CN105603077A - Detection method of wheat transgenic ingredients - Google Patents
Detection method of wheat transgenic ingredients Download PDFInfo
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Abstract
The invention discloses a detection method of wheat transgenic ingredients and belongs to the technical field of biology. The method comprises the steps that the transgenic ingredients, an endogenesis standard gene and an exogenous standard gene are determined; multiple amplification primers are prepared; sampling and mixing are carried out to obtain a mixed sample; genomes of the mixed sample are extracted and added into the exogenous standard genes to obtain mixed nucleic acid; a high-throughout sequencing library is constructed and sequenced in a high-throughout mode to obtain sequencing fragment sets; the number of sequencing fragments of the transgenic ingredients, the number of sequencing fragments of the endogenesis standard gene and the number of sequencing fragments of the exogenous standard gene in the sample to be detected are obtained; according to the number of the sequencing fragments of the exogenous standard gene, 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. The method can quantitatively detect the multiple transgenic ingredients in the sample to be detected at the same time.
Description
Technical field
The present invention relates to field of biological detection, particularly a kind of detection method of wheat transgenic composition.
Background technology
Wheat is the important crop of China and food, and the transgenic technology of wheat and products thereof is all ripe, turnsThe plantation of DNA triticum kind needs strict examination & approval with popularization, therefore needs transgenic wheat to supervise,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 inspection of nucleic acidThe main flow process of survey method is: extract the nucleic acid in testing sample, utilize PCR (PolymeraseChainReactIon, polymerase chain reaction) method amplification sample in transgene component, utilize real-time quantitative, agaroseWhether electrophoresis or chip technology detect transgene component and 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, because ofThis, need to detect one by one to possible multiple transgenosis composition, just can be defined as " non-transgenic " and produceProduct; What generally detect is the transgene component of cotransformation, instead of foreign gene itself, therefore, and for nothingMark transgenosis, adopting existing detection method to carry out detection can lose efficacy; Detecting is qualitative inspection substantiallySurvey, but quantitatively detection has again its current demand, for example, a small amount of flower of the transgenic wheat kind of adjacent fieldPruinescence has passed in non-transgenic kind, and in the time of qualitative detection, non-transgenic kind will be mistaken for transgenosisKind and will not authorize or carry out wrong administrative penalty.
Summary of the invention
In order to solve the problem of prior art, the embodiment of the present invention provides a kind of inspection of wheat transgenic compositionSurvey method. Described technical scheme is as follows:
The embodiment of the present invention provides a kind of detection method of wheat transgenic composition, and described method comprises:
Determine the endogenous standard gene in transgene component, the described testing sample that needs to detect in testing sampleWith the external source standard gene of described testing sample, described testing sample is wheat plant or described wheat plantA part;
For the preparation of amplification described transgene component, described endogenous standard gene and described external source standard geneThe multiplex amplification primer of test zone;
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 instituteState amplified production and build 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 sampleThe quantity of the order-checking fragment of quantity, described endogenous standard gene and the order-checking fragment of described external source standard geneQuantity;
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, terminationSon and external source are inserted 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, the whether successful method of described judgment experiment is: when the order-checking sheet of described external source standard geneThe quantity of section and the quantity of the order-checking fragment of described endogenous standard gene all >=when α 1, Success in Experiment; Work as instituteState the quantity < α 1 of the quantity of order-checking fragment or the order-checking fragment of described endogenous standard gene of external source standard geneTime, the failure of an experiment; Wherein, α 1 is decision threshold.
Particularly, described judge method that whether described testing sample contain transgene component as: when needs inspectionDescribed in any one that survey, when the content >=α 2 of transgene component, judge that described testing sample contains transgenosisPoint; In the time of the content < α 2 of all described transgene components, 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: m kindThe computing formula of the content of described transgene component isWherein, i is described mI the test zone of planting transgene component, n1 is the described test zone of described m kind transgene componentNumber, bi is the described order-checking fragment of i the described test zone of described m kind transgene componentQuantity, k is endogenous standard gene described in k kind, the number that n3 is described endogenous standard gene, j is kJ test zone of kind of described endogenous standard gene, n2 be the endogenous standard gene of described k kind described inThe number of test zone, aj is the described survey of test zone described in the j kind of the endogenous standard gene of described k kindThe quantity of order 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 sampleExample is greater than 1/100000.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: detection method provided by the inventionCan be general between different testing samples, the different target gene detecting, can disposable detection multiple arbitrarilyNeed the transgene component detecting, thereby judge whether testing sample contains transgene component, and the method canRealize quantitatively and detecting, and testing result almost do not have 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, below will be to embodiment of the present inventionBe described in further detail. In the embodiment of the present invention, operating process or operation unreceipted or that describe in detail are advisedModel is the known operation of common Protocols in Molecular Biology personnel. Not marked reagent in the embodiment of the present inventionOr biomaterial is common agents or the biomaterial sold on market, be common Protocols in Molecular BiologyPersonnel are known, and can on market, buy.
Embodiment
By agrobacterium-mediated transformation, two alanyl phosphorus resistances (Biolaphosresistance, Bar) gene is imported" Anyue daughter wheat " wheat breed, after 3 generations of selfing purifying, obtains " Anyue daughter wheat " wheat of transformationVariety seeds, as the wheat breed to be measured of the present embodiment. Wherein " Anyue daughter wheat " wheat is transgenosisWheat acceptor kind, is the Wheat landraces of Anyue Sichuan, " the Anyue daughter wheat " using in the present embodimentWheat seed is preserved and is bred by Jianghan University.
Wheat breed detection of GMOs method, concrete steps are as follows:
Step 1, determine the endogenous standard base in transgene component, the testing sample that needs in testing sample to detectThe external source standard gene of cause and testing sample, concrete grammar is as follows: testing sample is that wheat plant or wheat are plantedA part for strain, wherein, wheat plant can be root, stem, leaf, flower, the fruits and seeds of wheat plantIn organ, can be also the mixture of 2 kinds and Different Organs of more than two kinds, as the mixture of leaf and stem,A part for wheat plant can be wheat plant root, stem, leaf, flower, fruit and seed etc. at least oneA part in organ. In the present embodiment, testing sample is improved " Anyue daughter wheat " wheatSeed, transgene component, endogenous standard gene and external source standard gene all >=1, transgene component can beExternal source functional gene, resistant maker gene, promoter, terminator and external source are inserted in flanking sequence at leastOne, is not present in " peace by transgenic technology means to proceeding in " Anyue daughter wheat " wheat breedDaughter Yue wheat " exogenous nucleic acid sequences in wheat breed; Endogenous standard gene is in the genome of testing sampleSingle copy gene. In the present embodiment, endogenous standard gene passes through NCBI(http://www.ncbi.nlm.nih.gov/) is unique sequence while comparison on testing sample genome; External source markAccurate gene is not present in all biologies, and in the present embodiment, the external source standard gene using is ERCC04Gene, its homology when comparison on NCBI, do not find homologous sequence, can judge that external source standard gene do not depositBe in all biologies. In the present embodiment, totally 5 kinds of the transgene components of detection, endogenous standard gene 1Kind, a kind of external source standard gene, its relevant information is in table 1, and in table 1, listed transgene component not only comprisesBar gene, it is external source functional gene, also comprises other transgene components, these transgene components are at wheatIn be widely used, therefore, the detection method that the present embodiment provides has versatility. Gene order in table 1Have two kinds of expression modes, one is directly to write out gene order, and another kind is the gene numbering of NBCI, table 1In order-checking region in the position of digitized representation base, 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 multiple expansionIncrease primer, concrete grammar is as follows:
The acquisition process of multiplex amplification primer is as follows: login match Mo Feishier company multiple PCR primer is established onlineMeter webpage https: //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,And the primer of single-pool only needs multiplex PCR one time, save cost, so the present embodiment is selectedSingle-pool. The sequence of each gene listed in table 1 is coupled together with 100 N, form oneArtificial reference genome, and selection " Custom " in " Selectthegenomeyouwishtouse " optionAfter, upload artificial reference genome. DNAType (type) option is selected " StandardDNA " (standardDNA). In AddHotspot option, select 1 for each gene in table 1 is random and pass through NCBIThe homology of the homologous sequence that homology comparison obtains is all lower than 80% test zone. Wherein, test zone isThe amplification region that refers to multiplex PCR is also the region of high-flux sequence, due to test zone and other species ratio, without obvious homologous sequence, therefore, can represent detected gene, after high-flux sequence, surveyThere is order-checking fragment in examination region, shows that detected gene exists, the quantity of the order-checking fragment of test zone,Represent the amount of detected gene. In addition, homologous sequence does not comprise in the primary source species of transgene componentSequence, for example, the primary source species of the CryIAc gene in table 1 are thuringiensis (BacillusThuringiensis, is called for short Bt), therefore, homology comparison picture is not comprised to the gene of thuringiensisGroup. In order to ensure the versatility of the embodiment of the present invention, on NCBI, download in the kind of endogenous standard gene notSame sequence, compares by homology, obtains the conservative region between sequence, the test zone of endogenous standard geneBe chosen as in kind of the conservative region of inner boundary sequence. Further fill in the initial sum of each test zone of acquisitionFinal position, finally clicks " Submittargets " button and submits to, obtains each gene in amplification table 1The sequence of amplimer. In multiplex amplification primer, the amplification efficiency of every heavy primer is all between 95%～105%,Therefore, need to verify the amplification efficiency of multiplex amplification primer. Concrete verification method is: by raw work bioengineeringThe test zone of the sequence of Bar gene and amplification Bar gene in (Shanghai) limited company synthetic table 1Amplimer sequence, taking the sequence of the Bar gene that synthesizes as template, according to match Mo Feishier company of the U.S.The operation manual (PartNumber4376784Rev.E) of StepOne real-time PCR detects amplificationThe amplification efficiency of the amplimer of the test zone of Bar gene, if amplification efficiency not between 95%-105%,Need to redefine the test zone of Bar gene, design and verify the effect of the new amplimer of Bar geneRate, until its efficiency is between 95%-105%. In the same way, other gene in proof list 1The amplification efficiency of amplimer. Amplification efficiency, sequence and the corresponding amplification of final selected amplimerRegion is in table 1, and all final selected amplimers are called multiplex amplification primer, and multiplex amplification primer is by the U.S.Sai Mo Fei Shier company is synthetic and offer user with the form of mixing material. The present embodiment adopts U.S.'s matchThe multiple PCR technique that Mo Feishier company provides, its 12000 test zones of as many as that can simultaneously increase,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 according to standard " sampling of genetically modified plants and products thereof composition detection " (standard No.: the Ministry of AgricultureNo. 2031 bulletin-19-2013) the methods of sampling of bulk goods sample, because testing sample is only 1Kind of simple source, therefore, sample point changes 1 into, the sample size of sampling sample > 1000 seeds, by instituteThe plant sample to be measured extracting fully mixes, and obtains 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.: agriculturalNo. 1485 bulletin-4-2010 of portion) in the method for solid-state sample carry out extracting after pretreatment the gene of biased sampleGroup, the genomic nucleic acids for biased sample extracting.
Step 5, in the genome of biased sample, add external source standard gene, obtain mixing nucleic acid, specifically sideMethod is as follows:
Utilize the double-stranded DNA in spectrophotometer (Quawell company of the U.S. produces, and model is Q5000)Program, the genomic concentration of the biased sample that detection obtains. The quality of external source standard gene and biased sampleThe ratio of genomic gross mass be greater than 1/100000, this ratio be used for ensureing normal sequencing throughput (>=The 1M fragment that checks order) under, in high-flux sequence data, can detect >=10 external source standard genes, if high passAmount sequencing throughput is lower or higher, can do corresponding adjustment to this ratio. In the present embodiment, biased sampleGenomic gross mass be 1577ng, the external source standard gene adding is 1.577ng, additional proportion is1/1000, obtain and mix nucleic acid.
Step 6, utilize multiplex amplification primer pair mixing nucleic acid to increase, obtain amplified production, utilize amplificationProduct builds high-throughput sequencing library, and concrete grammar is as follows:
(produced by LifeTechnology company of the U.S., article No. is to utilize library construction kit 2.04475345) build high-throughput sequencing library. This library construction kit comprises following reagent: 5 × IonAmpliSeqTMHiFiMix, FuPa reagent, conversion reagent, sequence measuring joints solution and DNA ligase.The method of library construction is by the operation manual " IonAmpliSeq of this library construction kitTMLibraryPreparation " (publication number: MAN0006735, version: A.0) carry out. The amplification system of multiplex PCRAs 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 minutes) × 25 circulations; 10 DEG C of insulations. Utilize FuPa reagent to digest multiplex PCRIn amplified production after unnecessary primer, then carry out phosphorylation, concrete grammar is: the amplification to multiplex PCR is producedIn thing, add 2 μ lFuPa reagent, after mixing, on PCR instrument, react by following program: 50 DEG C, 10 pointsClock; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is for containingThere is the solution through the amplified production of phosphorylation. The amplified production of phosphorylation is connected to upper sequence measuring joints, concreteMethod is: in 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, react by following program: 22 DEG C, 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of guarantorsDeposit, obtain mixed liquor b. After utilizing the ethanol intermediate processing purifying mixed liquor b of standard, be dissolved in 10 μ l without enzymeIn water. Utilize American I nvitrigen company to produce(article No. is dsDNAHSAssayKitQ32852) and according to its description detect, obtain after the mass concentration of mixed liquor b, by after purifyingMixed liquor b is diluted to 15ng/ml, obtains the high-throughput sequencing library of the about 100pM of concentration.
Step 7, high-throughput sequencing library is carried out to high-flux sequence, obtains the slice groups that checks order, concrete grammar asUnder:
Utilize the high-throughput sequencing library and the kit IonPITemplateOT2200Kitv2 (U.S. that obtainInvirtrigen company produces, and article No. is 4485146) ePCR (EmulsionPCR, breast before checking orderChange polymerase chain reaction) amplification, method of operating is undertaken by the operation manual of this kit. Utilize ePCR product(invirtrigen company of the U.S. produces, and article No. is with kit IonPISequencing200Kitv24485149) on Proton bis-generations high-flux sequence instrument, carry out high-flux sequence, method of operating is by this kitOperation manual carry out. In the present embodiment, high-flux sequence amount is set to 1M order-checking fragment (1M=100Ten thousand), order-checking length is set to 500cycle (circulation), after order-checking finishes, obtains order-checking slice groups.
Step 8, analyze order-checking slice groups, obtain the order-checking fragment of transgene component in testing sample quantity,The quantity of the quantity of the order-checking fragment of endogenous standard gene and the order-checking fragment of external source standard gene, concrete grammarAs follows:
According to the primer of order-checking fragment, utilize blastall (version2.2.26) software, by the parameter of its acquiescenceArrange, the slice groups that will check order is compared each corresponding detection on gene in table 1, and the successful order-checking of comparison fragmentRepresentative gene detected, thereby obtain respectively the order-checking fragment of various transgene components in testing sample quantity,The quantity of the quantity of the order-checking fragment of various endogenous standard genes and the order-checking fragment of external source standard gene, its knotFruit is in table 1.
Step 9, according to the quantity of the order-checking fragment of external source standard gene, whether judgment experiment success, specifically sideMethod 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; When the quantity of order-checking fragment or the order-checking of endogenous standard gene of external source standard geneWhen quantity < the α 1 of fragment, the failure of an experiment, needs to re-start experiment after the failure of an experiment, until Success in ExperimentTill; Wherein, α 1 is decision threshold, and its size artificially judges according to the strict degree and the experience that require, oneAs, if in high-flux sequence data, 10 above order-checking fragments can be detected, show examineThe gene of surveying exists. In the present embodiment, α 1 can get 10 order-checking fragments, can from table 1Go out, the quantity of the quantity of the order-checking fragment of external source standard gene and the order-checking fragment of endogenous standard gene all >=10Bar, therefore confirms, this experiment is successful.
If step 10 Success in Experiment, calculates the content of transgene component in testing sample kind, concrete grammarAs follows:
The computing formula of the content of m kind transgene component isWherein, i isI test zone of m kind transgene component, n1 be m kind transgene component test zoneNumber, bi is the quantity of the order-checking fragment of i test zone of m kind transgene component, k is in k kindSource standard gene, the number that n3 is endogenous standard gene, j is j test of the endogenous standard gene of k kindRegion, n2 is the number of the test zone of the endogenous standard gene of k kind, aj is the endogenous standard gene of k kindThe quantity of order-checking fragment of j kind test zone; N is the sum of the test zone of all endogenous standard genes.
As can be seen from Table 1, in the present embodiment, detect altogether 5 kinds of transgene components, thus m=5,Each transgene component has detected respectively 1 test zone, so, n1=1, detected altogether 1 endogenousStandard gene, so n3=1, each endogenous standard gene has detected 1 test zone altogether, so, n2=1,N=3, table 1 has been listed the quantity of the order-checking fragment of each transgene component and each endogenous standard gene,To in the computing formula of the content of their substitution transgene components, can obtain the content of each transgene component,It the results are shown in Table 1. As can be seen from Table 1, the present embodiment is by 5 pairs of multiplex amplification primers, fixed onceAmount has detected the content of 5 kinds of transgene components. On the basis of the present embodiment, only need to increase moreMultiplex amplification primer, 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 the test mistake causing because of the different amplification efficiencies of different genes or different test zones by averagePoor, the present embodiment is due to the amplification efficiency of each test zone of the gene of each detection is controlled atBetween 95%-105%, error is less, for saving cost, so each gene has only been selected a testRegion.
Step 11, judge in testing sample, whether to contain transgene component, tool according to the content of transgene componentBody method is as follows:
In the time of the content >=α 2 of any one transgene component that needs detect, judge that testing sample contains to turnGene element; 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 threshold.
In GB " transgenic product detects nucleic acid quantification PCR detection method " (standard No.: GB/T19495.5-2004) in set transgenosis composition detect under be limited to 0.1%. In the present embodiment, by the value of α 2Be set as 0.1%, for judging whether testing sample is transgenic product. Because adopting order-checking, the present invention detectsTransgene component, is data signal and sequencing technologies obtains, measures and do not measure two kinds of situations, because ofThis, almost do not have technology lower limit, with traditional chip detection technology or comparison of real-time quantitative PCR detection technique,The problem that does not have background noise to disturb, therefore, judges that whether testing sample is the decision threshold of transgenic productThe strict degree free setting that can require as the case may be. As can be seen from Table 1, in the present embodiment,Among the transgene component that 5 kinds are detected, except CryIAc, the content of all the other 4 kinds of transgene components all >=α 2=0.1%, therefore, judges that the testing sample in the present embodiment contains transgene component, and this testing sample is for turningGene plant.
Result verification: according to " genetically modified plants and products thereof composition detection Bar gene or pat gene are qualitativePCR method " (standard No.: No. 1782 bulletin-6-2012 of the Ministry of Agriculture) method of providing enters testing sampleGone detection, testing result shows that testing sample contains transgene component, and this is consistent with the result of the present embodiment,As can be seen here, the detection method that the present embodiment provides is correct.
Detection method provided by the invention can be general between different testing samples, the different target gene detecting,Can detect any multiple transgene component in testing sample by simultaneous quantitative, fully meet transgenosis detectionCurrent demand, be that prior art does not reach.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all of the present inventionWithin spirit and principle, any amendment of doing, be equal to replacement, improvement etc., all should be included in of the present inventionWithin protection domain.
Claims (8)
1. a detection method for wheat transgenic composition, is characterized in that, described method comprises:
Determine the endogenous standard gene in transgene component, the described testing sample that needs to detect in testing sampleWith the external source standard gene of described testing sample, described testing sample is wheat plant or described wheat plantA part;
For the preparation of amplification described transgene component, described endogenous standard gene and described external source standard geneThe multiplex amplification primer of test zone;
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 instituteState amplified production and build 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 sampleThe quantity of the order-checking fragment of quantity, described endogenous standard gene and the order-checking fragment of described external source standard geneQuantity;
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 sourceFunctional gene, resistant maker gene, promoter, terminator and external source are inserted at least one in flanking sequence.
3. detection method according to claim 1, is characterized in that, described endogenous standard gene is instituteState the single copy gene in the genome of testing sample.
4. detection method according to claim 1, is characterized in that, described external source standard gene is not depositedBe in all biologies.
5. detection method according to claim 1, is characterized in that, whether described judgment experiment is successfulMethod be: when the quantity of order-checking fragment and the order-checking of described endogenous standard gene of described external source standard geneThe quantity of fragment all >=when α 1, Success in Experiment; When the quantity of the order-checking fragment of described external source standard gene orWhen quantity < the α 1 of the order-checking fragment of described endogenous standard gene, the failure of an experiment; Wherein, α 1 is decision threshold.
6. detection method according to claim 1, is characterized in that, the described testing sample of described judgementThe method that whether contains transgene component is: the content of transgene component described in any one detection when needsWhen>=α 2, judge that described testing sample contains transgene component; As the content<α 2 of all described transgene componentsTime, judge that described testing sample does not contain transgene component; Wherein, α 2 is decision threshold.
7. detection method according to claim 1, is characterized in that, calculates in described testing sample kindThe method of the content of described transgene component is: described in m kind, the computing formula of the content of transgene component isWherein, i is i described test section of described m kind transgene componentTerritory, n1 is the number of the described test zone of described m kind transgene component, bi is that described m kind turns baseBecause of the quantity of the described order-checking fragment of the i of composition described test zone, k is endogenous standard described in k kindGene, the number that n3 is described endogenous standard gene, j is j survey of endogenous standard gene described in k kindExamination region, n2 is the number of the described test zone of the endogenous standard gene of described k kind, aj is described kThe quantity of the described order-checking fragment of test zone described in the j kind of the endogenous standard gene of kind; N be all described inThe sum of the described test zone of source standard gene.
8. detection method according to claim 1, is characterized in that, the matter of described external source standard geneAmount is greater than 1/100000 with the ratio of the genomic gross mass of described biased sample.
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