel analysis imaging system or Ultraviolet Detector record.
2.4 the order-checking of PCR product
The PCR product is given birth to the worker PCR of biotech company product glue with Shanghai and is reclaimed test kit recovery purifying, and the PCR product is served the sea and given birth to the order-checking of worker biotech company.
The preparation of embodiment 1, the antiviral papaya standard reference of commentaries on classics PRSVR gene sample
The present invention extracts DNA with the CTAB method respectively with pure antiviral papaya of commentaries on classics PRSVR gene and non-transgenic papaya, and all be diluted to 50ng/ul, by volume ratio is mixed two kinds of DNA, be made into the antiviral papaya dna content of transgenosis and account for 5%, 2%, 1%, 0.5%, 0.1%, 0.05% dna solution respectively, as detection by quantitative with reference to sample.
The test kit of antiviral papaya of PRSVR gene and converted products thereof is changeed in embodiment 2, detection
Test kit contains:
(1) Auele Specific Primer of amplification CAR gene is right:
Upstream primer 5 ' AGT GGC TCA ATA TGG TAT TCA CTA CAG A 3 '
Downstream primer 5 ' AAA ATG TAG ATA TAC CTC CCT TGA GCG 3 '.
Each 100 μ l of 100uM;
(2) with CAR specificity of gene amplification product bonded probe:
5’ATA CTT ACC CAT ATG AGG GAG TGC AAC GTT ATT G 3’
(3) its Auele Specific Primer of amplification PRSVR gene is right:
Upstream primer 5 ' TTG TCC CCT CTT CCG GAG TT 3 '
Downstream primer 5 ' CTT CCT TGC TTA GAA CGC TTT TCA 3 ',
Each 100 μ l of 100uM;
(4) with PRSVR specificity of gene amplification product bonded probe:
5’CCT GGA GTG TAA TGA GGA AGC CAA GAC TTT CTT T 3’.
(5) its Auele Specific Primer of amplification PRSVCP gene is right:
Upstream primer 5 ' TCT AAC ACT CGC GCC ACT CA 3 '
Downstream primer 5 ' CCG TTT AAC ATT ACT TGC ATT TCG 3 ',
Each 100 μ l of 100uM;
(6) with PRSVCP specificity of gene amplification product bonded probe:
5’ACG AGG GAG TGA GGA ATG ATT ACG GCC T 3’.
(7) standard reference sample:
By the antiviral papaya standard reference of the commentaries on classics PRSVCP gene sample of embodiment 1 preparation, DNA concentration is 30ng/ μ l, each 200 μ l.
Wherein the probe of (2), (4), (6) is a Taqman type fluorescent probe, and 5 ' end all adopts fluorescence excitation dyestuff FAM mark, and 3 ' end all adopts fluorescent quenching dyestuff TAMRA mark.
Can also contain in the test kit:
10 * PCR reaction buffer, 10 * 25mM MgCl
2, components such as 10mM dNTP, 1U/ μ l UNG enzyme, 5U/ μ l Taq archaeal dna polymerase, sterilization ultrapure water.
Embodiment 3, carry out qualitative detection with the test kit of embodiment 2
Step is as follows:
(1) the paramagnetic particle method DNA extraction test kit of producing with Promega company extracts DNA in the cottonseed to be measured as masterplate;
(2) embodiment 2 test kits detect, and masterplate DNA is added amplification CAR gene, PRSVCP gene and PRSVR gene reaction system separately respectively, hatch the CAR gene that increases respectively, PRSVCP gene and PRSVR gene on the pcr amplification instrument;
The qualitative PCR reaction system (25 μ l reaction system) of amplification CAR gene, PRSVCP gene and PRSVR gene, wherein component is: 10 * PCR reaction buffer, 2.5 μ l, 10 * 25mM MgCl
22.5 the masterplate DNA 1 μ l (about 30ng DNA) of μ l, 10mM dNTP 2.5 μ l, Taq archaeal dna polymerase 0.2 μ l, 5 μ M upstream primers, 0.5 μ l, 5 μ M downstream primers, 0.5 μ l, sterilization ultrapure water 15.3 μ l, adding;
The qualitative PCR loop parameter is:
95 ℃ of pre-sex change 33min; 94 ℃ of sex change 20s; 54 ℃ of annealing 40s; 72 ℃ are extended 40s, circulate 40 times;
Extend 10min at 72 ℃ at last;
(3) the qualitative PCR product is carried out detected through gel electrophoresis; Result gel analysis imaging system or Ultraviolet Detector record; PCR product segment CAR gene amplification length is 91bp as a result; PRSVCP gene and PRSVR gene amplification length are 100bp; Meet the design of primers requirement of quantitative PCR detection.
(4) the PCR product is given birth to the worker PCR of biotech company product glue with Shanghai and is reclaimed test kit recovery purifying, and the PCR product is served the sea and given birth to the order-checking of worker biotech company.
Embodiment 4, carry out detection by quantitative with the test kit of embodiment 2
Step is as follows:
(1) the paramagnetic particle method DNA extraction test kit of producing with Promega company extracts DNA in the cottonseed to be measured as masterplate;
(2) detect with embodiment 2 test kits, masterplate DNA is added amplification CAR gene, PRSVCP gene and PRSVR gene quantitative reaction system separately respectively, on the quantitative pcr amplification instrument, hatch the CAR gene that increases respectively, PRSVCP gene and PRSVR gene;
The quantitative reaction system (50 μ l reaction system) of amplification CAR gene, PRSVCP gene and PRSVR gene, wherein component is: 10 * PCR reaction buffer, 5 μ l, 10 * 25mMMgCl
2The masterplate DNA 2 μ l (about 30ng DNA) of 10 μ l, 10mM dATP 1 μ l, 1mM dGTP 1 μ l, 1mM dUTP 1 μ l, 1mM dCTP 1 μ l, 1U/ μ l UNG enzyme 0.5 μ l, 5U/ μ l Taq archaeal dna polymerase 0.5 μ l, 5 μ M upstream primers, 4 μ l, 5 μ M downstream primers, 4 μ l, 20 μ M probes, 1 μ l, sterilization ultrapure water 19 μ l, adding;
The quantitative PCR loop parameter is:
50 ℃ of 10min; 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min circulate 50 times.
(3) respectively with amplified production and probe hybridization separately, detect the fluorescent signal that hybridization is produced, obtain data, result's (seeing accompanying drawing 1 and 3);
This type of result also can embody with list data, but form can not embody the Treatment Analysis result to these data.Accompanying drawing 1 and 3 not only can embody data results and itself but also embody the quality of these group data and problem such as repeatability.
(4) production standard curve:
As template, increase (2) set by step with marker; (3) are measured set by step, obtain data, make typical curve (seeing accompanying drawing 2 and 4);
Primer and probe with PRSVCP gene and PRSVR gene carry out the collection of illustrative plates of quantitative PCR test shown in Fig. 2 and 5 to the reference sample;
The collection of illustrative plates that the reference sample is carried out the quantitative PCR test with the primer of CAR gene and probe as illustrated in fig. 1 and 2;
Carry out the Δ Ct value of quantitative PCR reaction gained through statistical analysis by primer and probe to above-mentioned PRSVR gene, PRSVCP gene and SAD1 gene, can draw the typical curve (seeing Fig. 2,4 and 6) and the linear equation Y=-3.2311X+24.325 that obtain the transgenic papaya Quantitative Monitoring; R
2=0.9991; Y=-2.5435X+19.482; R
2=0.9953.
(5) data and the typical curve that step (3) is measured compares transgene component in detection by quantitative cottonseed or its converted products.
| Sample number | A | B | C | D |
| Gm content | 11.85 | 17.23 | 4.72 | 58.23 |
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1, a kind of test kit that is used to detect transgenic papaya and converted products thereof is characterized in that it contains:
(1) Auele Specific Primer of amplification CAR gene is right;
(2) with CAR specificity of gene amplification product bonded probe;
(3) Auele Specific Primer of amplification foreign gene is right;
(4) with foreign gene amplified production specificity bonded probe;
(5) standard reference sample.
2, test kit according to claim 1
Wherein increase the Auele Specific Primer of CAR gene to being:
Upstream primer 5 ' AGT GGC TCA ATA TGG TAT TCA CTA CAG A 3 '
Downstream primer 5 ' AAAATG TAGATATAC CTC CCT TGA GCG 3 ';
Wherein be with CAR specificity of gene amplification product bonded probe:
5’ATA CTT ACC CAT ATG AGG GAG TGC AAC GTT ATT G 3’。
3, test kit according to claim 1, wherein foreign gene is selected from one or more in PRSVR gene, PRSVCP gene, the FMV35S gene.
4, according to claim 1 or 3 described test kits
When wherein foreign gene was the PRSVR gene, its Auele Specific Primer was to being:
Upstream primer 5 ' TTG TCC CCT CTT CCG GAG TT 3 '
Downstream primer 5 ' CTT CCT TGC TTA GAA CGC TTT TCA 3 ',
Accordingly, with PRSVR specificity of gene amplification product bonded probe be:
5’CCT GGA GTG TAA TGA GGAAGC CAA GAC TTT CTT T 3’.
When wherein foreign gene was the PRSVCP gene, its Auele Specific Primer was to being:
Upstream primer 5 ' TCT AAC ACT CGC GCC ACT CA 3 '
Downstream primer 5 ' CCG TTT AAC ATT ACT TGC ATT TCG 3 ',
Accordingly, with PRSVCP specificity of gene amplification product bonded probe be:
5’ACG AGG GAG TGA GGA ATG ATT ACG GCC T 3’.
5, test kit according to claim 1, its middle probe are that wherein the probe mark thing is selected from one or more in biotin-avidin, digoxin, radio isotope, the fluorescent mark with the probe of marker mark.
6, test kit according to claim 5, its middle probe are Taqman type fluorescent probes.
7, test kit according to claim 1, wherein the standard reference sample is that one group of transgenic papaya DNA and non-transgenic papaya DNA are in gradient ratio blended mixture.
8, test kit according to claim 7, wherein the standard reference sample is the mixture of one group of transgenic papaya DNA and non-transgenic papaya DNA, and the content of transgenic papaya DNA is respectively 0.05%, 0.1%, 0.5%, 1%, 2%, 5% in each mixture.
9, the method for a kind of qualitative detection transgenic papaya and converted products thereof, it may further comprise the steps:
(1) extracts the DNA of papaya or its converted products as masterplate;
(2) detect with the described arbitrary test kit of arbitrary claim 1-8, masterplate DNA is added amplification CAR gene and foreign gene reaction system separately respectively, hatch on the pcr amplification instrument, CAR gene and foreign gene increase respectively;
Each the component volume proportion of qualitative PCR reaction system of CAR gene and foreign gene of wherein increasing is: 10 * PCR reaction buffer, 5 parts by volume, 10 * 25mM MgCl
2Masterplate DNA 2 parts by volume of 5 parts by volume, 10mM dNTP 5 parts by volume, Taq archaeal dna polymerase 0.4 parts by volume, 5 μ M upstream primers, 1 parts by volume, 5 μ M downstream primers, 1 parts by volume, sterilization ultrapure water 30.6 parts by volume, adding;
The qualitative PCR loop parameter is:
95 ℃ of pre-sex change 34min; 94 ℃ of sex change 20s; 549 ℃ of annealing 430s; 72 ℃ are extended 430s, circulate 40 times;
Extend 10min at 72 ℃ at last;
(3) the qualitative PCR product is carried out detected through gel electrophoresis;
(4) the PCR product is reclaimed purifying, order-checking.
10, the method for a kind of detection by quantitative transgenic papaya and converted products thereof, it may further comprise the steps:
(1) extracts the DNA of papaya or its converted products as masterplate;
(2) detect with the described arbitrary test kit of arbitrary claim 1-8, masterplate DNA is added amplification CAR gene and foreign gene quantitative reaction system separately respectively, hatch on the quantitative pcr amplification instrument, CAR gene and foreign gene increase respectively;
Each the component volume proportion of quantitative reaction system of CAR gene and foreign gene of wherein increasing is: 10 * PCR reaction buffer, 5 parts by volume, 10 * 25mM MgCl
2Masterplate DNA 2 parts by volume of 10 parts by volume, 10mM dATP 1 parts by volume, 1mM dGTP 1 parts by volume, 1mM dUTP 1 parts by volume, 1mM dCTP 1 parts by volume, 1U/ μ l UNG enzyme 0.5 parts by volume, 5U/ μ l Taq archaeal dna polymerase 0.5 parts by volume, 5 μ M upstream primers, 4 parts by volume, 5 μ M downstream primers, 4 parts by volume, 20 μ M probes, 1 parts by volume, sterilization ultrapure water 19 parts by volume, adding;
The quantitative PCR loop parameter is:
50 ℃ of 10min; 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min circulate 50 times;
(3) respectively with amplified production and probe hybridization separately, detect the signal surveyed that hybridization is produced, obtain data;
(4) production standard curve:
As template, increase (2) set by step with marker; (3) are measured set by step, obtain data, make typical curve;
(5) data and the typical curve that step (3) is measured compares transgene component in detection by quantitative papaya or its converted products.
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| CN 200610112099 CN101045942A (en) | 2006-08-31 | 2006-08-31 | Transgenic papaya and detection of transgenic component in transgenic papaya product |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694634A (en) * | 2015-02-12 | 2015-06-10 | 暨南大学 | Method for detecting genetically modified papayas by utilizing multiple PCR technology |
| CN108220397A (en) * | 2018-02-26 | 2018-06-29 | 杭州更蓝生物科技有限公司 | A kind of method for detecting transgenosis pawpaw |
| CN112210622A (en) * | 2020-11-12 | 2021-01-12 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group, application, kit and method for rapidly identifying transgenic papaya |
| CN114574629A (en) * | 2022-05-06 | 2022-06-03 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit weight and application thereof |
-
2006
- 2006-08-31 CN CN 200610112099 patent/CN101045942A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694634A (en) * | 2015-02-12 | 2015-06-10 | 暨南大学 | Method for detecting genetically modified papayas by utilizing multiple PCR technology |
| CN104694634B (en) * | 2015-02-12 | 2017-06-13 | 暨南大学 | A kind of method that utilization multiple PCR technique detects transgenic papaya |
| CN108220397A (en) * | 2018-02-26 | 2018-06-29 | 杭州更蓝生物科技有限公司 | A kind of method for detecting transgenosis pawpaw |
| CN112210622A (en) * | 2020-11-12 | 2021-01-12 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group, application, kit and method for rapidly identifying transgenic papaya |
| CN112210622B (en) * | 2020-11-12 | 2021-09-10 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Primer group, application, kit and method for rapidly identifying transgenic papaya |
| CN114574629A (en) * | 2022-05-06 | 2022-06-03 | 中国热带农业科学院三亚研究院 | SNP molecular marker related to papaya fruit weight and application thereof |
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