CN105624298A - Method for detecting genetically modified components of rape - Google Patents

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

A detection method for genetically modified components of rapeseed

technical field

The invention relates to the field of biological detection, in particular to a method for detecting genetically modified components of rapeseed.

Background technique

Rapeseed is the main crop in my country. Rapeseed transgenic technology and products have matured, and the promotion of genetically modified rapeseed needs to be supervised. The detection of genetically modified ingredients is the technical support for the supervision of genetically modified plants.

The existing genetically modified component detection technology mainly detects nucleic acid or protein. Among them, the main process of the nucleic acid-based detection method is: extract the nucleic acid in the sample to be tested, and use PCR (Polymerase Chain Reaction, polymerase chain reaction) method to amplify the transgene in the sample. Components, using real-time quantitative, agarose electrophoresis or chip technology to detect the presence of genetically modified components.

In the process of realizing the present invention, the inventor found that the prior art has at least one of the following problems:

There are many kinds of genetically modified ingredients, and the existing technology can only detect one or a few of them at a time. Therefore, it is necessary to test the possible multiple genetically modified ingredients one by one before they can be determined as "non-genetically modified" products; The transformed genetically modified components are not the exogenous gene itself. Therefore, for unmarked transgenes, the existing detection methods will be invalid; detection is basically qualitative detection, but quantitative detection has its practical needs, for example, A small amount of pollen from transgenic rapeseed varieties in adjacent fields has been transferred to non-transgenic varieties, and non-transgenic varieties will be misjudged as transgenic varieties during qualitative testing.

Contents of the invention

In order to solve the problems in the prior art, an embodiment of the present invention provides a method for detecting genetically modified components of rapeseed. Described technical scheme is as follows:

The embodiment of the present invention provides a method for detecting genetically modified components of rapeseed, the method comprising:

Determining the transgenic components to be detected in the sample to be tested, the endogenous standard gene in the sample to be tested and the exogenous standard gene in the sample to be tested, the sample to be tested is a rape plant or a part of the rape plant;

preparing multiplex amplification primers for amplifying the test regions of the transgenic component, the endogenous standard gene and the exogenous standard gene;

Sampling and mixing the samples to be tested to obtain a mixed sample;

extracting the genome of the mixed sample;

adding the exogenous standard gene to the genome of the mixed sample to obtain a mixed nucleic acid;

Using the multiple amplification primers to amplify the mixed nucleic acid to obtain an amplification product, using the amplification product to construct a high-throughput sequencing library;

performing high-throughput sequencing on the high-throughput sequencing library to obtain a sequencing fragment set;

analyzing the sequenced fragment group to obtain the number of sequenced fragments of the transgenic component, the number of sequenced fragments of the endogenous standard gene and the number of sequenced fragments of the exogenous standard gene in the sample to be tested;

According to the number of sequencing fragments of the exogenous standard gene, judge whether the experiment is successful;

If the experiment is successful, then calculate the content of the genetically modified component in the sample species to be tested;

According to the content of the genetically modified component, it is judged whether the sample to be tested contains the genetically modified component.

Specifically, the transgene component is at least one of an exogenous functional gene, a resistance marker gene, a promoter, a terminator, and an exogenous insertion flanking sequence.

Specifically, the endogenous standard gene is a single-copy gene in the genome of the sample to be tested.

Specifically, the exogenous standard gene is not present in all organisms.

Specifically, the method for judging whether the experiment is successful is: when the number of sequenced fragments of the exogenous standard gene and the number of sequenced fragments of the endogenous standard gene are both ≥ α1, the experiment is successful; When the number of sequenced fragments of the source standard gene or the number of sequenced fragments of the endogenous standard gene < α1, the experiment fails; where α1 is the threshold for determination.

Specifically, the method for determining whether the sample to be tested contains genetically modified components is: when the content of any one of the genetically modified components to be detected is greater than or equal to α2, it is determined that the sample to be tested contains genetically modified components; When the content of the genetically modified component is <α2, it is determined that the sample to be tested does not contain the genetically modified component; wherein, α2 is the judgment threshold.

Specifically, the method for calculating the content of the genetically modified component in the sample species to be tested is: the calculation formula for the content of the mth genetically modified component is: Wherein, i is the i-th test area of the m-th transgenic component, n1 is the number of the test areas of the m-th transgenic component, and bi is the i-th test area of the m-th transgenic component. The number of the sequenced fragments in the test area, k is the kth endogenous standard gene, n3 is the number of the endogenous standard gene, and j is the jth of the kth endogenous standard gene Test area, n2 is the number of the test area of the kth endogenous standard gene, aj is the number of the sequencing fragments of the jth test area of the kth endogenous standard gene; N is the total number of said test regions for all said endogenous standard genes.

Specifically, the ratio of the mass of the exogenous standard gene to the total mass of the genome of the mixed sample is greater than 1/100000.

The beneficial effects brought by the technical solutions provided by the embodiments of the present invention are: the detection method provided by the present invention can be used universally among different samples to be tested and different detected target genes, and can detect any number of transgenic components that need to be detected at one time, thereby To determine whether the sample to be tested contains genetically modified ingredients, this method can realize quantitative detection, and the detection result has almost no lower limit, and the result is accurate and reliable, which is beyond the reach of the existing technology.

detailed description

In order to make the purpose, technical solution and advantages of the present invention clearer, the implementation manners of the present invention will be further described in detail below. The operating procedures or operating specifications not indicated or described in detail in the embodiments of the present invention are operations well known to those skilled in molecular biology. Reagents or biological materials not indicated in the examples of the present invention are common reagents or biological materials sold in the market, are well known to ordinary molecular biology technicians, and can be purchased in the market.

Example

The biolaphos resistance (Bar) gene was introduced into Guiyouhui 15 rape variety by Agrobacterium-mediated method, and after selfing and purification for 3 generations, the transformed Guiyouhui 15 rape variety seeds were obtained as this example Rapeseed varieties to be tested. Among them, Guiyouhui 15 rapeseed is a transgenic rapeseed recipient variety, bred by the Rapeseed Research Laboratory of Guizhou Agricultural College, and Guiyouhui 15 rapeseed seeds used in this example were preserved and propagated by Jianghan University.

The method for detecting genetically modified components of rape plant varieties, the specific steps are as follows:

Step 1. Determine the transgenic components to be detected in the sample to be tested, the endogenous standard gene in the sample to be tested, and the exogenous standard gene of the sample to be tested. The specific method is as follows: the sample to be tested is a rape plant or a part of a rape plant, wherein Rapeseed plants can be organs such as roots, stems, leaves, flowers, fruits and seeds of rapeseed plants, or a mixture of two or more different organs, such as the mixture of leaves and stems, and a part of rapeseed plants can be A part of at least one organ such as roots, stems, leaves, flowers, fruits and seeds of rapeseed plants. In this embodiment, the sample to be tested is the seed of Guiyouhui 15 rapeseed after transformation, and the transgenic component, endogenous standard gene and exogenous standard gene are all ≥ 1, and the transgenic component can be exogenous functional gene, resistance marker At least one of genes, promoters, terminators, and foreign-inserted flanking sequences, that is, the introduction of exogenous nucleic acid sequences that do not exist in Guiyouhui 15 rapeseed varieties into Guiyouhui 15 rapeseed varieties by means of transgenic technology ; The endogenous standard gene is a single-copy gene in the genome of the sample to be tested. In this embodiment, the endogenous standard gene is a single sequence when compared on the genome of the sample to be tested by NCBI (http://www.ncbi.nlm.nih.gov/); the exogenous standard gene does not exist in all organisms Among them, in this example, the exogenous standard gene used is the ERCC04 gene, and no homologous sequence was found when it was homologously compared on NCBI, so it can be determined that the exogenous standard gene does not exist in all organisms. In this example, there are 5 types of genetically modified components detected, 1 type of endogenous standard gene, and 1 type of exogenous standard gene. Exogenous functional genes also include other transgenic components, which are widely used in transgenic crops, therefore, the detection method provided in this example is universal. The gene sequence in Table 1 has two representations, one is to write the gene sequence directly, and the other is the gene number of NBCI. The numbers in the sequencing area in Table 1 represent the position of the base, and the first The position of a base is defined as 1.

Table 1 is the relevant information and detection results of the detected genes in Example 1

"/" in Table 1 means none.

Step 2, preparing multiple amplification primers for amplifying transgenic components, endogenous standard genes and exogenous standard genes, the specific method is as follows:

The process of obtaining multiplex amplification primers is as follows: log in to Thermo Fisher’s multiplex PCR primer online design website https://ampliseq.com/, and select “DNAHotspotdesigns (single-pool)” in the “Application type” option. If multi-pool is selected, the multiplex PCR will be carried out in multiple tubes, and the cost will increase, while the primers of single-pool only need one multiplex PCR, which saves cost, so single-pool is selected in this embodiment. Connect the sequences of each gene listed in Table 1 with 100 Ns to form an artificial reference genome, and upload the artificial reference genome after selecting "Custom" in the "Selectthegenomeyouwishtouse" option. DNAType (type) option select "StandardDNA" (standard DNA). In the AddHotspot option, for each gene in Table 1, randomly select a test region whose homology of homologous sequences obtained through NCBI homologous alignment is lower than 80%. Among them, the test area refers to the amplification area of multiplex PCR, which is also the area of high-throughput sequencing. Compared with other species, the test area has no obvious homologous sequence, so it can represent the detected gene. Finally, the presence of sequenced fragments in the test area indicates the presence of the detected gene, and the number of sequenced fragments in the test area represents the amount of the detected gene. In addition, the homologous sequence does not include the sequence in the original source species of the transgenic component, for example, the original source species of the CryIAc gene in Table 1 is Bacillus thuringiensis (abbreviated as Bt), therefore, the object of the homologous comparison is not Includes the genome of Bacillus thuringiensis. In order to ensure the versatility of the embodiments of the present invention, download the different sequences of the endogenous standard gene in the species on NCBI, and obtain the conserved regions between the sequences through homologous comparison. The test region of the endogenous standard gene is selected as the A region where the inner boundary sequence is conserved. Further fill in the start and end positions of each test region obtained, and finally click the "Submit targets" button to submit, and obtain the sequence of the amplification primers for amplifying each gene in Table 1. The amplification efficiency of each primer in the multiple amplification primers is between 95% and 105%. Therefore, the amplification efficiency of the multiple amplification primers needs to be verified. The specific verification method is as follows: the sequence of the Bar gene in Table 1 and the amplification primer sequence for amplifying the test region of the Bar gene are synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the sequence of the synthesized Bar gene is used as a template. The operating manual (PartNumber4376784Rev.E) of the StepOne real-time quantitative PCR instrument of Thermo Fisher Corporation detects the amplification efficiency of the amplification primer of the test region of amplifying the Bar gene, if the amplification efficiency is not between 95%-105%, Then it is necessary to re-determine the test region of the Bar gene, design and verify the efficiency of the new amplification primers for the Bar gene until the efficiency is between 95% and 105%. In the same way, verify the amplification efficiencies of the amplification primers for other genes in Table 1. The amplification efficiency, sequence and corresponding amplification region of the final selected amplification primers are shown in Table 1. All the final selected amplification primers are called multiple amplification primers, and the multiple amplification primers were synthesized by Thermo Fisher Corporation of the United States and produced Provided to the user in the form of a mixed liquid. This embodiment adopts the multiplex PCR technology provided by Thermo Fisher Corporation of the United States, which can simultaneously amplify up to 12,000 test regions. Therefore, the present invention has the ability to detect all existing transgenic components that need to be detected at one time.

Step 3, sample and mix the sample to be tested to obtain a mixed sample, the specific method is as follows:

The sample to be tested is sampled according to the sampling method of rapeseed in bulk goods in the standard "Detection and Sampling of Genetically Modified Plants and Their Product Components" (Standard No.: Announcement No. 2031 of the Ministry of Agriculture-19-2013). Therefore, the sampling point is changed to 1, the sample size of the sampling sample is >1000 seeds, and the extracted plant samples to be tested are fully mixed to obtain a mixed sample.

Step 4, extracting the genome of the mixed sample, the specific method is as follows:

Refer to the standard "DNA Extraction and Purification for Detection of Components of Transgenic Plants and Their Products" (Standard Number: Ministry of Agriculture Announcement No. 1485-4-2010) in which the solid sample is pretreated to extract the genome of the mixed sample, and the extracted genome of the mixed sample genomic nucleic acid.

Step 5, adding an exogenous standard gene to the genome of the mixed sample to obtain a mixed nucleic acid, the specific method is as follows:

Using the double-stranded DNA program in the spectrophotometer (manufactured by Quawell, USA, model Q5000), the concentration of the genome in the obtained mixed sample was detected. The ratio of the mass of the exogenous standard gene to the total mass of the genome of the mixed sample is greater than 1/100000, and this ratio is used to ensure that ≥10 If the throughput of high-throughput sequencing is lower or higher, the ratio can be adjusted accordingly. In this embodiment, the total mass of the genome of the mixed sample is 1003ng, the exogenous standard gene added is 1.003ng, and the addition ratio is 1/1000, and the mixed nucleic acid is obtained.

Step 6, using multiple amplification primers to amplify the mixed nucleic acid to obtain amplified products, and using the amplified products to construct a high-throughput sequencing library, the specific method is as follows:

A high-throughput sequencing library was constructed using the library construction kit 2.0 (produced by LifeTechnology, USA, product number 4475345). The library construction kit includes the following reagents: 5×IonAmpliSeq ^TM HiFiMix, FuPa reagent, conversion reagent, sequencing adapter solution and DNA ligase. The method of library construction was carried out according to the operation manual "IonAmpliSeq ^TM Library Preparation" (publication number: MAN0006735, version: A.0) of the library construction kit. The multiplex PCR amplification system was as follows: 5×IonAmpliSeq ^TM HiFiMix 4 μl, prepared multiple amplification primer mixed liquid 4 μl, mixed nucleic acid 10 ng and enzyme-free water 11 μl. The multiplex PCR amplification program is as follows: 99°C, 2 minutes; (99°C, 15 seconds; 60°C, 4 minutes) x 25 cycles; 10°C incubation. Use FuPa reagent to digest the redundant primers in the multiple PCR amplification products, and then phosphorylate them. The specific method is: add 2 μl FuPa reagent to the multiple PCR amplification products, mix well, and react on the PCR instrument according to the following procedure: 50° C. for 10 minutes; 55° C. for 10 minutes; 60° C. for 10 minutes; and 10° C. for storage to obtain mixture a, which is a solution containing phosphorylated amplification products. Connect the phosphorylated amplification product to the sequencing adapter. The specific method is: add 4 μl of conversion reagent, 2 μl of sequencing adapter solution and 2 μl of DNA ligase to mixture a, mix well, and react on the PCR machine according to the following procedure: 22°C , 30 minutes; 72 ° C, 10 minutes; 10 ° C storage, to obtain the mixture b. Mixture b was purified by standard ethanol precipitation and dissolved in 10 μl of enzyme-free water. Produced by Invitrigen Company of the United States dsDNAHSAssayKit (Cat. No. Q32852) was tested according to its instructions. After obtaining the mass concentration of the mixed solution b, the purified mixed solution b was diluted to 15 ng/ml to obtain a high-throughput sequencing library with a concentration of about 100 pM.

Step 7. Perform high-throughput sequencing on the high-throughput sequencing library to obtain the sequenced fragment group. The specific method is as follows:

The obtained high-throughput sequencing library and kit IonPITemplateOT2200Kitv2 (produced by Invirtrigen, USA, product number 4485146) were used for ePCR (EmulsionPCR, emulsion polymerase chain reaction) amplification before sequencing, and the operation method was carried out according to the operation manual of the kit. High-throughput sequencing was performed on a Proton second-generation high-throughput sequencer using the ePCR product and the kit IonPISequencing200Kitv2 (produced by Invirtrigen, USA, product number 4485149), and the operation method was carried out according to the operation manual of the kit. In this embodiment, the high-throughput sequencing volume is set to 1M sequencing fragments (1M=1 million), and the sequencing length is set to 500 cycles (cycles). After the sequencing is completed, a sequenced fragment group is obtained.

Step 8. Analyze the sequenced fragment group to obtain the number of sequenced fragments of the transgenic component in the sample to be tested, the number of sequenced fragments of the endogenous standard gene, and the number of sequenced fragments of the exogenous standard gene. The specific methods are as follows:

According to the primers of the sequenced fragments, use the blastall (version2.2.26) software, according to its default parameter settings, compare the sequenced fragments to the corresponding detection genes in Table 1, and the sequenced fragments that are successfully compared represent the detected genes. The number of sequenced fragments of various transgenic components, the number of sequenced fragments of various endogenous standard genes, and the number of sequenced fragments of exogenous standard genes in the sample to be tested were respectively obtained. The results are shown in Table 1.

Step 9. According to the number of sequenced fragments of the exogenous standard gene, judge whether the experiment is successful, the specific method is as follows:

When the number of sequenced fragments of the exogenous standard gene and the number of sequenced fragments of the endogenous standard gene are both ≥α1, the experiment is successful; when the number of sequenced fragments of the exogenous standard gene or the number of sequenced fragments of the endogenous standard gene< When α1, the experiment fails. After the experiment fails, the experiment needs to be repeated until the experiment succeeds; among them, α1 is the judgment threshold, and its size is judged manually according to the strictness of requirements and experience. Generally speaking, if the high-throughput sequencing data Among them, more than 10 sequencing fragments can be detected, indicating that the detected gene exists. In this embodiment, α1 can take 10 sequencing fragments. It can be seen from Table 1 that the number of sequencing fragments of exogenous standard genes and the number of sequencing fragments of endogenous standard genes are both ≥ 10, so it is confirmed that this The experiment was successful.

Step 10, if the experiment is successful, then calculate the content of the transgenic component in the sample species to be tested, the specific method is as follows:

The formula for calculating the content of the mth genetically modified ingredient is Among them, i is the i-th test area of the m-th genetically modified component, n1 is the number of test areas of the m-th genetically modified component, bi is the number of sequencing fragments in the i-th test area of the m-th genetically modified component, k is the kth endogenous standard gene, n3 is the number of endogenous standard genes, j is the jth test area of the kth endogenous standard gene, n2 is the number of test areas of the kth endogenous standard gene , aj is the number of sequenced fragments of the jth test region of the kth endogenous standard gene; N is the total number of test regions of all endogenous standard genes.

It can be seen from Table 1 that in this embodiment, 5 kinds of genetically modified components were detected, so m=5, and one test area was detected for each kind of genetically modified component, so, n1=1, a total of 1 test area was detected Endogenous standard gene, so n3=1, each endogenous standard gene detected a total of 1 test area, so, n2=1, N=3, Table 1 lists each transgenic ingredient and each endogenous standard The number of sequenced fragments of the gene can be substituted into the calculation formula of the content of the genetically modified ingredients to obtain the content of each genetically modified ingredient. The results are shown in Table 1. It can be seen from Table 1 that in this example, the contents of five transgenic components were quantitatively detected at one time through five pairs of multiple amplification primers. On the basis of this example, it is only necessary to add more multiplex amplification primers to achieve one-time quantitative detection of the contents of any number of transgenic components that need to be detected. In addition, multiple test areas of transgenic components, endogenous standard genes, and exogenous standard genes can also be detected, and the test error caused by different amplification efficiencies of different genes or different test areas can be eliminated by averaging. The amplification efficiency of each test region of each detected gene is controlled between 95%-105%, and the error is small. In order to save costs, only one test region is selected for each gene.

Step 11. Determine whether the sample to be tested contains a genetically modified ingredient according to the content of the genetically modified ingredient. The specific method is as follows:

When the content of any genetically modified ingredient to be detected is ≥ α2, it is determined that the sample to be tested contains genetically modified ingredients; when the content of all genetically modified ingredients is < α2, it is judged that the sample to be tested does not contain genetically modified ingredients; where α2 is the judgment threshold.

The lower limit of detection of genetically modified ingredients set in the national standard "Nucleic acid quantitative PCR detection method for detection of genetically modified products" (standard number: GB/T19495.5-2004) is 0.1%. In this embodiment, the value of α2 is set to 0.1%, which is used to determine whether the sample to be tested is a genetically modified product. Since the present invention uses sequencing to detect transgenic components, and the sequencing technology obtains digital signals, that is, detected and not detected, there is almost no technical lower limit. Compared with traditional chip detection technology or real-time quantitative PCR detection technology, There is no problem of background noise interference, therefore, the judgment threshold for judging whether the sample to be tested is a genetically modified product can be freely set according to the strictness required by the specific situation. It can be seen from Table 1 that in this example, among the five detected transgenic components, except for CryIAc, the contents of the other four transgenic components are all ≥α2=0.1%. Therefore, it is determined that the tested components in this embodiment The sample contains genetically modified components, and the sample to be tested is a genetically modified plant.

Result verification: According to the method provided by "Qualitative PCR method for Bar gene or pat gene detection of transgenic plants and their product components" (standard number: Ministry of Agriculture Announcement No. 1782-6-2012), the test samples were tested, and the test results showed that The test sample contains genetically modified components, which is consistent with the results of this example, so it can be seen that the detection method provided by this example is correct.

The detection method provided by the present invention can be commonly used among different samples to be tested and different target genes to be detected, and can quantitatively detect any variety of transgenic components in the samples to be tested at the same time, which fully meets the actual needs of genetically modified detection, and is an achievement of the prior art. less than.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

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.