CN105603078A - Detection method for transgenic ingredients in tomato sauce - Google Patents
Detection method for transgenic ingredients in tomato sauce Download PDFInfo
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- CN105603078A CN105603078A CN201610061018.1A CN201610061018A CN105603078A CN 105603078 A CN105603078 A CN 105603078A CN 201610061018 A CN201610061018 A CN 201610061018A CN 105603078 A CN105603078 A CN 105603078A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a detection method for transgenic ingredients in tomato sauce, and belongs to the technical field of biology. The method includes the steps that the transgenic ingredients, endogenous standard genes and exogenous standard genes are determined; a multiple amplification primer is prepared; sampling and mixing are conducted to obtain a mixed sample; a genome in the mixed sample is extracted, and the exogenous standard genes are added to obtain mixed nucleic acid; a high-throughput sequencing library is constructed; high-throughput sequencing is conducted on the high-throughput sequencing library to obtain a sequencing fragment set; the number of sequencing fragments of the transgenic ingredients in the sample to be detected, the number of sequencing fragments of the endogenous standard genes and the number of the sequencing fragments of the exogenous standard genes are obtained; according to the number of the sequencing fragments of the exogenous standard genes, whether an experiment is successful or not is judged, and the content of the transgenic ingredients in the sample to be detected is calculated; according to the content of the transgenic ingredients, whether the sample to be detected contains the transgenic ingredients or not is judged. Multiple transgenic ingredients in the sample to be detected can be quantitatively detected at the same time.
Description
Technical field
The present invention relates to field of biological detection, particularly a kind of detection method of catsup transgene component.
Background technology
Tomato is the important vegetables of China, and catsup is the concentrated tomato products of making of fresh tomato, is conventional flavouring,Because the transgenic technology of tomato products is ripe, and transgene tomato product needed strictly examines, and therefore needs transgenosisCatsup is supervised, and detection of GMOs is the technical support of transgenosis supervision.
Existing detection of GMOs technology mainly detects nucleic acid or protein, wherein, and based on the detection method of nucleic acidMain flow process is: extract the nucleic acid in testing sample, (PolymeraseChainReaction, polymerase chain is anti-to utilize PCRShould) method amplification sample in transgene component, utilize real-time quantitative, agarose electrophoresis or chip technology to detect transgenosis and becomeDivide and whether exist.
Realizing in process of the present invention, inventor finds that prior art at least exists the one in following problem:
Transgene component is varied, and prior art once can only detect wherein a kind of or a few, therefore, needPossible multiple transgenosis composition is detected one by one, just can be defined as " non-transgenic " product; What generally detect is corotationThe transgene component of changing, instead of foreign gene itself, therefore, for marker-free transgenic, adopt existing detection sideMethod can lose efficacy; Detecting is qualitative detection substantially, but quantitatively detection has again its current demand, for example, and the transgenosis of adjacent fieldA small amount of pollen envelop of tomato variety has passed in non-transgenic kind, and in the time of qualitative detection, non-transgenic food will be mistaken forGM food.
Summary of the invention
In order to solve the problem of prior art, the embodiment of the present invention provides a kind of detection side of catsup transgene componentMethod. Described technical scheme is as follows:
The embodiment of the present invention provides a kind of detection method of catsup transgene component, and described method comprises:
Determine endogenous standard gene in transgene component, the described testing sample that needs in testing sample to detect and described inThe external source standard gene of testing sample, described testing sample is the catsup processing by tomato;
For the preparation of the test section of amplification described transgene component, described endogenous standard gene and described external source standard geneThe multiplex amplification primer in territory;
Described testing sample is sampled and mixed, obtain biased sample;
Extract the genome of described biased sample;
In the genome of described biased sample, add described external source standard gene, obtain mixing nucleic acid;
Utilize and mix nucleic acid described in described multiplex amplification primer pair and increase, obtain amplified production, utilize described amplificationProduct builds high-throughput sequencing library;
Described high-throughput sequencing library is carried out to high-flux sequence, obtain the slice groups that checks order;
Analyze described order-checking slice groups, obtain the order-checking fragment of transgene component described in described testing sample quantity,The quantity of the quantity of the order-checking fragment of described endogenous standard gene and the order-checking fragment of described external source standard gene;
According to the quantity of the order-checking fragment of described external source standard gene, whether judgment experiment is successful;
If described Success in Experiment, calculates the content of transgene component described in described testing sample kind;
Judge in described testing sample, whether to contain transgene component according to the content of described transgene component.
Particularly, described transgene component is external source functional gene, resistant maker gene, promoter, terminator and external sourceInsert at least one in flanking sequence.
Particularly, the single copy gene in the genome that described endogenous standard gene is described testing sample.
Particularly, described external source standard gene is not present in all biologies.
Particularly, described judgment experiment whether successfully method be: when the number of the order-checking fragment of described external source standard geneThe quantity of amount and the order-checking fragment of described endogenous standard gene is equal >=when α 1, and Success in Experiment; When described external source standard geneWhen quantity < the α 1 of the order-checking quantity of fragment or the order-checking fragment of described endogenous standard gene, the failure of an experiment; Wherein, α 1 is for sentencingDetermine threshold value.
Particularly, described judge method that whether described testing sample contain transgene component as: when appointing that needs detectWhile anticipating a kind of content >=α 2 of described transgene component, judge that described testing sample contains transgene component; Described in all, turnWhen content < the α 2 of gene element, judge that described testing sample does not contain transgene component; Wherein, α 2 is decision threshold.
Particularly, the method for calculating the content of transgene component described in described testing sample kind is: described in m kind, turn baseBecause the computing formula of the content of composition isWherein, i is described m kind transgene componentI test zone, n1 is the number of the described test zone of described m kind transgene component, bi is described m kind transgenosisThe quantity of the described order-checking fragment of the i of composition described test zone, k is endogenous standard gene described in k kind, described in n3 isThe number of endogenous standard gene, j is j test zone of endogenous standard gene described in k kind, n2 is that described k kind is endogenousThe number of the described test zone of standard gene, aj is the institute of test zone described in the j kind of the endogenous standard gene of described k kindState the quantity of order-checking fragment; N is the sum of the described test zone of all described endogenous standard genes.
Particularly, the ratio of the quality of described external source standard gene and the genomic gross mass of described biased sample is greater than1/100000。
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: detection method provided by the invention can beGeneral between different testing samples, the different target gene detecting, can any multiple transgenosis that needs detection of disposable detectionComposition, thus judge whether testing sample contains transgene component, and the method can realize quantitative detection, and testing result is almostThere is no lower limit, result accurately and reliably, is that prior art does not reach.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, will do into one embodiment of the present invention belowStep ground is described in detail. In the embodiment of the present invention, operating process or working specification unreceipted or that describe in detail are common molecule lifeThe thing known operation of personnel that learns a skill. In the embodiment of the present invention, not marked reagent or biomaterial are and sell on marketCommon agents or biomaterial, be common Protocols in Molecular Biology personnel known, and can on market, buy.
Embodiment
Two alanyl phosphorus resistances (Biolaphosresistance, Bar) gene is imported strong by agrobacterium-mediated transformationRich tomato variety, after 3 generations of selfing purifying, obtains the strong rich tomato of transformation, is processed into catsup, as the test sample for the treatment of of the present embodimentProduct. Wherein, strong rich tomato is transgene tomato acceptor kind, is cultivated by the Chinese Academy of Agricultural Sciences vegetables, in the present embodiment, usesStrong rich tomato seeds is preserved and is bred by Jianghan University.
Catsup detection of GMOs method, concrete steps are as follows:
Step 1, determine the endogenous standard gene in transgene component, the testing sample that needs in testing sample to detect and treatThe external source standard gene of test sample product, concrete grammar is as follows: testing sample is catsup, and in the present embodiment, testing sample is for changingThe catsup that strong rich tomato after making processes, transgene component, endogenous standard gene and external source standard gene all >=1,Transgene component can be that external source functional gene, resistant maker gene, promoter, terminator and external source are inserted in flanking sequenceAt least one proceeds to the external source not being present in strong rich tomato variety by transgenic technology means in strong rich tomato varietyNucleotide sequence; Endogenous standard gene is the single copy gene in the genome of testing sample. In the present embodiment, endogenous standard baseCause is unique sequence while comparison on testing sample genome by NCBI (http://www.ncbi.nlm.nih.gov/); OutwardSource standard gene is not present in all biologies, and in the present embodiment, the external source standard gene using is ERCC04 gene, itsWhen homology is compared on NCBI, do not find homologous sequence, can judge that external source standard gene is not present in all biologies. At thisIn embodiment, totally 5 kinds of the transgene components of detection, a kind of endogenous standard gene, a kind of external source standard gene, its relevant information is in Table1, in table 1, listed transgene component not only comprises Bar gene, and it is external source functional gene, also comprises other transgene components, thisA little transgene components are widely used in tomato, and therefore, the detection method that the present embodiment provides has versatility. Base in table 1Because sequence has two kinds of expression modes, one is directly to write out gene order, and another kind is the gene numbering of NBCI, the survey in table 1The position of the digitized representation base in order region, the position of the 1st base of this gene is defined as 1.
Table 1 is relevant information and the testing result of detected gene in embodiment mono-
In table 1 "/" indicate without.
Step 2, for the preparation of amplification transgene component, endogenous standard gene and external source standard gene multiplex amplification drawThing, concrete grammar is as follows:
The acquisition process of multiplex amplification primer is as follows: login match Mo Feishier company multiple PCR primer designs webpage onlineHttps: //ampliseq.com/, selects " DNAHotspotdesigns at " Applicationtype " option(single-pool) ". If select multi-pool, multiplex PCR will divide multitube to carry out, and cost can increase to some extent, andThe primer of single-pool only needs multiplex PCR one time, saves cost, so the present embodiment is selected single-pool.The sequence of each gene listed in table 1 is coupled together with 100 N, form an artificial reference genome, andIn " Selectthegenomeyouwishtouse " option, select, after " Custom ", to upload artificial reference genome.DNAType (type) option is selected " StandardDNA " (standard DNA). In AddHotspot option, every in table 1The homology that gene is random selects 1 homologous sequence obtaining by the comparison of NCBI homology is all lower than 80% test sectionTerritory. Wherein, test zone refers to the amplification region of multiplex PCR, is also the region of high-flux sequence, due to test zone and otherSpecies comparison, without obvious homologous sequence, therefore, can represent detected gene, and after high-flux sequence, test zone is depositedIn order-checking fragment, show that detected gene exists, the quantity of the order-checking fragment of test zone, has represented detected geneAmount. In addition, homologous sequence does not comprise the sequence in the primary source species of transgene component, for example, and the CryIAc gene in table 1Primary source species be thuringiensis (Bacillusthuringiensis, be called for short Bt), therefore, homology comparisonPicture is not comprised to the genome of thuringiensis. In order to ensure the versatility of the embodiment of the present invention, in downloading on NCBIDifferent sequences in the kind of source standard gene, compare by homology, obtain the conservative region between sequence, endogenous standard geneTest zone is chosen as in kind of the conservative region of inner boundary sequence. Further fill in the initial sum end of each test zone of acquisitionStop bit is put, and finally clicks " Submittargets " button and submits to, obtains the order of the amplimer of each gene in amplification table 1Row. In multiplex amplification primer, the amplification efficiency of every heavy primer, all between 95%～105%, therefore, need to be verified multiplex amplificationThe amplification efficiency of primer. Concrete verification method is: by Bar base in Sangon Biotech's synthetic table 1The amplimer sequence of the test zone of the sequence of cause and amplification Bar gene, taking the sequence of the Bar gene that synthesizes as template, pressesMatch the operation manual (PartNumber4376784 of the StepOne real-time PCR of Mo Feishier company according to the U.S.Rev.E) amplification efficiency of the amplimer of the test zone of detection amplification Bar gene, if amplification efficiency is not at 95%-105%Between, need to redefine the test zone of Bar gene, design and verify the efficiency of the new amplimer of Bar gene, straightTo its efficiency till between 95%-105%. In the same way, the amplification of the amplimer of other gene effect in proof list 1Rate. The amplification efficiency of final selected amplimer, sequence and corresponding amplification region be in table 1, all final selected expansionsIncrease primer and be called multiplex amplification primer, multiplex amplification primer is synthetic and with the form of mixing material by U.S. match Mo Feishier companyOffer user. The multiple PCR technique that the present embodiment adopts match Mo Feishier company of the U.S. to provide, it can increase many simultaneouslyTo 12000 test zones, therefore, the existing all transgene components that need detection of the capable disposable detection of the present invention.
Step 3, testing sample sampled and mixed, obtaining biased sample, concrete grammar is as follows:
By testing sample reference standard " sampling of genetically modified plants and products thereof composition detection " (standard No.: the Ministry of Agriculture 2031Number bulletin-19-2013) bulk goods in the methods of sampling of soya-bean oil sample, extracted sample to be tested is fully mixed,Obtain biased sample.
The genome of step 4, extraction biased sample, concrete grammar is as follows:
Reference standard " genetically modified plants and products thereof composition detection DNA extracts and purifying " (standard No.: No. 1485, the Ministry of AgricultureBulletin-4-2010) in the method for non-grease fluid-like state sample carry out extracting after pretreatment the genome of biased sample, extractBe the genomic nucleic acids of biased sample.
Step 5, in the genome of biased sample, add external source standard gene, obtain mixing nucleic acid, concrete grammar is as follows:
Utilize the double-stranded DNA program in spectrophotometer (Quawell company of the U.S. produces, and model is Q5000), detection obtainsThe genomic concentration of the biased sample obtaining. The ratio of the quality of external source standard gene and the genomic gross mass of biased sampleBe greater than 1/100000, this ratio is used for ensureing under normal sequencing throughput (>=1M check order fragment), can in high-flux sequence dataWith detect >=10 external source standard genes, if high-flux sequence flux is lower or higher, can do corresponding tune to this ratioWhole. In the present embodiment, the genomic gross mass of biased sample is 5564ng, and the external source standard gene adding is 5.564ng, addsEntering ratio is 1/1000, obtains and mixes nucleic acid.
Step 6, utilize multiplex amplification primer pair mixing nucleic acid to increase, obtain amplified production, utilize amplified production structureBuild high-throughput sequencing library, concrete grammar is as follows:
Utilize library construction kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345) to buildHigh-throughput sequencing library. This library construction kit comprises following reagent: 5 × IonAmpliSeqTMHiFiMix, FuPa examinationAgent, conversion reagent, sequence measuring joints solution and DNA ligase. The method of library construction is by the operator of this library construction kitVolume " IonAmpliSeqTMLibraryPreparation " (publication number: MAN0006735, version: A.0) carry out. Multiplex PCRAmplification system as follows: 5 × IonAmpliSeqTMThe mixing material 4 μ l of the multiplex amplification primer of HiFiMix4 μ l, preparation,Mix nucleic acid 10ng and without enzyme water 11 μ l. The amplification program of multiplex PCR is as follows: 99 DEG C, and 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 pointsClock) × 25 circulations; 10 DEG C of insulations. Utilize FuPa reagent to digest after primer unnecessary in multiplex PCR amplified production, then carry outPhosphorylation, concrete grammar is: in the amplified production of multiplex PCR, add 2 μ lFuPa reagent, after mixing, on PCR instrument by asLower program reaction: 50 DEG C, 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a isContain the solution through the amplified production of phosphorylation. The amplified production of phosphorylation is connected to upper sequence measuring joints, and concrete grammar is: toIn mixture a, add conversion reagent 4 μ l, sequence measuring joints solution 2 μ l and DNA ligase 2 μ l, after mixing, on PCR instrument, press as followsProgram reaction: 22 DEG C, 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed liquor b. Utilize the ethanol intermediate processing of standardAfter purifying mixed liquor b, be dissolved in 10 μ l without in enzyme water. Utilize American I nvitrigen company to producedsDNAHSAssayKit (article No. is Q32852) also detects according to its description, obtains after the mass concentration of mixed liquor b, by purifyingAfter mixed liquor b be diluted to 15ng/ml, obtain the high-throughput sequencing library of the about 100pM of concentration.
Step 7, high-throughput sequencing library is carried out to high-flux sequence, obtain the slice groups that checks order, concrete grammar is as follows:
Utilize the high-throughput sequencing library and the kit IonPITemplateOT2200Kitv2 (U.S. that obtainInvirtrigen company produces, and article No. is 4485146) ePCR (EmulsionPCR, emulsion polymerization enzyme chain before checking orderReaction) amplification, method of operating is undertaken by the operation manual of this kit. Utilize ePCR product and kit IonPISequencing200Kitv2 (invirtrigen company of the U.S. produces, and article No. is 4485149) is in Proton bis-generations high passOn amount sequenator, carry out high-flux sequence, method of operating is undertaken by the operation manual of this kit. In the present embodiment, high fluxOrder-checking amount is set to 1M order-checking fragment (1M=,100 ten thousand), and order-checking length is set to 500cycle (circulation), after order-checking finishes, obtainsSlice groups must check order.
Step 8, analysis order-checking slice groups, quantity, the endogenous mark of the order-checking fragment of transgene component in acquisition testing sampleThe quantity of the quantity of the order-checking fragment of accurate gene and the order-checking fragment of external source standard gene, concrete grammar is as follows:
According to the primer of order-checking fragment, utilize blastall (version2.2.26) software, establish by the parameter of its acquiescencePut, the slice groups that will check order is compared each corresponding detection on gene in table 1, and the successful order-checking of comparison fragment represents and base detectedCause, thus the quantity of the order-checking fragment of various transgene components in testing sample, the survey of various endogenous standard genes obtained respectivelyThe quantity of the order-checking fragment of the quantity of order fragment and external source standard gene, it the results are shown in Table 1.
Step 9, according to the quantity of the order-checking fragment of external source standard gene, whether judgment experiment success, concrete grammar is as follows:
When the quantity of order-checking fragment of external source standard gene and the quantity of the order-checking fragment of endogenous standard gene all >=when α 1,Success in Experiment; In the time of the quantity of order-checking fragment of external source standard gene or the quantity < α 1 of the order-checking fragment of endogenous standard gene,The failure of an experiment, needs to re-start experiment after the failure of an experiment, until Success in Experiment; Wherein, α 1 is decision threshold, and it is largeStrict degree and experience that little foundation requires are artificially judged, in general, if in high-flux sequence data, can detect 10Order-checking fragment more than bar, shows that detected gene exists. In the present embodiment, α 1 can get 10 order-checking fragments,As can be seen from Table 1, the quantity of the quantity of the order-checking fragment of external source standard gene and the order-checking fragment of endogenous standard gene is equalArticle >=10,, therefore confirm, this experiment is successful.
If step 10 Success in Experiment, calculates the content of transgene component in testing sample kind, concrete grammar is as follows:
The computing formula of the content of m kind transgene component isWherein, i is m kindI test zone of transgene component, n1 is the number of the test zone of m kind transgene component, bi is m kind transgenosisThe quantity of the order-checking fragment of i test zone of composition, k is the endogenous standard gene of k kind, what n3 was endogenous standard gene is individualNumber, j is j test zone of the endogenous standard gene of k kind, n2 is the number of the test zone of the endogenous standard gene of k kind,Aj is the quantity of the order-checking fragment of the j kind test zone of the endogenous standard gene of k kind; N is the survey of all endogenous standard genesThe sum in examination region.
As can be seen from Table 1, in the present embodiment, detected altogether 5 kinds of transgene components, so m=5, each turns baseBecause composition has detected respectively 1 test zone, so n1=1, has detected 1 endogenous standard gene altogether, so n3=1 is eachEndogenous standard gene has detected 1 test zone altogether, so, n2=1, N=3, table 1 has been listed each transgene component with everyThe quantity of the order-checking fragment of an endogenous standard gene, will can obtain in the computing formula of the content of their substitution transgene componentsThe content of each transgene component, it the results are shown in Table 1. As can be seen from Table 1, the present embodiment passes through 5 pairs of multiplex amplification primers,Quantitatively detect once the content of 5 kinds of transgene components. On the basis of the present embodiment, only need to increase more manyHeavy amplimer, can realize and once quantitatively detect any multiple content that needs the transgene component detecting. In addition also can,Detect multiple test zones of transgene component, endogenous standard gene and external source standard gene, eliminate because of difference by averageThe different amplification efficiencies of gene or different test zones and the test error that causes, the present embodiment is due to by the base of each detectionThe amplification efficiency of each test zone of cause has been controlled between 95%-105%, and error is less, for saving cost, soEach gene has only been selected a test zone.
Step 11, judge in testing sample, whether to contain transgene component, concrete grammar according to the content of transgene componentAs follows:
In the time of the content >=α 2 of any one transgene component that needs detect, judge that testing sample contains transgenosisPoint; In the time of the content < α 2 of all transgene components, judge that testing sample does not contain transgene component; Wherein, α 2 is decision thresholdValue.
In GB " transgenic product detects nucleic acid quantification PCR detection method " (standard No.: GB/T19495.5-2004)The transgenosis composition of setting is limited to 0.1% under detecting. In the present embodiment, the value of α 2 is set as to 0.1%, treats test sample for judgingWhether product are transgenic product. Detecting transgene component because the present invention adopts order-checking, is numeral letter and sequencing technologies obtainsNumber, measure and do not measure two kinds of situations, therefore, almost there is no technology lower limit, with traditional chip detection technology or in real timeQuantitative PCR detection technique comparison, the problem that does not have background noise to disturb, therefore, judges whether testing sample is transgenic productThe decision threshold strict degree free setting that can require as the case may be. As can be seen from Table 1, in the present embodiment, 5Among the transgene component kind detecting, except CryIAc, the content of all the other 4 kinds of transgene components all >=α 2=0.1%, because ofThis, judge that the testing sample in the present embodiment contains transgene component, and this testing sample is genetically modified plants.
Result verification: according to " genetically modified plants and products thereof composition detection Bar gene or pat gene qualitative PCR method "The method that (standard No.: No. 1782 bulletin-6-2012 of the Ministry of Agriculture) provides detects testing sample, and testing result showsTesting sample contains transgene component, and this is consistent with the result of the present embodiment, as can be seen here, and the detection method that the present embodiment providesCorrect.
Detection method provided by the invention can be general between different testing samples, the different target gene detecting, canSimultaneous quantitative detects any multiple transgene component in testing sample, has fully met the current demand that transgenosis detects, and isPrior art does not reach.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all in spirit of the present invention andWithin principle, any amendment of doing, be equal to replacement, improvement etc., within protection scope of the present invention all should be included in.
Claims (8)
1. a detection method for catsup transgene component, is characterized in that, described method comprises:
Determine endogenous standard gene in transgene component, the described testing sample that needs in testing sample to detect and described to be measuredThe external source standard gene of sample, described testing sample is the catsup processing by tomato;
For the preparation of the test zone of amplification described transgene component, described endogenous standard gene and described external source standard geneMultiplex amplification primer;
Described testing sample is sampled and mixed, obtain biased sample;
Extract the genome of described biased sample;
In the genome of described biased sample, add described external source standard gene, obtain mixing nucleic acid;
Utilize and mix nucleic acid described in described multiplex amplification primer pair and increase, obtain amplified production, utilize described amplified productionBuild high-throughput sequencing library;
Described high-throughput sequencing library is carried out to high-flux sequence, obtain the slice groups that checks order;
Analyze described order-checking slice groups, obtain the order-checking fragment of transgene component described in described testing sample quantity, described inThe quantity of the quantity of the order-checking fragment of endogenous standard gene and the order-checking fragment of described external source standard gene;
According to the quantity of the order-checking fragment of described external source standard gene, whether judgment experiment is successful;
If described Success in Experiment, calculates the content of transgene component described in described testing sample kind;
Judge in described testing sample, whether to contain transgene component according to the content of described transgene component.
2. detection method according to claim 1, is characterized in that, described transgene component is external source functional gene, anti-Property marker gene, promoter, terminator and external source insert at least one in flanking sequence.
3. detection method according to claim 1, is characterized in that, described endogenous standard gene is described testing sampleSingle copy gene in genome.
4. detection method according to claim 1, is characterized in that, described external source standard gene is not present in all biologiesIn.
5. detection method according to claim 1, is characterized in that, whether successfully described judgment experiment method is: whenThe quantity of the quantity of the order-checking fragment of described external source standard gene and the order-checking fragment of described endogenous standard gene all >=when α 1,Success in Experiment; When the quantity of order-checking fragment of described external source standard gene or the quantity of the order-checking fragment of described endogenous standard geneWhen < α 1, the failure of an experiment; Wherein, α 1 is decision threshold.
6. detection method according to claim 1, is characterized in that, whether the described testing sample of described judgement contains turns baseBecause the method for composition is: described in any one detection when needs when the content >=α 2 of transgene component, treat test sample described in judgementProduct contain transgene component; In the time of the content < α 2 of all described transgene components, judge that described testing sample does not contain transgenosisComposition; Wherein, α 2 is decision threshold.
7. detection method according to claim 1, is characterized in that, calculates transgenosis described in described testing sample kindThe method of the content dividing is: described in m kind, the computing formula of the content of transgene component isWherein, i is i the described test zone of described m kind transgene component, n1 be described m kind transgene component described inThe number of test zone, bi is the number of the described order-checking fragment of i described test zone of described m kind transgene componentAmount, k is endogenous standard gene described in k kind, the number that n3 is described endogenous standard gene, j is endogenous standard base described in k kindJ test zone of cause, n2 is the number of the described test zone of the endogenous standard gene of described k kind, aj is described k kindThe quantity of the described order-checking fragment of test zone described in the j kind of endogenous standard gene; N is all described endogenous standard genesThe sum of described test zone.
8. detection method according to claim 1, is characterized in that, the quality of described external source standard gene is mixed with describedThe ratio of the genomic gross mass of sample is greater than 1/100000.
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