CN101575648A - Method for testing multi-PRC reaction of transgenic fruit - Google Patents

Abstract

The invention discloses a method for testing the multi-PRC reaction of a transgenic fruit. The method is characterized by including the following steps: (1) proper primer sequences are designed and selected; (2) the hexadecyl triethyl ammonium bromide method is used for extracting the genome DNA of a fruit to test and for determining the concentration of DNA; in the primer sequence of the step (1), the extracted DNA is used as a template for the multi-PCR amplification of 35S promoter, NOS terminator, plant endogenous rbcl gene; and (3) restriction enzyme is used for the restriction endonuclease reaction of the result of the multi-PCR amplification, and the agarose gel electrophoresis analysis and the DNA sequencing are used for judging whether the fruit contains transgenic ingredients or not. The method can successfully distinguish a transgenic fruit from a non-transgenic fruit, and can quickly, conveniently and accurately determine the transgenic ingredients in the fruits.

Description

The detection method of the multi-PRC reaction of transgenic fruit

Technical field

The invention belongs to transgenic fruit safety and detection technique field, be specifically related to a kind of multi-PCR detection method of transgenic fruit; The invention still further relates to the test kit that detects transgenic fruit.

Background technology

By genetic engineering technique foreign gene is directed in other specific organism, make it produce the forward genetic mutation, transformed biological genetic material, make its at aspects such as shape, nutritional quality, consumption qualities to the needed target transition of people, become a kind of new bio body with brand-new characteristic.This organism is called transgenic organism or genetic modified organism body, and (Genetically Modified Organisms GMOs), comprises transgenic animal, plant and microorganism.Genetically modified organism is directly edible, perhaps as the food that processes raw material and produce, be referred to as " genetically modified food " (Genetically Modified Foods, GMF).Yet transgenic organism brings risk also may for human health or ecotope when bringing welfare to the mankind.The Biosafety problem that transgenic organism causes comprises that transgenic organism is to the safety issue of human health with to the safety issue of natural ecological environment.Under the market economy background, genetically modified organism technology and products thereof exploitation, research and the marketization are existed bigger Economic Stimulus and driving factors, thereby cause the development of genetically modified organism technology and products thereof to have the tendency that is subjected to the commercial benefits domination.Because the extensive environment of transgenic organism discharges and genetically modified food commercialization production may bring certain environmental risk and healthy hidden danger, so need do further research to genetically modified crops.Though can't determine the transgenic technology safety issue of (comprising genetically modified food) at last now, its potential risk to ecotope and human health should not underestimated, and takes precautions against in possible trouble necessary.

Because the genetically modified organism security is paid close attention to by people, various countries all require the genetically modified food of import is carried out strict safety detection, guarantee Biosafety and human consumer's interests.China has begun genetically modified organism is implemented safety evaluation and identity management, but at present genetically modified food safety evaluation and management system also are in the starting stage, existing check authentication technique can't satisfy feeler mechanisies at different levels the various factors that influence security in the genetically modified food are carried out comprehensive and accurate mensuration requirement.

The technological line that the vegetalitas methods for detecting transgenic foods adopts has two, the one, to use PCR, Southern hybridization and detect the foreign gene that inserts, the hybridization detection technique of main PCR-based technology of these means and nucleic acid probe can detect accurately and fast whether alien gene (comprising goal gene, marker gene and primer) is arranged among the GMO; The 2nd, use ELISA, Western hybridization and detect the recombinant protein of expressing, mainly adopt the method for chemical analysis, gel electrophoresis and enzyme linked immunological, testing is comparatively numerous and diverse.At present, the U.S. and European Union member countries mainly adopt the pcr amplification technology that transgenic product is detected, and use the less of chemical analysis method.Round pcr is applied to methods for detecting transgenic foods, and its responsive fast and convenient characteristics are that other detection technique is incomparable.In addition, methods for detecting transgenic foods has also developed quantitative PCR, is used for determining sample GMO per-cent.Though fluorescent quantitative PCR technique can obtain the accurate quantitative result of dna profiling, quantitative PCR detector costs an arm and a leg, and considerable laboratory can only be to hang back.Along with the development of modern transgenic technology, transgene component contained in the genetically modified food is more and more, when containing unknown transgene component in the food, just need detect respectively every kind of rake sequence, could finally determine contained transgene component in the food.This checkout procedure need be carried out repeatedly the PCR reaction, not only spends the more time, and inspection cost is also very high.In recent years, the multiple PCR technique that grows up, two or more target gene sequences simultaneously can increase in a reaction.

About the genetically modified organism body detecting method can be divided into to measure nucleic acid and to measure protein substantially is two kinds on basis.Diverse ways has different characteristics, and is comprehensive, can be used for processed food based on the PCR method of measuring nucleic acid, and sensing range is than wide based on the immunological method of measuring protein, so use more extensive.

(Polymerase Chain Reaction PCR), is a kind of Protocols in Molecular Biology, is used to the specific dna fragmentation that increases in the polymerase chain reaction.The ultimate principle of round pcr is similar to the natural reproduction process of DNA, and its specificity depends on and target sequence two ends complementary Oligonucleolide primers.PCR is by sex change---annealing---extends three primitive reaction steps formations.Template DNA dissociates template DNA double-stranded DNA double-stranded or that form through pcr amplification after being heated to 93 ℃ of left and right sides certain hours, makes it to become strand, so that it combines with primer, for the lower whorl reaction is prepared; This is the denaturing step of template DNA; Template DNA is after heat denatured becomes strand, and temperature is reduced to about 55 ℃, and primer combines with the complementary sequence pairing of template DNA strand, and this is annealing (renaturation) step of template DNA and primer; Dna profiling---the primer binding substances is under the effect of TaqDNA polysaccharase, with dNTP is reaction raw materials, and target sequence is a template, presses base complementrity pairing and semiconservative replication principle, synthetic new and a template DNA chain complementary semiconservative replication chain, this is the extension step of primer.By the recirculation sex change---annealing---extend three processes, just can obtain more " semiconservative replication chain ", and this new chain can become next round-robin template again.Whenever finish a circulation and need 2～4 minutes, just can will wait to expand the goal gene amplification and amplify millions of times in 2～3 hours.

Hot resistant DNA polymerase---the Taq enzyme has milestone inthe for the application of PCR, this enzyme can tolerate high temperature more than 90 ℃ and non-inactivation, do not need each circulation enzyme-added, make round pcr become very simple and direct, also greatly reduce cost simultaneously, make round pcr be widely applied, and progressively be applied to clinical.Measuring transgenic organism based on the PCR method of measuring nucleic acid also is because of in this.

The gene element that transgenic cell changes in the transgenic organism generally comprises promotor (Promotor), reporter gene (Reporter Gene), goal gene (Target Gene), terminator (Terminator), and wherein promotor and terminator are necessary for expressing goal gene.What so far, transgenic plant were commonly used is cauliflower mosaic virus promoter (CaMV 35S) and agrobacterium tumefaciens terminator (NOS).By these special promotors of pcr amplification and terminator sequence, be the method that has or not transgene component in the at present the most frequently used evaluation food.

Be used for DNA extraction test kit and a series of detection method that genetically modified organism detects although developed, and the detection of transgene component in the fruit is still lacked research.Current both at home and abroad for the research of transgenic plant detection, mainly concentrate on the transgenic crops such as soybean, corn rare research for transgenic fruit., realized the screening of resistance glyphosate soybean Roundup Ready transgene component is detected by detection as Shirai etc. to 35S promoter, NOS terminator.Cao Jijuan etc. are to transgenic corns and roughing food thereof, have carried out the PCR qualitative detection as the gene of puffed rice, cooked maize rod, instant corn sheet.But PCR method is lower slightly to the raw-material remolding sensitivity genetically engineered soybean of transgenic corns starting material qualitatively, as the false negative quantity that detects the 35S promoter of 0.1% transgenic corns kind is 14, and the false negative quantity of the 35S promoter of corresponding genetically engineered soybean is 5.PCR method has developed into the method that detects transgenosis Roundup Ready soybean, presents high degree of specificity and susceptibility (0.01%GMF), is applicable to from the soybean to bean product, the work in-process and the finished product sample of soy-protein, Yelkin TTS and soya-bean oil.

In addition, to the detection of transgenic fruit, the domestic method that standard is not also arranged.In view of fruit often people directly eat, wherein contain transgene component and mostly do not pass through any processing treatment, its security more merits attention.The detection of transgenic fruit at present mainly contains the qualitative PCR method.The qualitative PCR method is widely adopted with its high sensitivity, better specificity and fast and convenient property, but the PCR method also exists problem: the gene efficient amplification may produce false positive results and be difficult to carry out detection by quantitative.Along with the development of modern transgenic technology, transgene component contained in the genetically modified food is more and more, when containing unknown transgene component in the food, just need detect respectively every kind of target sequence, could finally determine contained transgene component in the food.This checkout procedure need be carried out repeatedly the PCR reaction, not only spends the more time, and inspection cost is also very high.In order to make comprehensive evaluation and to implement effectively supervision transgenic fruit, foundation and popularization are accurately, the transgene component detection technique is extremely urgent fast and efficiently.

In recent years, the multiple PCR technique that grows up, two or more target gene sequences simultaneously can increase in a reaction.Multiplex PCR (multiplex PCR), claim multi-primers PCR or composite PCR again, it is to add primer more than two pairs in same PCR reaction system, amplifies the PCR reaction of a plurality of nucleic acid fragments simultaneously, its reaction principle, reaction reagent is identical with general PCR with operating process.Multiplex PCR be mainly used in multiple pathogenic micro-organism the time detection or evaluation and pathogenic micro-organism, the somatotype of some inherited disease and oncogene is identified.It at home and abroad still is blank that present stage is carried out transgenic research to fruit, and the present invention comes therefrom.

Summary of the invention

Main purpose of the present invention is to provide a kind of multi-PCR detection method of transgenic fruit, has solved that transgenic fruit is difficult to detect or easily cause problems such as false positive by PCR method commonly used in the prior art.

In order to solve these problems of the prior art, technical scheme provided by the invention is:

A kind of multi-PCR detection method of transgenic fruit is characterized in that said method comprising the steps of:

(1) designs and choose and comprise two pairs of following primer sequences;

35SFZMP1： SEQ ID No：1；

35SFZMP2： SEQ ID No：2；

nos FZMP1：SEQ ID No：3；

nos FZMP2：SEQ ID No：4；

(2) extract fruit genomic dna to be measured and measure DNA concentration with the cetyltriethylammonium bromide method; With the DNA that is extracted is that masterplate carries out the multiplex PCR amplification to 35S promoter, NOS terminator, plant endogenous rbcl gene in the PCR reaction system of the primer sequence that comprises step (1);

(3) restriction enzyme carries out endonuclease reaction to the multiplex PCR amplification, whether contains transgene component through agarose gel electrophoresis analysis and dna sequencing proof fruit.

Preferably, described primer sequence is three pairs of primer sequences:

35SFZMP1： SEQ ID No：1；

35SFZMP2： SEQ ID No：2；

nos FZMP1：SEQ ID No：3；

nos FZMP2：SEQ ID No：4；

rbcL-F： SEQ ID No：5；

rbcL-R： SEQ ID No：6。

Preferably, described fruit to be measured is selected from pawpaw, pears, hami melon, banana.

Preferably, measuring DNA concentration in the described step (2) comprises after the fixed doubly amount of the DNA dilution of extracting, with ddH ₂O is contrast, measures A by ultraviolet-visible pectrophotometer ₂₆₀, A ₂₈₀Value obtains.

DNA concentration (μ g/mL)=50 * A ₂₆₀* extension rate.From A ₂₆₀And A ₂₈₀Ratio Analysis DNA quality.

Preferably, PCR reaction system final concentration consists of in the step (2):

PCR damping fluid final concentration 10 *;

dNTPs 0.2～0.4mM；

The primer sequence final concentration is 0.2mM;

Template DNA 1～10ng/ μ L;

TaqDNA polysaccharase 0.04～0.08U/ μ L;

MgCl ₂Final concentration is 6mM;

Solvent is ddH ₂O;

The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 59 ℃ of annealing 1min, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min.

Preferably, the restriction enzyme site of endonuclease reaction is in the described step (3):

XmnI restriction enzyme site: 5 ' ... GAANN ↓ NNTTC...3 '

3’...CTTNN↑NNAAG...5’；

Mph restriction enzyme site: 5 ' ... ATGCA ↓ T...3 '

3’...T↑ACGTA...5’；

Van91 I restriction enzyme site: 5 ' ... CCANNNN ↓ NTGG...3 '

3’...GGTTN↑NNNNACC...5’；

EcoR V restriction enzyme site: 5 ' ... GAT ↓ ATC...3 '

3’...CT A↑TAG...5’；

Xba I restriction enzyme site: 5 ' ... T ↓ CTAGA...3 '

3’...AGATC↑T...5’。

Preferably, the restriction enzyme of endonuclease reaction is selected from the described step (3): PdmI (XmnI) restriction enzyme, EcoR V restriction enzyme, Mph restriction enzyme, Van91 I restriction enzyme, Xba I restriction enzyme.

Preferably, the agarose gel electrophoresis analysis is specific band whether to occur according to electrophoresis result behind 2% agarose gel electrophoresis in the described step (3), judges whether fruit to be measured contains corresponding foreign gene.

Preferably, in the described step (3) to whether being corresponding foreign gene by carrying out sequence homology analysis conclusive evidence after the DNA gene sequencing that amplifies.

Preferably, the step of extracting fruit genomic dna to be measured with the cetyltriethylammonium bromide method in the described step (2) may further comprise the steps:

Take by weighing an amount of sample, after being ground to homogenate on ice with the preextraction damping fluid, add CTAB and extract damping fluid, through fracturing cell walls, lysing cell film, remove steps such as secondary substances such as polysaccharide and polyphenol, sex change isolated protein, precipitate nucleic acids, removal RNA and concentration of DNA, obtain the DNA of sample tissue.

More preferred, may further comprise the steps:

After sample and preextraction buffered soln was ground to homogenate, the CTAB that adds 65 ℃ of preheatings extracted damping fluid and an amount of beta-mercaptoethanol, and 65 ℃ of mixings insulation 1h are cooled to room temperature, add chloroform/primary isoamyl alcohol, and are centrifugal to phase-splitting; Supernatant liquor adds Rnase A storage liquid, and 37 ℃ of insulation 30min add the Virahol of precooling, places more than the 20min for-20 ℃; Carrying out refrigerated centrifugation precipitation DNA.After abandoning supernatant liquid, add 70% washing with alcohol precipitation; Recentrifuge deposit D NA.

Highly preferred, may further comprise the steps:

Sample is moved into centrifuge tube with a small amount of quartzy after being ground to homogenate with preextraction buffered soln, the CTAB that adds 65 ℃ of preheatings extracts damping fluid and an amount of beta-mercaptoethanol, 65 ℃ of mixing insulation 1h, be cooled to room temperature, add chloroform/primary isoamyl alcohol, centrifugal to phase-splitting, supernatant liquor adds Rnase A storage liquid, 37 ℃ of insulation 30min; The Virahol that adds precooling is placed more than the 20min for-20 ℃; Carrying out refrigerated centrifugation precipitation DNA.After abandoning supernatant liquid, add 70% washing with alcohol precipitation 2-3 time; Recentrifuge deposit D NA on thieving paper, removes mouth of pipe back-off to residual ethanol with the suction of rifle head, and room temperature is air-dry; The DNA resolution of precipitate is in 1 * TE damping fluid; Remove part DNA and be used for the detection of 0.8% agarose gel electrophoresis, all the other DNA are standby in-20 ℃ of preservations.

Preferably, when sample is plant leaf, directly gets blade and grind; When sample is pulp organization, grind after sample made lyophilized powder.

Preferably, the preextraction damping fluid by its final concentration component is in the described method:

Tris·HCl 50mmol/L(pH 8.0)；

NaCl 0.7mol/L；

EDTA 10mmol/L(pH 8.0)。

Preferably, CTAB extraction damping fluid final concentration component is in the described method:

CTAB 2％(m/V)；

PVP 2％(m/V)；

Beta-mercaptoethanol 20mL/L.

Preferably, extracting fruit genomic dna to be measured with the cetyltriethylammonium bromide method in the described method steps (2) may further comprise the steps:

(A) according to the tissue for the treatment of measuring plants sample is carried out pre-treatment after, with preextraction damping fluid cryogrinding to homogenate, the CTAB that adds 65 ℃ of preheatings extracts damping fluid and an amount of beta-mercaptoethanol, mixing insulation 30min～1h; Be cooled to room temperature;

(B) add chloroform/primary isoamyl alcohol, centrifugal to phase-splitting, get supernatant liquor and add Rnase A storage liquid, 37 ℃ of insulation 30min; The Virahol that adds precooling is placed more than the 20min for-20 ℃; Carrying out refrigerated centrifugation precipitation;

(C) precipitation adds 70% washing with alcohol precipitation, the centrifugation precipitation; Adding the dissolving of TE damping fluid preserves.

Another object of the present invention is to provide a kind of multiple PCR detection kit of transgenic fruit, described test kit comprises the above-mentioned primer sequence of effective detection limit.

The present invention has enlarged the scope that detects external source goal gene in the transgenic fruit by selecting universal primer for use, and a PCR reaction can detect transgenic fruit 35S promoter, NOS terminator commonly used.According to the difference of fruit to be measured, can detect plant endogenous rbcl gene, the external source PRSV gene of transgenosis pawpaw.Simultaneously, the present invention select for use primer be each primer concentration be 0.2 μ M etc. increase under the molar conditions, amplified production can effectively separate by once common agarose gel electrophoresis, method is simple.The method that the present invention set up only just can detect above-mentioned foreign gene simultaneously by PCR reaction, and the sensitivity of detection reaches 0.1% level, and superiority is very obvious.

PCR damping fluid final concentration is 10 *, be meant that each component concentration of PCR damping fluid in the reaction system is 10 times of each concentration of component of 1 * PCR damping fluid; In the practical application, 10 * PCR damping fluid of desirable certain volume dilutes according to the concentration of each component in reaction system, as require PCR damping fluid final concentration be 2 *, then negate answers 10 * PCR damping fluid of 1/5 volume of system cumulative volume to get final product.10 * PCR damping fluid composition is: 100mM Tris-HCl pH 8.0,500mM KCl, 15mM MgCl ₂

The present invention is in the prior art on the regular-PCR technical foundation of transgenic fruit, adopts the concentration of the combination of at least two pairs of different primer sequences, different PCR damping fluids to set up with different primer sequence concentration and optimizes the multiple PCR technique system that detects transgenic fruit.

The target fragment of the primer amplification all was the short-movie section less than 500bp during transgenic product detected, because the DNA major part that the transgenic product after processing is extracted is the fragment of having degraded.In multi-PRC reaction, under high salt concn amplification is better for the primer of short-movie section amplified production, and high salt concentration can make the long segment amplified production be difficult to sex change and unwind, and is difficult to effectively be increased.Multiplex PCR of the present invention detects 35s, use during Nos the PCR buffer concentration be 10 *, primer concentration is 0.2mM, 60 ℃ of effective amplifications that both can guarantee each primer of annealing temperature have reduced the interference of primer dimer and the generation of non-special band again.Triple PCR and quadruple PCR detect 35s, select during Nos buffer concentration be 2 *, primer concentration is 0.2mM, 60 ℃ of effective amplifications that promptly can guarantee each primer of annealing temperature have reduced the generation of primer dimer again.

The fruit that the present invention detected is selected from pawpaw, pears, hami melon, banana; The external source goal gene of these fruit is comparatively common in present transgenic fruit, so the present invention has practicality.In addition, method of the present invention can shorten detection time, and single sample only detects from the specimen preparation to result needs 1 day time; It is conventional qualitative PCR and plain agar sugar detected through gel electrophoresis that the present invention detects used method, only needs conventional at present molecular biology reagent, and cost is lower; And detection sensitivity height of the present invention, 0.1% level that can be up to state standards and formulate detects than single primer PCR simultaneously, accuracy rate height of the present invention, and the false positive ratio is low.Operate comparison by different PCR instrument comparison and detection and different testing staff and all can obtain stable detected result.

Beneficial effect of the present invention is that only a PCR can fast, accurately detect current transgenic fruit, and sensitivity can reach 0.1%, and cost is low, the accuracy rate height, and false positive rate is low.

Description of drawings

Below in conjunction with drawings and Examples the present invention is further described:

Fig. 1 is embodiment of the invention evergreen tree leaflet tablet DNA (negative control) pcr amplification figure; Wherein 1 is λ Hind III marker, and 2,3 is the DNA with the extraction of evergreen tree tender leaf;

Fig. 2 is evergreen tree leaflet tablet DNA (negative control), the ddH of embodiment of the invention transgenosis pawpaw DNA, different weaker concns ₂O (blank) pcr amplification comparison diagram; Wherein 1 is 50bp DNAladder, and 2～7 are respectively with 10 times of transgenosis pawpaw DNA, evergreen tree tender leaf DNA, evergreen tree tender leaf DNA, evergreen tree tender leaf DNA dilutions, evergreen tree tender leaf DNA and dilute 20 times and ddH ₂O;

Fig. 3 is embodiment of the invention transgenosis pawpaw DNA (positive control), evergreen tree leaflet tablet DNA (negative control), ddH ₂O (blank), Hainan and safe pawpaw DNA, prosperous board pawpaw DNA are the pcr amplification comparison diagram of template; 1 is 50bp DNA ladder; 2 is Hainan and safe pawpaw DNA; 3 is prosperous board pawpaw DNA; 4 positive contrasts; 5 negative contrasts; 6 is blank;

Fig. 4 is a 35S gene comparison chart;

Fig. 5 is a NOS gene comparison chart;

Fig. 6 is a rbcl gene comparison chart;

Fig. 7 is embodiment of the invention hami melon DNA, Lao Xie board pawpaw DNA, banana DNA, pears DNA, positive control dna, evergreen tree leaflet tablet DNA (negative control), ddH2O (blank), is the pcr amplification comparison diagram of template; Wherein 1 is 50bp DNA ladder, and 2～9 template DNAs are respectively: 2-hami melon, 3-Lao Xie board pawpaw, 4-blank, 5-blank, 6-banana, 7-banana, 8-blank, 9-pears, 10-positive control, 11-negative control, 12-blank;

Fig. 8 is the contrast figure of the single pcr amplification of embodiment of the invention 35S gene and other template DNAs; 1 is 50bp DNA ladder in wherein being, 2～9 template DNAs are respectively: 2-hami melon, the prosperous board pawpaw of 3-, 4-Lao Xie board pawpaw, 5-and safe board pawpaw, 6-and safe pawpaw, 7-and safe pawpaw, 8-banana, the 9-pears, 10-positive control, 11-negative control, 12-blank;

Fig. 9 cuts contrast figure with other template DNAs for the embodiment of the invention to the pcr amplification product enzyme; 1 is 50bp DNA ladder, and 2～9 template DNAs are respectively: 2-hami melon, the prosperous board pawpaw of 3-, 4-Lao Xie board pawpaw, 5-and safe board pawpaw, 6-and safe pawpaw, 7-and safe pawpaw, 8-banana, 9-pears, 10-positive control, 11-negative control, 12-blank;

Figure 10 is a PRSV-rp gene gene comparison chart;

Figure 11 is a 35S gene restriction enzyme site synoptic diagram;

Figure 12 is a NOS gene restriction enzyme site synoptic diagram;

Figure 13 is a rbcl gene restriction enzyme site synoptic diagram;

Figure 14 cuts figure as a result for the single pcr amplification product enzyme of 35S; Wherein 1 is 50bp DNA ladder, and 2,3 is the single pcr amplification product of 35S;

Figure 15 cuts figure as a result for the another enzyme of the single pcr amplification product of 35S; Wherein 1,2 is the single pcr amplification product of 35S, and 3 is the single pcr amplification product of NOS, and 4,5 is the single pcr amplification product of rbcl, adds XmnI, EcoRV, NsiI, PflMI and XbaI enzyme respectively in 1～5, and 6 is 50bpDNA ladder.

Embodiment

For the technical scheme of more detailed statement foregoing invention, the following inventor lists specific embodiment and comes bright technique effect; It is emphasized that these embodiment are used to the present invention is described and are not limited to limit the scope of the invention.

The double PCR detection validation of 1 pair of transgenosis pawpaw of embodiment

1, the PCR primer is synthetic

PCR primer sequence of the present invention is the synthetic primer sequence; Primer sequence information such as following table:

Table 1 primer sequence information table

2, the extraction of sample DNA

Plant and instrument: Bio-rad PCR thermal cycler; Bio-rad electrophoresis apparatus and gel imaging system; Ultraviolet-visible pectrophotometer; Micropipet; Eppendorf centrifuge and refrigerated centrifuge; DK-80 type electric heating constant temperature tank; Freeze drying equipment; Various glasswares commonly used.

The material preparation:

Preextraction damping fluid: 50mmol/L TrisHCl (pH 8.0), 0.7mol/L NaCl, 10mmol/L EDTA (pH 8.0).

CTAB extracts damping fluid: add 2% (m/V) CTAB in the preextraction damping fluid, 2%PVP, 20mL/L beta-mercaptoethanol (using preceding adding).

Chloroform/primary isoamyl alcohol (24: 1; 70% ethanol; 1 * TE damping fluid: 10mmol/L TrisHCl (pH 8.0), 1mmol/L EDTA (pH 8.0); RNase A (10mg/mL): use 0.15mol/LNaCl, the 0.015mol/L citrate three sodium is made into 10mg/mL; Use preceding 100 ℃ of thermal treatment 15min to remove residual Dnase activity; Agarose; DNA relative molecular mass standard substance (λ HindIII); PCR reaction: 4 kinds of dNTP, primer 1, primer 2, Taq enzyme, DNA relative molecular mass standard substance (50bp ladder).Adopt the evergreen tree leaflet tablet as negative control.

Experimental procedure:

(evergreen tree leaflet tablet tissue is got 20mg-40mg to take by weighing an amount of transgenosis pawpaw and evergreen tree leaflet tablet sample, the transgenosis pawpaw is removed the peel and made powder in 20 ℃ of lyophilize 1-2 of fresh melon and fruit days, get the 0.1g lyophilized powder), sample is moved into the 5mL centrifuge tube with a small amount of quartzy after being ground to homogenate with 500 μ L preextraction buffered soln, the CTAB that adds 65 ℃ of preheatings extracts the beta-mercaptoethanol of damping fluid 500 μ L and 40mmol/L, 65 ℃ of mixing insulation 1h, be cooled to room temperature, add 450 μ L chloroform/primary isoamyl alcohol, centrifugal 10min (12000r/min) is to phase-splitting, add 5 μ L Rnase A storage liquid, 37 ℃ of insulation 30min; The Virahol that adds 600 μ L precoolings is placed more than the 20min for-20 ℃; Frozen centrifugation 10min (12000r/min), deposit D NA.After abandoning supernatant liquid, add 800 μ L, 70% washing with alcohol precipitation 2-3 time; Recentrifuge (12000r/min) 5min, deposit D NA on thieving paper, removes mouth of pipe back-off to residual ethanol with the suction of rifle head, and room temperature is air-dry; The DNA resolution of precipitate is in 50 μ L, 1 * TE damping fluid; Get 5 μ L DNA and detect through 0.8% agarose gel electrophoresis, all the other DNA are standby in-20 ℃ of preservations.

Get 100 times of gained DNA20 μ L dilutions, with ddH ₂O measures A for contrast with ultraviolet-visible pectrophotometer ₂₆₀, A ₂₈₀Value, and calculate its concentration, by the two ratio Analysis DNA quality.DNA concentration (μ g/mL)=50 * A ₂₆₀* extension rate.

3, pcr amplification, endonuclease reaction and gel electrophoresis

Adopt 2 primers right, respectively to 35S promoter, the NOS terminator, plant endogenous gene rbcL increases.Two-fold PCR reaction system is: 10 * amplification buffer, 2.5 μ L, dNTP final concentration 0.4mM, primer final concentration are 0.2 μ M, and MgCl2 final concentration 6mM, Taq archaeal dna polymerase are 1.5U, and the template DNA amount is 1 μ L, add the dd sterilized water cumulative volume is adjusted into 25 μ L.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 59 ℃ of annealing 1min, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min.Get 5-6 μ LPCR product, detect through 2% agarose gel electrophoresis.

The restriction enzyme site of described endonuclease reaction and as follows:

(1) XmnI restriction enzyme site: 5 ' ... GAANN ↓ NNTTC...3 '

3’...CTTNN↑NNAAG...5’

Its used damping fluid component is 10 * Buffer Tango ^TM: 330mM Tris-acetate (pH 7.9), 100mM magnesium acetate, 660mM potassium acetate, 1.0mg/ml BSA.

(2) Mph restriction enzyme site: 5 ' ... ATGCA ↓ T...3 '

3’...T↑ACGTA...5’

Its used damping fluid component is 10 * Buffer R:100mM Tris-HCl (pH 8.5), 100mMMgCl2,1000mM KCl, 1.0mg/ml BSA.

(3) Van91 I restriction enzyme site: 5 ' ... CCANNNN ↓ NTGG...3 '

3’...GGTTN↑NNNNACC...5’

Its used damping fluid component is 10 * Buffer R: with the used buffered soln of (2) Mph restriction enzyme site.

(4) EcoR V restriction enzyme site: 5 ' ... GAT ↓ ATC...3 '

3’...CTA↑TAG...5’

Its used damping fluid component is 10 * Buffer H:500mM Tris-HCl (pH7.5), 100mMMgCl2,10mM Dithiothreitol, 1000mM NaCl.

(5) Xba I restriction enzyme site: 5 ' ... T ↓ CTAGA...3 '

3’...AGATC↑T...5’

Its used damping fluid component is 10 * Buffer M:100mM Tris-HCl (pH 7.5), 100mM MgCl2,10mM Dithiothreitol, 500mM NaCl.

The endonuclease reaction operation steps is as follows:

(1) adopts PdmI (XmnI) restriction enzyme that 35S gene substance pcr amplification result is carried out endonuclease reaction, add PCR reaction mixture 10 μ L successively, ddH ₂O 16 μ L, 10 * Buffer TangoTM2 μ L, Xmn I 1 μ L, light and slow mixing, 10 seconds of low-speed centrifugal, at 37 ℃ of insulation 1h down, then 65 ℃ down insulation 20min heat stop.

(2) adopt EcoR V restriction enzyme that 35S gene substance pcr amplification result is carried out endonuclease reaction, add PCR reaction mixture 10 μ L successively, ddH ₂O 16 μ L, 10 * Buffer H, 2 μ L, EcoR V 1 μ L, light and slow mixing, 10 seconds of low-speed centrifugal, at 37 ℃ of insulation 1h down, then 65 ℃ down insulation 20min heat stop.

(3) adopt the Mph restriction enzyme that NOS gene substance pcr amplification result is carried out endonuclease reaction, add PCR reaction mixture 10 μ L successively, ddH ₂O 18 μ L, 10 * Buffer R, 2 μ L, Mph1 μ L, light and slow mixing, 10 seconds of low-speed centrifugal, at 37 ℃ of insulation 1h down, then 65 ℃ down insulation 20min heat stop.

(4) adopt the Van91I restriction enzyme that RBCL gene substance pcr amplification result is carried out endonuclease reaction, add PCR reaction mixture 10 μ L successively, ddH ₂O 18 μ L, 10 * Buffer R, 2 μ L, Van91I 1 μ L, light and slow mixing, 10 seconds of low-speed centrifugal, at 37 ℃ of insulation 1h down, then 65 ℃ down insulation 20min heat stop.

(5) adopt Xba I restriction enzyme that RBCL gene substance pcr amplification result is carried out endonuclease reaction, add PCR reaction mixture 10 μ L successively, ddH ₂O 16L, 10 * Buffer M, 2 μ L, Xba I 1 μ L, light and slow mixing, 10 seconds of low-speed centrifugal, at 37 ℃ of insulation 1h down, then 65 ℃ down insulation 20min heat stop.

With λ Hind III marker, the DNA and 50bp DNAladder, GMO pawpaw DNA, evergreen tree tender leaf DNA, evergreen tree tender leaf DNA, 10 times of evergreen tree tender leaf DNA dilutions, the evergreen tree tender leaf DNA that extract with the evergreen tree tender leaf dilute 20 times and ddH respectively ₂O carries out 2% agarose gel electrophoresis after the triple PCR amplification carried out of template; Result such as Fig. 1, Fig. 2; Wherein in the GMO pawpaw group amplification place three bands, and be that template only amplifies a band (rbcl gene band) with the different extension rates of evergreen tree DNA, 35s and no band are not arranged.And the different extension rates of evergreen tree DNA, a band brightness that amplifies also has respective change.

The triple PCR of 2 pairs of Hainan of embodiment and safe pawpaw, prosperous board pawpaw detects

1, the PCR primer is synthetic

PCR primer sequence of the present invention is the synthetic primer sequence; Primer sequence information such as following table:

Table 2 primer sequence information table

2, the extraction of sample DNA

Laboratory apparatus and experiment reagent are with embodiment 1.With the negative contrast of DNA of evergreen tree leaflet tablet extraction, with ddH ₂O is a blank, with the positive contrast of transgenosis pawpaw DNA, Hainan and safe pawpaw, two kinds of products of prosperous board pawpaw is detected.

Experimental procedure is similar to embodiment 1, and wherein the CTAB of DNA extraction extracts damping fluid and 70 ℃ of preheatings of beta-mercaptoethanol.Adopt three primers right during pcr amplification, respectively to 35S promoter, the NOS terminator, plant endogenous gene rbcL increases.10 * amplification buffer, 2.5 μ L in the triple PCR reaction system, the dNTP final concentration is 0.4mM, and the primer final concentration is 0.2 μ M, and MgCl2 final concentration 6mM, Taq archaeal dna polymerase are 2.0U, and the template DNA amount is 1 μ L, adds the dd sterilized water cumulative volume is adjusted into 25 μ L.The PCR reaction conditions is: 92 ℃ of pre-sex change 5min; 97 ℃ of sex change 30s, 56 ℃ of annealing 1min, 75 ℃ are extended 30s, 35 circulations; 75 ℃ are extended 10min.Get 5-6 μ LPCR product, detect through 2% agarose gel electrophoresis.

Detected result can see as shown in Figure 3, with safe pawpaw and prosperous board pawpaw DNA be that template (being No. 2 samples among Fig. 3) has amplified triple purpose bands, illustrate and there are transgene component in safe pawpaw, prosperous board pawpaw, be transgenic product probably.In order to determine the gene order of these purpose bands, the gene of triple purpose bands of amplification is checked order.And carry out sequence homology analysis with the DNAMAN analysis software.Triple purpose bands are respectively 35S gene, NOS gene, rbcl gene.The following Fig. 4 of analytical results～6.

As follows to the 35S sequencing result: SEQ ID No:9; Through the Genbank inquiry, compare situation as shown in Figure 4 with standard sequence, homology is 97.44%.

Because the NOS gene fragment is too small, be difficult for directly order-checking, to the order-checking of PCR product purification rear clone, the following SEQ ID of sequencing result No:10; Through the Genbank inquiry, actual amplification part is compared situation as shown in Figure 5 with standard sequence, and homology is 100%.

To the following SEQ ID of the rbcl gene sequencing result No:11 that amplifies, through the Genbank inquiry, compare situation as shown in Figure 6 with standard sequence, homology is 100%.

The triple PCR of 3 pairs of Lao Xie boards of embodiment pawpaw, hami melon, banana, pears detects

Choosing Lao Xie board pawpaw, hami melon, banana, pears is that given the test agent detects, step similar embodiment 1, and the negative contrast of DNA of extracting with the evergreen tree leaflet tablet is with ddH ₂O is a blank, with the positive contrast of transgenosis pawpaw DNA, Lao Xie board pawpaw, hami melon, banana, four kinds of products of pears is detected.Wherein the CTAB of DNA extraction extracts damping fluid and 60 ℃ of preheatings of beta-mercaptoethanol.Adopt three primers right during pcr amplification, respectively to 35S promoter, the NOS terminator, plant endogenous gene rbcL increases.Primer sequence information such as following table:

Table 3 primer sequence information table

10 * amplification buffer, 2.5 μ L in the triple PCR reaction system, the dNTP final concentration is 0.4mM, and the primer final concentration is 0.2 μ M, and MgCl2 final concentration 6mM, Taq archaeal dna polymerase are 2.0U, and the template DNA amount is 1 μ L, adds the dd sterilized water cumulative volume is adjusted into 25 μ L.The PCR reaction conditions is: 97 ℃ of pre-sex change 5min; 92 ℃ of sex change 30s, 60 ℃ of annealing 1min, 70 ℃ are extended 40s, 40 circulations; 70 ℃ are extended 12min.Get 5-6 μ LPCR product, detect through 2% agarose gel electrophoresis.

The results are shown in Figure 7.As seen from Figure 7, (No. 3 sample) amplifies triple DNA bands simultaneously in the Lao Xie board pawpaw, and be identical with the positive control situation, so the Lao Xie pawpaw has transgene component.And be in the triple PCR reaction carried out of template with hami melon, banana, pears, only amplified plant endogenous gene rbcl gene, identical with negative control cases, so do not have transgene component in the hami melon of being bought, banana, pears.In order to determine further whether various fruit are transgenic product, the 35S gene are carried out single pcr amplification, result such as Fig. 8, Fig. 9.

Substance PCR reaction system is: 10 * amplification buffer, 2.5 μ L, and the dNTP final concentration is 0.2mM, and the primer final concentration is 0.2 μ M, and MgCl2 final concentration 6mM, Taq archaeal dna polymerase are 1U, and the template DNA amount is 1 μ L, adds the dd sterilized water cumulative volume is adjusted into 25 μ L.The PCR reaction conditions is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 59 ℃ of annealing 1min, 72 ℃ are extended 30s, 35 circulations; 72 ℃ are extended 10min.Get 5-6 μ LPCR product, detect through 2% agarose gel electrophoresis.

As can be seen from Figure 8, be that template is carried out single PCR reaction with prosperous board pawpaw, Lao Xie board pawpaw and safe board pawpaw, all amplified the 35S gene; And be that template does not have the purpose band to occur with hami melon, banana and pears.With PdmI (XmnI) restriction enzyme the 35S gene that amplifies is cut, experimental result as shown in Figure 9.Can see that sample number is to have the 100bp of being about enzyme to cut the product segment in 3～7 and 10 to exist, and the specificity of PCR reaction has been described.Present embodiment also checks order to the PRSV-rp gene, and the PRSV-rp gene order that records is SEQ No:12., through the Genbank inquiry, with standard sequence comparison situation, homology is 100%, as Figure 10.Can judge that in conjunction with Fig. 8 and Fig. 9 Lao Xie board pawpaw is a transgenic product, hami melon, pears, banana are the non-transgenic product.So, can judge more accurately whether the fruits product is transgenic product by triple PCR reaction and single PCR and endonuclease reaction bonded method.

The endonuclease reaction conclusive evidence of 4 pairs of amplified productions of embodiment

By the 35S gene order is analyzed, can cut by being limited property restriction endonuclease in following base site:

Table 4 restriction enzyme digestion sites

Site/bp	32	58	61	61	64	64	98
								Restriction enzyme	BsiI	XmnI	Bsc91I	BbvII	Bsc91I	BbvII	EcoRV

Can select XmnI and EcoRV to carry out endonuclease reaction, as Figure 11.XmnI can be cut into 58bp and 100bp two portions with 35S (158bp) gene in theory; EcoRV can be cut into 35S 98bp and 60bp.

The NOS gene order is analyzed, and in the 125bp base that amplifies, the NOS gene only can be cut by NsiI at the 83bp place, as shown in figure 12.Can be cut to 83bp and 42bp two portions in theory.

The rbcl gene order is analyzed, can be cut by being limited property restriction endonuclease in following base site:

Table 5 restriction enzyme digestion sites

Site/bp	172	212	224	279	384
						Restriction enzyme	RleAI	BssHII	XbaI	PflMI	BstD102

Select PflM I and Xba I to carry out endonuclease reaction, as Figure 13.PflM I can be cut into rbcl gene (433bp) 279bp and 154bp two portions in theory, and Xba I can be cut into the rbcl gene 224bp and 209bp two portions.

35S gene PCR amplified production is carried out endonuclease reaction, adopt PdmI (XmnI) restriction enzyme, be incubated 1h down at 37 ℃, be incubated 20min down at 65 ℃ then, behind 2% agarose gel electrophoresis, observe (result such as Figure 14), can see that 35S gene (being about 158bp) is cut into is about two sections of 58bp and 100bp, is consistent with hypothesis.Thereby further proved PCR method accurately and reliably, products therefrom really is the Camv35S promotor.

Cut 35S with XmnI, with EcoRV cutting Camv 35S, with Mph1103 (NsiI) cutting NOS, with Van91I (PflMI) cutting rbcl, rbcl carries out endonuclease reaction with the XbaI cutting, the gained result is as shown in figure 15: can see that 35S (158bp) can be cut into by the XmnI enzyme is about two sections of 58bp and 100bp, can be cut into about 98bp and 60bp by EcoRV; Nos (125bp) can be cut to 83bp by Mph1103 (AvaIII); Rbcl (433bp) can be cut to 279bp and 154bp by Van91I (PflMI), is cut to 224/209bp by XbaI.With predict and meet substantially, shown that the PCR product is non-false positive.

Extracted pawpaw by using the CTAB method in the embodiment of the invention, hami melon, banana, the DNA of pears etc., the multiplex PCR method that method is set up is to 35S promoter, the NOS terminator, plant endogenous rbcl gene, transgenosis pawpaw external source PRSV rdrp gene increases, by endonuclease reaction to 35S, NOS, the rbcl gene cuts, and to 35S, NOS, rbcl, the Prsv-rp gene checks order, the result has proved the specificity of PCR reaction, by single PCR product is carried out restriction analysis, can determine further whether the fruits product contains transgene component, demonstration this method can successfully be distinguished transgenic fruit and non-transgenic fruit.Its validity is embodied in, by to transgene component in the fruit quick, convenient, measure accurately.

Above-mentioned example only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.

SEQUENCE LISTING

＜110〉Chen Jun Xiao Lindai haze Chen Jun cutting edge of a knife or a sword Zhu Zhen China

＜120〉detection method of the multi-PRC reaction of transgenic fruit

<160>12

<170>PatentIn version 3.5

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<400>1

ccgacagtgg tcccaaagat g 21

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<400>2

agaggaaggg tcttgcgaag g 21

<210>3

<211>20

<212>DNA

＜213〉artificial sequence

<400>3

gaatcctgtt gccggtcttg 20

<210>4

<211>23

<212>DNA

＜213〉artificial sequence

<400>4

gcgggactct aatcataaaa acc 23

<210>5

<211>22

<212>DNA

＜213〉artificial sequence

<400>5

aatcttctac tggtacatgg ac 22

<210>6

<211>23

<212>DNA

＜213〉artificial sequence

<400>6

tcatcatctt tggtaaaatc aag 23

<210>7

<211>20

<212>DNA

＜213〉artificial sequence

<400>7

ataccaatgg tgatgatctc 20

<210>8

<211>20

<212>DNA

＜213〉artificial sequence

<400>8

ttacttagac tggtgaaaca 20

<210>9

<211>119

<212>DNA

＜213〉cauliflower mosaic virus (Cauliflower mosaic virus)

<400>9

agccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg 60

tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttggaccc actgtcgga 119

<210>10

<211>289

<212>DNA

＜213〉agrobacterium tumefaciens (Agrobacterium tumefaciens)

<400>10

tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 60

ttacgaattc gagctcggta cccggggatc ctctagagat tgaatcctgt tgccggtctt 120

gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 180

tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatc gtcgacctgc 240

aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggg 289

<210>11

<211>389

<212>DNA

＜213〉plant chloroplast (ribulose 1,5-bisphosphate carboxylose Large Gene)

<400>11

ccagccttga tcgttacaaa ggacgatgct acggcatcga gcccgttcct ggagaagaaa 60

gtcaatttat tgcttatgta gcttacccct tagacctttt tgaagaaggt tctgttacta 120

acatgtttac ttccattgtg ggtaatgtat ttgggttcaa agccctgcgc gctctacgtc 180

tagaggatct gcgaatccct cctgcttata ttaaaacttt ccagggacca cctcatggta 240

tccaagttga aagagataaa ttgaacaagt atggtcgtcc cctattagga tgtactatta 300

aacctaaatt ggggttatcc gctaaaaact acggtagagc ggtttatgaa tgtctacgcg 360

gtggacttga ttttaccaaa gatgatgaa 389

<210>12

<211>541

<212>DNA

＜213〉prv (Papaya ringspot virus)

<400>12

gaactgttct tgactcattc tctggttcat ttgctgagct tggacttaag tacgatttca 60

ctcaaaggca ccgaaataaa caagatttgt ggttcatgtc acatcgaggt attctgatcg 120

atgacatcta tatccccaaa cttgaacctg agagaattgt ggccatcctt gagtgggata 180

aatccaagct tccagaacat cgattggaag ctatcacagc agcaatgata gagtcatggg 240

gatatgagga gctaacgcac cagatccgca ggttctatca atgggtgctt gagcaggctc 300

cattcaatga attagcgaag cagggcaggg ctccatatgt gtctgaggtt ggattgaggc 360

gcttatacac tagtaagcgt gggtcaatgg atgaattgga ggcctacata gataaatatt 420

ttgagcgaga aaggggagat tcgcctgagc tactggtgta ttatgaatcg agaagcactg 480

atgatcatca gttaacttgc ggcagtaaaa cacatgtgtt tcaccagtcc taaagtaaaa 540

a 541

Claims (10)

1. the multi-PCR detection method of a transgenic fruit is characterized in that said method comprising the steps of:

(1) designs and choose and comprise following two pairs of primer sequences at least;

35SFZMP1： SEQ ID No：1；

35SFZMP2： SEQ ID No：2；

nos FZMP1： SEQ ID No：3；

nos FZMP2： SEQ ID No：4；

(2) extract fruit genomic dna to be measured and measure DNA concentration with the cetyltriethylammonium bromide method; With the DNA that is extracted is that masterplate carries out the multiplex PCR amplification to 35S promoter, NOS terminator, plant endogenous rbcl gene in the PCR reaction system of the primer sequence that comprises step (1);

(3) restriction enzyme carries out endonuclease reaction to the multiplex PCR amplification, whether contains transgene component through agarose gel electrophoresis analysis and dna sequencing proof fruit.

2, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that described primer sequence is to being three pairs of primer sequences:

35SFZMP1： SEQ ID No：1；

35SFZMP2： SEQ ID No：2；

nos FZMP1： SEQ ID No：3；

nos FZMP2： SEQ ID No：4；

rbcL-F： SEQ ID No：5；

rbcL-R： SEQ ID No：6。

3, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that described fruit to be measured is selected from pawpaw, pears, hami melon, banana.

4, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that measuring in the described step (2) DNA concentration and comprises after the fixed doubly amount of the DNA dilution of extracting, with ddH ₂O is contrast, calculates by ultraviolet-visible pectrophotometer mensuration A260, A280 value.

5, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that PCR reaction system final concentration consists of in the step (2):

PCR damping fluid final concentration 10 *;

dNTPs 0.2～0.4mM；

The primer sequence final concentration is 0.2mM;

Template DNA 1～10ng/ μ L;

TaqDNA polysaccharase 0.04～0.08U/ μ L;

MgCl ₂Final concentration is 6mM;

Solvent is ddH ₂O;

The PCR reaction conditions is: 92～97 ℃ of pre-sex change 4～9min; 92～97 ℃ of sex change 20～40s, 56～60 ℃ of annealing 40～70s, 70～75 ℃ are extended 20～40s, 30～40 circulations; 70～75 ℃ are extended 8～12min.

6, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that the restriction enzyme site of endonuclease reaction in the described step (3) is:

XmnI restriction enzyme site: 5 ' ... GAANN ↓ NNTTC...3 '

3’...CTTNN↑NNAAG...5’；

Mph restriction enzyme site: 5 ' ... ATGCA ↓ T...3 '

3’...T↑ACGTA...5’；

Van91 I restriction enzyme site: 5 ' ... CCANNNN ↓ NTGG...3 '

3’...GGTTN↑NNNNACC...5’；

EcoR V restriction enzyme site: 5 ' ... GAT ↓ ATC...3 '

3’...CTA↑TAG...5’；

Xba I restriction enzyme site: 5 ' ... T ↓ CTAGA...3 '

3’...AGATC↑T...5’。

7, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that the restriction enzyme of endonuclease reaction in the described step (3) is selected from: PdmI (XmnI) restriction enzyme, EcoR V restriction enzyme, Mph restriction enzyme, Van91 I restriction enzyme, Xba I restriction enzyme.

8, the multi-PCR detection method of multiple transgenic fruit according to claim 1, it is characterized in that the agarose gel electrophoresis analysis is specific band whether to occur according to electrophoresis result behind 2% agarose gel electrophoresis in the described step (3), judges whether fruit to be measured contains corresponding foreign gene.

9, the multi-PCR detection method of multiple transgenic fruit according to claim 1 is characterized in that extracting fruit genomic dna to be measured with the cetyltriethylammonium bromide method in the described method steps (2) may further comprise the steps:

(A) according to the tissue for the treatment of measuring plants sample is carried out pre-treatment after, with preextraction damping fluid cryogrinding to homogenate, the CTAB that adds 60～70 ℃ of preheatings extracts damping fluid and an amount of beta-mercaptoethanol, mixing insulation 30min～1h; Be cooled to room temperature;

(B) add chloroform/primary isoamyl alcohol, centrifugal to phase-splitting, get supernatant liquor and add Rnase A storage liquid, 37 ℃ of insulation 30min; The Virahol that adds precooling is placed more than the 20min for-20 ℃; Carrying out refrigerated centrifugation precipitation;

(C) precipitation adds 70% washing with alcohol precipitation, the centrifugation precipitation; Adding the dissolving of TE damping fluid preserves.

10, a kind of multiple PCR detection kit of transgenic fruit is characterized in that described test kit comprises the described primer sequence of claim 1 of effective detection limit.

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