CN114807406B

CN114807406B - Primer pair combination, kit and detection method for detecting soybean transgenic component

Info

Publication number: CN114807406B
Application number: CN202210224773.2A
Authority: CN
Inventors: 张静; 彭海; 陈利红; 肖华锋; 高利芬; 李甜甜; 李论; 方治伟; 万人静; 周俊飞
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2023-09-08
Anticipated expiration: 2042-03-07
Also published as: CN114807406A

Abstract

The application belongs to the technical field of biology, and particularly relates to a primer pair combination, a kit and a detection method for detecting soybean transgenic components. The nucleotide sequence of the primer pair combination is shown as SEQ ID NO.1-SEQ ID NO. 78. The amplification products combined by the primer pairs can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, so that more false positive or false negative results are avoided when multiple PCR is performed in the prior art; meanwhile, when the primer provided by the application is used for detecting samples, multiplex PCR amplification of more than 9 target molecules in one sample or multiplex PCR amplification of multiple targets in multiple samples can be realized, and then the detection result of transgenic components is obtained through one-time high-throughput sequencing and analysis.

Description

Primer pair combination, kit and detection method for detecting soybean transgenic component

Technical Field

The application belongs to the technical field of biology, and particularly relates to a primer pair combination, a kit and a detection method for detecting soybean transgenic components.

Background

Soybeans are crops planted generally worldwide, contain rich vegetable proteins and edible oil and fat, and are important edible, feeding and oil crops. Under the background that the soybean planting area is difficult to be greatly increased at present, the method for cultivating the novel variety of high-yield and high-quality transgenic soybean by utilizing the modern biotechnology has great significance for promoting the soybean industry to be happy. However, with the increasing concern of the direct or indirect production of large amounts of transgenic soybeans into food and the safety problems of transgenic products in the international society, the detection of transgenic components in agricultural products has been brought into the detection projects of inspection and quarantine departments at home and abroad and gradually strengthened. Therefore, development of efficient and convenient transgenic food detection technology is very important.

The detection technology of the transgenic products mainly comprises a protein-based detection method and a nucleic acid-based detection method. The current PCR detection method based on nucleic acid is still the most common and accurate transgene detection technology at present, and mainly comprises the methods of common qualitative PCR, nested PCR, loop-mediated isothermal amplification (LAMP), fluorescent quantitative PCR multiplex PCR and the like. Compared with the common qualitative PCR method, the nested PCR has higher detection sensitivity and is easy to cause false positive. LAMP is simple to operate and high in specificity, however, primer design is complex, DNA pollution is easy to cause, and subsequent experiments are affected. The fluorescent quantitative PCR method has the advantages of good repeatability, high sensitivity and less nucleic acid cross contamination, but has high cost and needs a special detection instrument. The common multiplex PCR method can detect a plurality of genes simultaneously in one reaction, but the number of the genes is not more than six, otherwise, the interference among primers is large, and the detection effect is influenced. The gene chip and the digital PCR technology are also common transgenic product detection technologies, have the advantages of high flux, high sensitivity, strong specificity and the like, and can detect a plurality of genes in1 transgenic crop in parallel or detect a plurality of transgenic crops simultaneously; however, the cost is high, special instruments and equipment are required, operators are required to have high professional quality, and the factors limit the wide application of the technology in detection.

Therefore, developing a high-efficiency, sensitive and high-flux transgenic product detection method becomes a key problem to be solved urgently.

Disclosure of Invention

The application provides a primer pair combination, a kit and a detection method for detecting soybean transgenic components, which are used for solving the technical problem of how to efficiently detect the soybean transgenic components.

In a first aspect, the present application provides a primer pair combination for detecting a transgenic soybean component, the primer pair combination comprising:

a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;

a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;

a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;

a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;

a primer pair for specifically amplifying tPIN II, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;

a primer pair for specifically amplifying pRBCS4, the nucleotide sequence of which is shown in SEQ ID NO.11 to SEQ ID NO. 12;

a primer pair for specifically amplifying tE9, the nucleotide sequence of which is shown in SEQ ID NO.13 to SEQ ID NO. 14;

a primer pair for specifically amplifying t7s, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.15 to SEQ ID NO. 16;

a primer pair for specifically amplifying PAT, the nucleotide sequence of which is shown as SEQ ID NO.17 to SEQ ID NO. 18;

a primer pair for specifically amplifying pTsf1, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;

a primer pair for specifically amplifying tsf1, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22;

a primer pair for specifically amplifying GAT, the nucleotide sequence of which is shown in SEQ ID NO.23 to SEQ ID NO. 24;

a primer pair for specifically amplifying Cry1Ab-Ac, the nucleotide sequence of which is shown as SEQ ID NO.25 to SEQ ID NO. 26;

a primer pair for specifically amplifying Cry1A.105, the nucleotide sequence of which is shown as SEQ ID NO.27 to SEQ ID NO. 28;

a primer pair for specifically amplifying gm-hra, the nucleotide sequence of which is shown as SEQ ID NO.29 to SEQ ID NO. 30;

a primer pair for specifically amplifying gm-als, the nucleotide sequence of which is shown in SEQ ID NO.31 to SEQ ID NO. 32;

a primer pair for specifically amplifying Cry2Ab, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.33 to SEQ ID NO. 34;

a primer pair for specifically amplifying pCISV, the nucleotide sequence of which is shown as SEQ ID NO.35 to SEQ ID NO. 36;

a primer pair for specifically amplifying ATcsr1-2, the nucleotide sequence of which is shown as SEQ ID NO.37 to SEQ ID NO. 38;

the nucleotide sequence of the primer pair for specifically amplifying tAtAHASL is shown as SEQ ID NO.39 to SEQ ID NO.40;

the nucleotide sequence of the primer pair for specifically amplifying DMO is shown as SEQ ID NO.41 to SEQ ID NO. 42;

a primer pair for specifically amplifying tORF23, the nucleotide sequence of which is shown in SEQ ID NO.43 to SEQ ID NO. 44;

a primer pair for specifically amplifying the AtRbcs-transit-peptide, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 45-SEQ ID NO. 46;

a primer pair for specifically amplifying AAD-12, the nucleotide sequence of which is shown as SEQ ID NO.47 to SEQ ID NO. 48;

a primer pair for specifically amplifying tORF1, the nucleotide sequence of which is shown as SEQ ID NO.49 to SEQ ID NO. 50;

a primer pair for specifically amplifying pFMV35S, the nucleotide sequence of which is shown as SEQ ID NO.51 to SEQ ID NO. 52;

a primer pair for specifically amplifying cry1F, the nucleotide sequence of which is shown as SEQ ID NO.53 to SEQ ID NO. 54;

a primer pair for specifically amplifying CTP2, the nucleotide sequence of which is shown as SEQ ID NO.55 to SEQ ID NO. 56;

a primer pair for specifically amplifying the Tev-5UTR, the nucleotide sequence of which is shown as SEQ ID NO.57 to SEQ ID NO. 58;

a primer pair for specifically amplifying hppdPf, the nucleotide sequence of which is shown in SEQ ID NO.59 to SEQ ID NO. 60;

a primer pair for specifically amplifying the H4A-terminator, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.61 to SEQ ID NO. 62;

a primer pair for specifically amplifying H3At-intron, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.63 to SEQ ID NO. 64;

a primer pair for specifically amplifying pH4A748, the nucleotide sequence of which is shown as SEQ ID NO.65 to SEQ ID NO. 66;

a primer pair for specifically amplifying 2mepsps, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.67 to SEQ ID NO. 68;

a primer pair for specifically amplifying cp4epsps, the nucleotide sequence of which is shown as SEQ ID NO.69 to SEQ ID NO. 70;

a primer pair for specifically amplifying pAtUbiQuittin10, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.71 to SEQ ID NO. 72;

and/or a primer pair for specifically amplifying pCSVMV, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.73 to SEQ ID NO. 74.

Optionally, the primer pair combination further comprises a primer pair for amplifying the soybean internal reference gene Gm_lectin_control.

Alternatively, two pairs of primers of a primer pair for amplifying the soybean reference gene Gm_Lectin_control have nucleotide sequences shown in SEQ ID NO.75-SEQ ID NO. 78.

Optionally, the primer pair combination comprises a primer pair that specifically amplifies a soybean transgenic element selected from the group consisting of: p35S, t35S, pNOS, tNOS, tPIN II, pRBCS4, tE9, t7s, PAT, pTsf1, tsf1, GAT, cry1Ab-Ac, cry1A.105, gm-hra, gm-als, cry2Ab, pCISV, ATcsr1-2, tAtAHASL, DMO, tORF, atRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, cry1F, CTP2, tev-5UTR, hppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, pAtUbiQuittin10 and pCSVMV.

In a second aspect, a kit for detecting a transgenic soybean component, the kit comprising the primer pair combination of the first aspect for detecting a transgenic soybean component.

Optionally, the kit includes a first container, and the first container contains the primer pair combination therein.

Optionally, the kit further comprises a multiplex PCR premix.

In a third aspect, there is provided a primer pair combination according to the first aspect, and an application of the detection kit according to the second aspect in detecting transgenic soybeans and related products thereof.

In a fourth aspect, a method of detecting a transgenic soybean component, the method comprising the steps of:

obtaining DNA of soybean to be tested and the primer pair combination in the first aspect;

taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing amplification reaction to obtain an amplification product;

carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;

and analyzing the gene sequences in the high-throughput library to obtain the result of detecting the soybean transgenic components.

Optionally, the reaction system comprises: the total system is 30-50 μl; primer pair: 2-5 μl;2 Xbuffer: 15-30ul; multiplex amplification enzyme: 0.5-1 μl; the balance being water.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the nucleotide sequence of the primer pair combination is shown as SEQ ID NO.1-SEQ ID NO. 78. The amplification product of the primer pair combination can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, wherein the detection results comprise transgenic components, judging whether a sample to be detected contains target molecules or not, and determining the copy numbers of reference genes and target molecules in the sample to be detected so as to determine the content of exogenous genes; the method has the advantages that the traditional Real-time PCR technology is avoided, only one purpose can be achieved at a time, multiple amplification and detection are needed to cover multiple target transgene components in a sample, more false positive and false negative results are avoided when multiple PCR is carried out in the prior art, more than 9 PCR reactions can be carried out, simultaneous detection of multiple target molecules in the sample can be completed through one-time high-throughput sequencing and analysis, the detection efficiency is greatly improved, and meanwhile, the detection flux and the cost are simultaneously taken into consideration.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a schematic flow chart of a method for detecting transgenic soybean components according to an embodiment of the present application;

FIG. 2 is a schematic diagram of the transgenic line DAS-44406-6 according to an embodiment of the application.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification will control. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present application. For example, room temperature may refer to a temperature in the range of 10 to 35 ℃.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

according to an exemplary embodiment of the present application, there is provided a primer pair combination for detecting a soybean transgenic ingredient, the primer pair combination comprising: a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;

The specific correspondence between the above primers and the nucleotide sequences of the soybean transgenic elements amplified by the primers, i.e., the numbers of the target molecules and the corresponding primer pairs, and the nucleotide sequences of the primers are shown in Table 1.

Table 1 numbering of target molecules and corresponding primer pairs and nucleotide sequences of primers.

In the process of primer design, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be normally amplified in one amplification reaction, and the use proves that the primers have high sensitivity and strong applicability.

In some embodiments, the primer pair combination further comprises a primer pair for amplifying the soybean reference gene gm_lectin_control.

In order to realize the aim of quantitatively detecting the soybean transgenic component in the sample, when the soybean transgenic element is selected, a detection primer for the soybean reference gene is added, so that the quantitative detection of the transgenic component content in the sample is realized.

In some embodiments, the two pairs of primers of the primer pair for amplifying soybean reference gene Gm_Lectin_control have the nucleotide sequences shown in SEQ ID NO.75-SEQ ID NO. 78.

The reason for using 2 primer pairs is to avoid instability of the reference gene and that the DNA content cannot be detected effectively at low levels.

In some embodiments, the primer pair combination comprises a primer pair that specifically amplifies a soybean transgenic element selected from the group consisting of: p35S, t35S, pNOS, tNOS, tPIN II, pRBCS4, tE9, t7s, PAT, pTsf1, tsf1, GAT, cry1Ab-Ac, cry1A.105, gm-hra, gm-als, cry2Ab, pCISV, ATcsr1-2, tAtAHASL, DMO, tORF, atRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, cry1F, CTP2, tev-5UTR, hppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, pAtUbiQuittin10 and pCSVMV.

In the embodiment of the application, the nucleotide sequence of a detection element of a common soybean transgenic product, namely a target molecule and an internal reference gene, are screened as detection targets. Including 37 transgenic elements commonly used for detection: p35S, t35S, pNOS, tNOS, tPIN II, pRBCS4, tE9, t7s, PAT, pTsf1, tsf1, GAT, cry1Ab-Ac, cry1A.105, gm-hra, gm-als, cry2Ab, pCISV, ATcsr1-2, tAtAHASL, DMO, tORF, atRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, cry1F, CTP2, tev-5UTR, hppdPf, H A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, pAtUbiQuittin and pCSVMV, and the sequence GmLectin_control including 1 soybean reference gene.

Next, the present application developed multiplex PCR primer compositions for detecting the transgenic elements and soybean reference genes, wherein 37 pairs are directed against 37 transgenic elements and 2 pairs are directed against 1 reference gene. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR. The multiplex PCR primer composition can be used for developing a transgenic element detection kit. In the existing primer pair combination: 1-39 pairs, may further comprise primer pairs that specifically amplify soybean transgenic elements selected from the group consisting of, but not limited to, 39 pairs or more. The logarithmic combination of the multiplex PCR primers can be increased periodically according to the newly collected transgenic elements in the later period, and can reach 3000 pairs through verification, so that the amplification effect is excellent; compared with the conventional 8-pair specific multiplex PCR, the method has the advantages of high detection flux and high sensitivity.

And is appropriately adjusted according to the specific detection sample. The later period can be increased periodically according to the newly collected transgenic elements, 3000 pairs of primer combinations are tried, and the amplification effect is still good. To achieve detection of transgenic components in soybean, we collected 39 pairs of commonly used soybean transgenic elements and sequences of internal reference genes, the logarithmic range of the multiplex PCR primers was: 1-39 pairs, compared with the conventional 8-pair specific multiplex PCR, have the advantages of high detection flux and sensitivity.

In particular, high throughput sequencing can be second generation sequencing or third generation sequencing, and the resulting high throughput library can analyze the components of the transgene from multiple dimensions, including but not limited to transgenic elements in our embodiment.

In some embodiments, the kit comprises a first container containing the primer pair combination therein.

In some embodiments, the kit further comprises a multiplex PCR premix.

Specifically, when the components of the multiplex PCR premix include the combination of the transgenic element of soybean and each primer group of the reference gene, each primer is premixed according to a ratio of 1:1, and the mixture of each primer is carried out according to different experimental purposes, and in a specific implementation example, each primer concentration is 2nM.

In a fourth aspect, a method for detecting a transgenic soybean component, as shown in FIG. 1, comprises the steps of:

s1, obtaining DNA and primer pair combination of soybean to be detected;

s2, taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing an amplification reaction to obtain an amplification product;

s3, carrying out high-flux sequencing on the amplification product to obtain a high-flux library;

s4, analyzing the gene sequences in the high-flux library to obtain a result of detecting the soybean transgenic components.

In some embodiments, the reaction system comprises: the total system is 30-50 μl; primer pair: 2-5 μl;2 Xbuffer: 15-30ul; multiplex amplification enzyme: 0.5-1 μl; the balance being water.

Preferably, the reaction system comprises: 30 μl of the total system, primer pair: 2 μl, 2 Xbuffer: 15ul, multiplex amplification enzyme: 0.5 μl; the rest water is used for supplementing; the high throughput library is qualified at a concentration greater than 2 ng/ul.

Preferably, the high throughput library is qualified at a concentration greater than 2 ng/ul.

Preferably, the environment/procedure of the amplification reaction comprises: pre-denaturation at 94 ℃ for 15 min; the first amplification step, denaturation at 94℃for 20 seconds, annealing at 65℃to 57℃and extension for 60 seconds, 10 Touch Down cycles, (annealing and extension temperatures for each cycle reduced by 0.8 ℃); the second amplification step was performed by denaturation at 94℃for 20 seconds, annealing at 57℃and extension for 60 seconds, 26 cycles.

The kit provided by the application can sensitively detect the transgenic component with the content of 0.05% in the sample.

In the reproducibility test of the application, the reproducibility r=100% of detection results between different libraries and different library-building batches of each sample and the accuracy a=99.2%.

The kit has high specificity for detecting various transgenic components in complex templates.

The method of the present application will be described in detail with reference to examples, comparative examples and experimental data.

Example 1 screening of target transgenic Components and design of multiplex PCR amplification primers

The target transgene components, namely the transgene element and the reference gene, are comprehensively collected from a common transgene database, a national standard, an industry standard or the existing literature as far as possible so as to ensure the specificity and the accuracy of detection. Wherein the names of the selected transgenic elements and reference genes are as shown in Table 1 above.

The Primer3Plus is used for designing multiple PCR primers, the length of the primers is 18-30bp, the primers are not interfered with each other, the dimer between the primers, or the hairpin structure inside the primers and the nonspecific amplification of non-target sequences are mainly evaluated, and all the evaluated primers can be combined into a Primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction. Specific primer sequences include: SEQ ID NO.1-SEQ ID NO. 78.

Example 2 detection of whether a soybean sample contains transgenic Components

1. Experimental materials: transgenic soybean line DAS-44406-6 (FIG. 2). The experimental materials were transferred into H4A-terminator, 2mepsps, pH4A748, pAtUbiQuittin10, tORF23, pCSVMV, AAD-12, PAT and tORF1 with transgene content of 10% and used as our study materials.

Preparation of DNA templates: the extraction of plant genome adopts a high-efficiency plant genome DNA extraction kit (DP 350) of CTAB or Tiangen biochemical technology (Beijing) limited company. In this example, three biological replicates were performed for each sample of sample DNA extracted using the root DNA extraction kit.

PCR amplification, library construction and sequencing

Amplifying genomic DNA of the sample using 39 pairs of multiplex PCR amplification primers; connecting the amplified product of each sample with a sequencing joint and a specific sample DNA bar code, and then mixing to form a high-throughput sequencing library; and detecting the high-throughput sequencing library by using a high-throughput sequencing platform and performing quality control on the high-throughput sequencing data. The step is to research and adjust key parameters such as amplification cycle number, sequencing depth and the like according to the requirements of detection accuracy, sensitivity and the like; the step can also be connected with the step of the third generation sequencing related task so as to realize the complementary advantages between the second generation sequencing and the third generation sequencing.

4. Determination of results

1) Determining whether the contamination is acceptable based on the signal index S of the transgene component in the test sample and the signal index P of the transgene component in the blank, wherein: the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total number of sequenced fragments of the transgenic component, respectively, in the control. The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments of the transgenic component and the total number of sequenced fragments, respectively, in the test sample. Signal to noise ratio = S/P

2) Determination of transgene outcome

And (3) distributing each sequencing fragment to each target position of each target species by using the DNA bar code of the sample to be tested and homology comparison, wherein the targets comprise transgenic elements and internal reference genes. Absolute quantification of the transgene component is achieved based on the number of sequenced sequences at each target location. Qualitatively judging that the sample contains transgenic components when the sequencing sequences on the reference gene and the transgenic element are compared to exceed a specified threshold value; when the sample contains the transgenic component, the content of the exogenous gene in the sample is quantitatively determined according to the ratio of the sequence of the transgenic component to the sequence of the internal reference gene.

The calculation formula of the transgene content in this embodiment is shown in (a):

CtestDNA-transgenic content of test sample

tTi-number of sequenced sequences of each transgenic element in test sample

tRi number of sequenced sequences of each reference gene fragment detected in the test sample

m-total number of internal Gene fragments detected in test sample

n-total number of transgene element fragments detected in standard

According to this example we detected a total of 2 samples, each with three biological replicates, the results are shown in table 2: promoters and terminators commonly used in negative samples also detect several sequences in negative soybeans, in this example we require that sequences with a number of sequencing reads less than 5 be filtered out. The application provides that when the signal to noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable. And when the signal to noise ratio of the transgenic component in the sample is greater than 10, judging the nucleic acid of the detected transgenic component in the sample.

TABLE 2 transgene test results for the test sample of example 2

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As can be seen from Table 2, all the transgenic elements in the sample of soybean transgenic line DAS-44406-6 were effectively detected in three repeated experiments and the content was close to 10%; from this table it is demonstrated that this soybean transgenic kit of our application can be used to detect transgenic products.

Example 3 accuracy, specificity and sensitivity assessment

Transgenic soybean lines DAS-44406-6 and MON89788 transgenic standards transgenic samples of different mass percentages were prepared to evaluate the accuracy and sensitivity of the developed techniques. Specifically, the transgene content of each sample was diluted in mass percent, specifically transgenic soybean DAS-44406-6 and MON89788 were diluted with negative soybean to 10%,1%,0.1%,0.05%,0.025% and 0.01% samples, respectively, corresponding to diluted sample numbers (A1, A2, A3, A4, A5, A6) and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line DAS-44406-6 and MON89788, respectively. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree of coincidence of the average value of multiple determinations with a true value, and is expressed by errors. The specificity is also called true negative rate, and the percentage of true negative detected by multiple detection is the percentage of all negative. Sensitivity refers to the lowest amount of transgene component that can be detected at 95% confidence, i.e., the lower detection limit. The assay was performed as in example 2, with three replicates per sample, and the results are shown in table 3. Meanwhile, a genetic structure diagram of the soybean strain DAS-44406-6 is obtained.

Table 3 accuracy and sensitivity assessment of the method of the present application.

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Note that: + for detected, -for undetected, A1 and B1 for transgene content of 10%, A2 and B2 for transgene content of 1%,

a3 and B3 represent a transgene content of 0.1%, A4 and B4 represent a transgene content of 0.05%, A5 and B5 represent a transgene content of 0.025%, and A6 and B6 represent a transgene content of 0.01%.

As can be seen from Table 3, the kit can stably detect each transgenic element in a sample with a transgenic content of 0.05%, while no transgenic component is detected in a negative sample, indicating that the specificity is strong, and the kit can obviously distinguish a sample with a transgenic content of 0.05% from a negative sample, and has technical stability and detection sensitivity with a transgenic content of 0.05%.

Example 4 application of our inventive method to practical detection of samples

To verify the accuracy of the application and the role in the transgene testing of bulk samples, the laboratory selected 263 soybean leaf samples of unknown genotypes from a company for testing, tested the same as that of example 2, and compared the test results with the preservation type of the company, and the consistency of the statistical results. The analysis result shows that in 263 test samples, only 2 samples are inconsistent in result, and the consistency of the detection result is as high as 99.2%, so that the accuracy and the good application prospect of the method are better proved.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

1) The method is simple to operate, multiple transgenic components in multiple samples or one sample can be synchronously detected by single-tube PCR amplification, library construction and sequencing through primary sample pretreatment, and the method has the characteristics of parallel analysis and multiple judgment, so that the detection efficiency of transgenic products is greatly improved;

2) The whole test object comprises the transgene element sequence and the transgene strain which are common at present, and a new detection sequence can be conveniently added, so that single target amplification failure is avoided, and the specificity, accuracy and sensitivity of detection are improved;

3) The kit fuses a second generation sequencing platform to sequence the amplified product, so that the detection flux and repeatability of the system are improved, the detection result can be directly digitized, and the kit is suitable for large-scale detection of transgenic soybeans and products thereof. Therefore, the application overcomes the defects of time and labor waste and high cost in the prior art, and the provided soybean transgenic detection kit is simple in operation, quick and sensitive, large in detection flux, good in repeatability of detection results, low in cost of multi-sample multi-target sequence detection, and has important application to detection of transgenic products in and out of ports of a seed station and a customs.

It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The foregoing is only a specific embodiment of the application to enable those skilled in the art to understand or practice the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aacatagatg acaccgcgcg 20

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tgtgggcatc aaagttgtgt g 21

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tgaaatgcat ctggttcatc aaaga 25

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ccactccacc atcacacaat 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cgctggaata atgccacgtg 20

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cgcacacacc agaatcctac t 21

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

cggtcattag aggccacgat 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gagcgaggaa agtatcgggc 20

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gtgccaagtg ccaaccatat c 21

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gtctatggag gcgcaaggtt 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

tcatgccatc caccatgctt 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

agcgttgaat ccgaaaccaa 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cacggctaaa caggggcata 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

agcaacgatc agcagacgaa 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gccgctacac acttttctgc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ggaatggcta ccttggaggg 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gaggaatgtc gtacacgcca 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gccgatccta ctaacccagc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

cggctattga tggttgcagc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ctgtccgctg gtacaacact 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

cacaatgtcc aggacggtga 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

tgaccgtgtt actgggaagc 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

acttcaaatc cgcgcaaacc 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

tatctccgat gctgtgctcg 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

ctcatagtcc acgttggcct 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

tggacatcaa cgtgaccctg 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

atcagtacag cggcgagatg 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

tgacgatgtg acccaacgag 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

cgacaggtca catcagcctt 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

tcgtcacgaa caaggaggtg 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

tcgaacgcgt tacctcaaca 20

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

tgtgactggc tcagtttggt 20

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

ggaaaccacg gcccatatga 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

tgatctggat ctgccctgga 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

tagttgcagt ccacgtgacc 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

gctctgtgtg ggcctacttt 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

tcgattctcc agcaagcctg 20

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

acagtcagcc gtgcttctac 20

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 46

actcttccac cattgcttgt 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 47

tcaccaaagg tctgctcgtc 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 48

cccattggtg agcatggact 20

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 49

accttagacc aagcgcttcc 20

<210> 50

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 50

tgggcaaggc tctctttcag 20

<210> 51

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 51

cttcggtgga tgtctttttc tgaaa 25

<210> 52

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 52

aattctcagt ccaaagcctc aacaa 25

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 53

aggaggcttt ggcaatcctc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 54

gctacgatgc gtccaagaga 20

<210> 55

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 55

gcatccacga gcttatccga 20

<210> 56

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

ggacgaagct cagagccaat 20

<210> 57

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 57

ggctatcgtt cgtaaatggt ga 22

<210> 58

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 58

acgaatctca agcaatcaag ca 22

<210> 59

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 59

gactttgaga cctgcaccga 20

<210> 60

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 60

gcccagccga tccatattga 20

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 61

tgaggataac gaaagggcgg 20

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 62

tgtccgcgtt tcagtagctt 20

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 63

actgtctccg cgatctgatt 20

<210> 64

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 64

ccctagttct cgatacacgg c 21

<210> 65

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 65

gtactcgagt cgcgacgtac 20

<210> 66

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 66

taccaaccgg cactcacaaa 20

<210> 67

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 67

agttgttggc tgtggtggaa 20

<210> 68

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 68

tccaccagca gcagtaacag 20

<210> 69

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 69

aaatcctctg gcctttccgg 20

<210> 70

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 70

atcgatgatc caggtgtcgc 20

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 71

agctaaggcg gtgaagcaat 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 72

tgtggttgtc gacgagtcag 20

<210> 73

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 73

ccgcccttac attttccacc 20

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 74

gcagatcaat atgcggcaca 20

<210> 75

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 75

ggcaaactca gcggaaactg 20

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 76

tggatggggg tggagtagag 20

<210> 77

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 77

actccccatg catcacagtg 20

<210> 78

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 78

gcacgaactt gttccagctg 20

Claims

1. A primer pair composition for detecting a transgenic soybean component, the primer pair composition comprising:

a primer pair for specifically amplifying tAtAHASL, the nucleotide sequence of which is shown as SEQ ID NO.39 to SEQ ID NO.40;

and a primer pair for specifically amplifying pCSVMV, the nucleotide sequences of which are shown in SEQ ID NO.73 to SEQ ID NO. 74.

2. The primer pair composition according to claim 1, further comprising a primer pair for amplifying the soybean reference gene gm_lectin_control.

3. The primer pair composition according to claim 2, wherein the nucleotide sequence of the primer pair for amplifying the soybean reference gene gm_lectin_control is shown as SEQ ID No.75-SEQ ID No. 78.

4. A kit for detecting a soybean transgenic component, comprising the primer pair composition for detecting a soybean transgenic component according to any one of claims 1 to 3.

5. The kit of claim 4, wherein the kit comprises a first container containing the primer pair composition therein.

6. The kit of claim 5, further comprising a multiplex PCR premix.

7. Use of a primer pair composition according to any one of claims 1-3 or a kit according to any one of claims 4-6 for the detection of transgenic soybeans.

8. A method for detecting a transgenic soybean component, comprising the steps of:

obtaining the DNA of the soybean to be tested and the primer pair composition of any one of claims 1-3;

adding the primer pair composition into a reaction system by taking the DNA as a template, and performing an amplification reaction to obtain an amplification product;

9. The method of claim 8, wherein the reaction system comprises: the total system is 30-50 μl; primer pair: 2-5 μl;2 Xbuffer: 15-30ul; multiplex amplification enzyme: 0.5-1 μl; the balance being water.