CN105624298A - Method for detecting genetically modified components of rape - Google Patents
Method for detecting genetically modified components of rape Download PDFInfo
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- CN105624298A CN105624298A CN201610061083.4A CN201610061083A CN105624298A CN 105624298 A CN105624298 A CN 105624298A CN 201610061083 A CN201610061083 A CN 201610061083A CN 105624298 A CN105624298 A CN 105624298A
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- 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
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a method for detecting the genetically modified components of rape and belongs to the technical field of biology. The method includes: determining the genetically modified components, endogenous standard genes and exogenous standard genes; preparing multiple amplification primers; sampling and mixing to obtain a mixed sample; extracting the genome of the mixed sample, and adding the exogenous standard genes to obtain mixed nucleic acid; building a high-throughput sequencing library; performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment group; acquiring the number of the sequencing fragments of the genetically modified components in the to-be-detected sample, the numbers of the sequencing fragments of the endogenous standard genes in the to-be-detected sample and the sequencing fragments of the exogenous standard genes in the to-be-detected sample; judging whether the experiment is successful or not according to the number of the sequencing fragments of the exogenous standard genes, and calculating the content of the genetically modified components in the to-be-detected sample; judging whether the to-be-detected sample contains the genetically modified components or not according to the content of the genetically modified components. By the method, optional multiple genetically modified components in the to-be-detected sample can be quantitatively detected at the same time.
Description
Technical field
The present invention relates to field of biological detection, particularly to the detection method of a kind of Brassica campestris L transgene component.
Background technology
Brassica campestris L is China chief crop, and Brassica campestris L transgenic technology and product are all ripe, it is necessary to the popularization of transgene rape is supervised, and detection of GMOs is the technical support of transgenic supervision.
Existing detection of GMOs technology predominantly detects nucleic acid or protein, wherein, detection method main flow based on nucleic acid is: extract the nucleic acid in testing sample, utilize PCR (PolymeraseChainReaction, polymerase chain reaction) method amplification sample in transgene component, utilize real-time quantitative, sepharose electrophoresis or chip technology detection transgene component whether exist.
In the process realizing the present invention, inventor have found that prior art at least exists the one in problems with:
Transgene component is varied, and prior art once can only detect one of which or a few, accordingly, it would be desirable to possible multiple transgenic composition is detected one by one, just can be defined as " non-transgenic " product; What generally detect is the transgene component of cotransformation, rather than exogenous gene itself, and therefore, for marker-free transgenic, adopting existing detection method to carry out detection can lose efficacy; Detection is qualitative detection substantially, but detection by quantitative has again its current demand, for instance, a small amount of pollen envelop of the transgene rape kind of adjacent field has passed in non-transgenic kind, and when qualitative detection, non-transgenic kind will be mistaken for transformed variety.
Summary of the invention
In order to solve problem of the prior art, embodiments provide the detection method of a kind of Brassica campestris L transgene component. Described technical scheme is as follows:
Embodiments providing the detection method of a kind of Brassica campestris L transgene component, described method includes:
Determining the external source standard gene of the endogenous standard gene and described testing sample needed in testing sample in the transgene component of detection, described testing sample, described testing sample is rapeseed plants or a part for described rapeseed plants;
Preparation is for expanding the multiplex amplification primer of the test zone of described transgene component, described endogenous standard gene and described external source standard gene;
Described testing sample it is sampled and mixes, obtaining 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 mixing nucleic acid described in described multiplex amplification primer pair to expand, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
Described high-throughput sequencing library is carried out high-flux sequence, obtains order-checking fragment group;
Analyze described order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of transgene component described in described testing sample, the quantity of order-checking fragment of described endogenous standard gene and described external source standard gene;
The quantity of the order-checking fragment according to described external source standard gene, it is judged that whether experiment is successful;
If described Success in Experiment, then calculate the content of transgene component described in described testing sample kind;
Content according to described transgene component judges whether contain transgene component in described testing sample.
Specifically, described transgene component is at least one that external source functional gene, resistant maker gene, promoter, terminator and external source insert in flanking sequence.
Specifically, described endogenous standard gene is the single copy gene in the genome of described testing sample.
Specifically, described external source standard gene is not present in all biologies.
Specifically, described judgment experiment whether successfully method is: when the quantity of the quantity of order-checking fragment of described external source standard gene and the order-checking fragment of described endogenous standard gene equal>=�� 1 time, then Success in Experiment; When the quantity of order-checking fragment of described external source standard gene or the order-checking fragment of described endogenous standard gene quantity<during �� 1, then the failure of an experiment; Wherein, �� 1 is decision threshold.
Specifically, described judge described testing sample whether contain the method for transgene component as: as the content>=�� 2 of any one described transgene component of needs detection, it is determined that described testing sample contains transgene component; When the content of all described transgene components is<during �� 2, it is determined that described testing sample does not contain transgene component; Wherein, �� 2 is decision threshold.
Specifically, the method for the content of transgene component described in described testing sample kind that calculates is: described in m kind, the computing formula of the content of transgene component isWherein, i is the i-th test zone of described m kind transgene component, n1 is the number of the described test zone of described m kind transgene component, bi be described m kind transgene component i-th described in the quantity of described order-checking fragment of test zone, k is endogenous standard gene described in kth kind, n3 is the number of described endogenous standard gene, j is the jth test zone of endogenous standard gene described in kth kind, n2 is the number of the described test zone of the described endogenous standard gene of kth kind, aj be the described endogenous standard gene of kth kind jth kind described in the quantity of described order-checking fragment of test zone, N is the sum of the described test zone of all described endogenous standard genes.
Specifically, the quality of described external source standard gene and the ratio of the genomic gross mass of described biased sample are more than 1/100000.
The technical scheme that the embodiment of the present invention provides has the benefit that detection method provided by the invention can be general between different testing samples, the different target gene detected, can disposable detection any multiple need detection transgene component, thus judging whether testing sample contains transgene component, the method can realize detection by quantitative, and testing result is almost without 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 clearly, below embodiment of the present invention is described further in detail. In the embodiment of the present invention, unreceipted or detailed description operating process or working specification are the operation that common molecular biology known to the skilled person which. In the embodiment of the present invention, not marked reagent or biomaterial are common agents or the biomaterial of sale on market, are what common molecular biology known to the skilled person which, and can commercially buy.
Embodiment
By agrobacterium-mediated transformation by double; two alanyl phosphorus resistance (Biolaphosresistance, Bar) extensive 15 rape varieties of the expensive oil of channel genes, after selfing purification 3 generation, it is thus achieved that the extensive 15 rape variety seeds of your oil of transformation, as the rape variety to be measured of the present embodiment. Wherein, extensive 15 Brassica campestris Ls of your oil are transgene rape receptor kinds, Brassica campestris L research department of Guizhou Agriculture College be bred as, and extensive 15 Semen Brassicae campestris of your oil used in the present embodiment are preserved by Jianghan University and breed.
Rapeseed plant kind detection of GMOs method, specifically comprises the following steps that
Step 1, determine the transgene component needing detection in testing sample, endogenous standard gene in testing sample and the external source standard gene of testing sample, concrete grammar is as follows: testing sample is a part for rapeseed plants or rapeseed plants, wherein, rapeseed plants can be the root of rapeseed plants, stem, leaf, flower, the organ such as fruit and seed, it can also be the mixture of 2 kinds and Different Organs of more than two kinds, mixture such as leaf and stem, a part for rapeseed plants can be the root of rapeseed plants, stem, leaf, flower, a part at least one organ such as fruit and seed. in the present embodiment, testing sample is the seed of extensive 15 Brassica campestris Ls of your oil improved, transgene component, endogenous standard gene and external source standard gene all >=1, transgene component can be at least one that external source functional gene, resistant maker gene, promoter, terminator and external source insert in flanking sequence, namely proceeds to, in extensive 15 rape varieties of your oil, the exogenous nucleic acid sequences being not present in extensive 15 rape varieties of your oil by transgenic technology means, endogenous standard gene is the single copy gene in the genome of testing sample. in the present embodiment, endogenous standard gene is unique sequence during comparison by NCBI (http://www.ncbi.nlm.nih.gov/) on testing sample genome, external source standard gene is not present in all biologies, and in the present embodiment, the external source standard gene used is ERCC04 gene, and it during homology comparison, does not find homologous sequence on NCBI, it is possible to determine that external source standard gene is not present in all biologies. in the present embodiment, the transgene component totally 5 kinds of detection, endogenous standard gene a kind, external source standard gene a kind, its relevant information is in Table 1, in table 1, listed transgene component not only includes Bar gene, Bar gene is external source functional gene, also includes other transgene components, and these transgene components widely use in genetically modified crops, therefore, the detection method that the present embodiment provides has versatility. gene order in table 1 has two kinds of representations, and one is directly to write out gene order, and another kind is the gene numbering of NBCI, and the position of the digitized representation base in order-checking region in table 1, 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 one
In table 1 "/" indicate without.
Step 2, preparation are for expanding the multiplex amplification primer of transgene component, endogenous standard gene and external source standard gene, and concrete grammar is as follows:
The acquisition process of multiplex amplification primer is as follows: logs in match Mo Feishier company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/, selects " DNAHotspotdesigns (single-pool) " at " Applicationtype " option. if selecting multi-pool, then a point multitube is carried out by multiplex PCR, and cost can increase to some extent, and the primer of single-pool has only to a multiplex PCR, saves cost, so the present embodiment selects single-pool. the sequence of each gene listed in table 1 is coupled together with 100 N, forms an artificial reference genome, and in " Selectthegenomeyouwishtouse " option after selection " Custom ", upload artificial reference genome. DNAType (type) option selects " StandardDNA " (standard DNA). in AddHotspot option, randomly choose 1 homology of homologous sequence obtained by NCBI homology comparison for each gene in table 1 and be below the test zone of 80%. wherein, test zone refers to the amplification region of multiplex PCR, is also the region of high-flux sequence, owing to test zone compares with other species, without obvious homologous sequence, therefore, it can represent detected gene, after high-flux sequence, there is order-checking fragment in test zone, then show that the gene being detected exists, the quantity of the order-checking fragment of test zone, represent the amount of detected gene. in addition, homologous sequence does not include the sequence in the primary source species of transgene component, such as, the primary source species of the CryIAc gene in table 1 are thuringiensis (Bacillusthuringiensis, it is called for short Bt), therefore, the genome that picture is not included thuringiensis of homology comparison. in order to ensure the versatility of the embodiment of the present invention, NCBI downloads the different sequences in the kind of endogenous standard gene, by homology comparison, it is thus achieved that the conservative region between sequence, the test zone of endogenous standard gene is chosen as in kind of a region for inner boundary sequence preservative. fill in the initial and final position of each test zone of acquisition further, finally click " Submittargets " button and submit to, it is thus achieved that the sequence of the amplimer of each gene in amplification table 1. in multiplex amplification primer, often the amplification efficiency of weight primer is all between 95%��105%, accordingly, it would be desirable to verify the amplification efficiency of multiplex amplification primer. concrete verification method is: by the amplimer sequence of the sequence of Bar gene in Sangon Biotech's synthetic table 1 and the test zone of amplification Bar gene, with the sequence of the Bar gene of synthesis for template, the amplification efficiency of the amplimer of the test zone of workbook (PartNumber4376784Rev.E) the detection amplification Bar gene of the StepOne real-time PCR of Mo Feishier company is matched according to the U.S., if amplification efficiency is not between 95%-105%, then need to redefine the test zone of Bar gene, design and verify the efficiency of new amplimer of Bar gene, till its efficiency is between 95%-105%. in the same way, the amplification efficiency of the amplimer of other gene in proof list 1. the amplification region of the final selected amplification efficiency of amplimer, sequence and correspondence is in Table 1, all final selected amplimers are called multiplex amplification primer, and multiplex amplification primer is matched the synthesis of Mo Feishier company by the U.S. and the form to mix liquid is supplied to user. the present embodiment adopt the U.S. match Mo Feishier company provide multiple PCR technique, it can expand up to 12000 test zones simultaneously, therefore, the present invention have the ability disposable detection existing be there is a need to detection transgene component.
Step 3, testing sample being sampled and mixes, obtaining biased sample, concrete grammar is as follows:
Testing sample is sampled according to the sampling approach of Semen Brassicae campestris in the bulk goods of standard " sampling of transgenic plant and products thereof composition detection " (standard No.: No. 2031 bulletin-19-2013 of the Ministry of Agriculture), due to testing sample only a kind of simple source, therefore, sample point changes 1 into, the sample size of sample > 1000 seeds, the plant sample to be measured extracted is sufficiently mixed, obtains biased sample.
Step 4, extract biased sample genome, concrete grammar is as follows:
In reference standard " transgenic plant and products thereof composition detection DNA extraction and purification " (standard No.: No. 1485 bulletin-4-2010 of the Ministry of Agriculture), solid sample extracts the genome of biased sample after carrying out pretreatment, the genomic nucleic acids for biased sample extracted.
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), the genomic concentration of the biased sample that detection obtains. The ratio of the genomic gross mass of the quality of external source standard gene and biased sample is more than 1/100000, this ratio is used for ensureing under normal sequencing throughput (>=1M check order fragment), high-flux sequence data can detect >=10 external source standard genes, if high-flux sequence flux is lower or higher, it is possible to this ratio is done corresponding adjustment. In the present embodiment, the genomic gross mass of biased sample is 1003ng, and the external source standard gene of addition is 1.003ng, and additional proportion is 1/1000, it is thus achieved that mixing nucleic acid.
Step 6, utilizing multiplex amplification primer pair mixing nucleic acid to expand, obtain amplified production, utilize amplified production to build high-throughput sequencing library, concrete grammar is as follows:
Library construction Kit 2.0 (being produced by LifeTechnology company of the U.S., article No. is 4475345) is utilized to build high-throughput sequencing library. This library construction Kit includes following reagent: 5 �� IonAmpliSeqTMHiFiMix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase. The method of library construction is by the workbook " IonAmpliSeq of this library construction KitTMLibraryPreparation " (publication number: MAN0006735, version: A.0) carry out. The amplification system of multiplex PCR is as follows: 5 �� IonAmpliSeqTMHiFiMix4 �� l, the mixing liquid 4 �� l of multiplex amplification primer of preparation, mixing nucleic acid 10ng and without enzyme water 11 �� l. The amplification program of multiplex PCR is as follows: 99 DEG C, 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) �� 25 circulations; 10 DEG C of insulations. After utilizing FuPa reagent to digest primer unnecessary in multiplexed PCR amplification product, then carry out phosphorylation, method particularly includes: in the amplified production of multiplex PCR, add 2 �� lFuPa reagent, after mixing, by the reaction of following program in PCR instrument: 50 DEG C, 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtaining mixture a, mixture a is the solution containing the amplified production through phosphorylation. The amplified production of phosphorylation is connected upper sequence measuring joints, method particularly includes: in mixture a, add transferring reagent 4 �� l, sequence measuring joints solution 2 �� l and DNA ligase 2 �� l, after mixing, by the reaction of following program in PCR instrument: 22 DEG C, 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed liquor b. It is dissolved in 10 �� l without in enzyme water after utilizing the ethanol precipitation methods purification mixed liquor b of standard. American I nvitrigen company is utilized to produceDsDNAHSAssayKit (article No. is Q32852) also detects according to its description, it is thus achieved that after the mass concentration of mixed liquor b, the mixed liquor b after purification is diluted to 15ng/ml, obtains concentration and be about the high-throughput sequencing library of 100pM.
Step 7, high-throughput sequencing library being carried out high-flux sequence, obtain order-checking fragment group, concrete grammar is as follows:
(invirtrigen company of the U.S. produces to utilize the high-throughput sequencing library obtained and test kit IonPITemplateOT2200Kitv2, article No. is 4485146) check order before ePCR (EmulsionPCR, emulsion polymerization enzyme chain reaction) amplification, operational approach is undertaken by the workbook of this test kit. (invirtrigen company of the U.S. produces to utilize ePCR product and test kit IonPISequencing200Kitv2, article No. is 4485149) on the secondary high-flux sequence instrument of Proton, carry out high-flux sequence, operational approach is undertaken by the workbook of this test kit. In the present embodiment, high-flux sequence amount is set to 1M order-checking fragment (1M=,100 ten thousand), and order-checking length is set to 500cycle (circulation), after order-checking terminates, it is thus achieved that order-checking fragment group.
Step 8, analyzing order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of the quantity of the order-checking fragment of transgene component, endogenous standard gene and external source standard gene in testing sample, concrete grammar is as follows:
Primer according to order-checking fragment, utilize blastall (version2.2.26) software, arrange by the parameter of its acquiescence, order-checking fragment group comparison is detected on gene to correspondence each in table 1, namely the comparison fragment that successfully checks order represents and gene detected, thus obtaining the quantity of the quantity of the order-checking fragment of the quantity of order-checking fragment of various transgene components, various endogenous standard gene in testing sample and the order-checking fragment of external source standard gene respectively, its result is in Table 1.
Step 9, quantity according to the order-checking fragment of external source standard gene, it is judged that experiment whether success, concrete grammar is as follows:
When the quantity of order-checking fragment of external source standard gene and the order-checking fragment of endogenous standard gene quantity all>=�� 1 time, then Success in Experiment; When the quantity of order-checking fragment of external source standard gene or the order-checking fragment of endogenous standard gene quantity<during �� 1, then the failure of an experiment, need after the failure of an experiment to re-start experiment, till Success in Experiment; Wherein, �� 1 is decision threshold, and its size artificially judges according to the Stringency required and experience, in general, if in high-flux sequence data, it is possible to the order-checking fragment of more than 10 detected, it was shown that the gene detected is to exist. In the present embodiment, �� 1 can take 10 order-checking fragments, from table 1 it follows that the quantity of the order-checking fragment of the quantity of the order-checking fragment of external source standard gene and endogenous standard gene equal>=10, hence it was demonstrated that this experiment is successful.
If step 10 Success in Experiment, then calculating 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 the i-th test zone of m kind transgene component, n1 is the number of the test zone of m kind transgene component, bi is the quantity of the order-checking fragment of the i-th test zone of m kind transgene component, k is the endogenous standard gene of kth kind, and n3 is the number of endogenous standard gene, and j is the jth test zone of the endogenous standard gene of kth kind, n2 is the number of the test zone of the endogenous standard gene of kth kind, and aj is the quantity of the order-checking fragment of the jth kind test zone of the endogenous standard gene of kth kind; N is the sum of the test zone of all endogenous standard genes.
As can be seen from Table 1, in the present embodiment, have detected 5 kinds of transgene components altogether, so m=5, each transgene component have detected 1 test zone respectively, so, n1=1, have detected 1 endogenous standard gene altogether, so n3=1, each endogenous standard gene have detected 1 test zone altogether, so, n2=1, N=3, table 1 lists the quantity of each transgene component and the order-checking fragment of each endogenous standard gene, substitute them in transgene component content computing formula in can obtain the content of each transgene component, its result is in Table 1. as it can be seen from table 1 the present embodiment is by 5 pairs of multiplex amplification primers, the detection by quantitative content of 5 kinds of transgene components once. on the basis of the present embodiment, it is only necessary to increase more multiplex amplification primer, the content of any multiple transgene component needing detection of detection by quantitative can be realized. in addition, multiple test zones of transgene component, endogenous standard gene and external source standard gene can also be detected, the test error caused because of the different amplification efficiencies of different genes or different test zone is eliminated by average, the present embodiment is owing to controlling between 95%-105% by the amplification efficiency of each test zone of the gene of each detection, error is less, for saving cost, so each gene only have selected a test zone.
Step 11, content according to transgene component judge whether to contain in testing sample transgene component, and concrete grammar is as follows:
When needing the content>=�� 2 of any one transgene component of detection, then judge that testing sample contains transgene component; When the content of all transgene components is<during �� 2, it is determined that testing sample does not contain transgene component; Wherein, �� 2 is decision threshold.
The transgenic ingredient analysis lower limit set in GB " GMO detection nucleic acid quantification PCR detection method " (standard No.: GB/T19495.5-2004) is as 0.1%. In the present embodiment, the value of �� 2 is set as 0.1%, is used for judging that testing sample is whether as transgenic product. Owing to the present invention adopts order-checking detection transgene component, and sequencing technologies obtains is digital signal, namely measure and do not measure two kinds of situations, therefore, almost without technology lower limit, compare with traditional chip detection technology or real-time quantitative PCR detection technique, it does not have the problem of background noise interference, therefore, it is determined that whether testing sample is that the Stringency that the decision threshold of transgenic product can require as the case may be freely sets. As it can be seen from table 1 in the present embodiment, among 5 kinds of transgene components detected, except CryIAc, the content of all the other 4 kinds of transgene components all >=�� 2=0.1%, therefore, the testing sample judged in the present embodiment contains transgene component, and this testing sample is transgenic plant.
Result verification: testing sample has been detected according to the method that " transgenic plant and products thereof composition detection Bar gene or pat gene qualitative PCR method " (standard No.: No. 1782 bulletin-6-2012 of the Ministry of Agriculture) provides, testing result shows that testing sample contains transgene component, 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 detected, any multiple transgene component in testing sample can be detected by simultaneous quantitative, fully meet the current demand of detection GMOs, be that prior art does not reach.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (8)
1. the detection method of a Brassica campestris L transgene component, it is characterised in that described method includes:
Determining the external source standard gene of the endogenous standard gene and described testing sample needed in testing sample in the transgene component of detection, described testing sample, described testing sample is rapeseed plants or a part for described rapeseed plants;
Preparation is for expanding the multiplex amplification primer of the test zone of described transgene component, described endogenous standard gene and described external source standard gene;
Described testing sample it is sampled and mixes, obtaining 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 mixing nucleic acid described in described multiplex amplification primer pair to expand, obtain amplified production, utilize described amplified production to build high-throughput sequencing library;
Described high-throughput sequencing library is carried out high-flux sequence, obtains order-checking fragment group;
Analyze described order-checking fragment group, it is thus achieved that the quantity of the order-checking fragment of the quantity of the order-checking fragment of transgene component described in described testing sample, the quantity of order-checking fragment of described endogenous standard gene and described external source standard gene;
The quantity of the order-checking fragment according to described external source standard gene, it is judged that whether experiment is successful;
If described Success in Experiment, then calculate the content of transgene component described in described testing sample kind;
Content according to described transgene component judges whether contain transgene component in described testing sample.
2. detection method according to claim 1, it is characterised in that described transgene component is at least one that external source functional gene, resistant maker gene, promoter, terminator and external source insert in flanking sequence.
3. detection method according to claim 1, it is characterised in that described endogenous standard gene is the single copy gene in the genome of described testing sample.
4. detection method according to claim 1, it is characterised in that described external source standard gene is not present in all biologies.
5. detection method according to claim 1, it is characterized in that, described judgment experiment whether successfully method is: when the quantity of the quantity of order-checking fragment of described external source standard gene and the order-checking fragment of described endogenous standard gene equal>=�� 1 time, then Success in Experiment; When the quantity of order-checking fragment of described external source standard gene or the order-checking fragment of described endogenous standard gene quantity<during �� 1, then the failure of an experiment; Wherein, �� 1 is decision threshold.
6. detection method according to claim 1, it is characterized in that, described judge described testing sample whether contain the method for transgene component as: as the content>=�� 2 of any one described transgene component of needs detection, it is determined that described testing sample contains transgene component; When the content of all described transgene components is<during �� 2, it is determined that described testing sample does not contain transgene component; Wherein, �� 2 is decision threshold.
7. detection method according to claim 1, it is characterised in that the method for the content of transgene component described in described testing sample kind that calculates is: described in m kind, the computing formula of the content of transgene component isWherein, i be described m kind transgene component i-th described in test zone, n1 is the number of the described test zone of described m kind transgene component, bi be described m kind transgene component i-th described in the quantity of described order-checking fragment of test zone, k is endogenous standard gene described in kth kind, n3 is the number of described endogenous standard gene, j is the jth test zone of endogenous standard gene described in kth kind, n2 is the number of the described test zone of the described endogenous standard gene of kth kind, aj be the described endogenous standard gene of kth kind jth kind described in the quantity of described order-checking fragment of test zone, N is the sum of the described test zone of all described endogenous standard genes.
8. detection method according to claim 1, it is characterised in that the ratio of the quality of described external source standard gene and the genomic gross mass of described biased sample is more than 1/100000.
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| CN108546737A (en) * | 2018-05-10 | 2018-09-18 | 湖南农业大学 | A kind of detection method of rape transgene component |
| CN114107552A (en) * | 2022-01-04 | 2022-03-01 | 江汉大学 | Primer pair combination, kit, detection method and application for detecting rape transgenic line |
| CN114277178A (en) * | 2022-01-04 | 2022-04-05 | 江汉大学 | Primer pair combination, kit, detection method and application for detecting transgenic components of rape |
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| CN114107552B (en) * | 2022-01-04 | 2023-09-08 | 江汉大学 | Primer pair combination, kit, detection method and application for detecting rape transgenic line |
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