CN114807406A

CN114807406A - Primer pair combination, kit and detection method for detecting soybean transgenic components

Info

Publication number: CN114807406A
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: 2022-07-29
Anticipated expiration: 2042-03-07
Also published as: CN114807406B

Abstract

The invention 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 product of the primer pair combination can be subjected to high-throughput sequencing and analysis once to obtain a plurality of detection results, so that the phenomenon that more false positive or false negative results appear when multiple PCR is carried out in the prior art is avoided; meanwhile, when the primers are used for sample detection, multiplex PCR amplification of more than 9 target molecules in one sample or multiplex PCR amplification of a plurality of targets in a plurality of samples can be realized, and then a detection result of the transgenic components is obtained through one-time high-throughput sequencing and analysis.

Description

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

Technical Field

The invention 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 which are generally planted globally, contain rich vegetable protein and edible oil and fat, and are important edible, feeding and oil crops. Under the background that the planting area of the soybean is difficult to greatly increase at present, a novel high-yield and high-quality transgenic soybean variety is cultivated by utilizing the modern biotechnology, and the method has very important significance for promoting the joy of the soybean industry. However, with the fact that a large number of transgenic soybeans are directly or indirectly made into food and the increasing concern of international society on the safety of transgenic products, the detection of transgenic components in agricultural products is incorporated into the detection projects of inspection and quarantine departments at home and abroad and is gradually strengthened. Therefore, the development of efficient and convenient transgenic food detection technology is very important.

The detection technology of transgenic products mainly comprises a protein-based detection method and a nucleic acid-based detection method. At present, the PCR detection method based on nucleic acid is still the most common and accurate transgene detection technology at present, and mainly comprises methods such as 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 method has high detection sensitivity and is easy to cause false positive. LAMP is simple to operate and strong in specificity, however, primer design is complex, DNA pollution is easily caused, 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 the cost is high and a special detection instrument is required. The common multiplex PCR method can detect a plurality of genes in one reaction at the same time, but generally the detection is not more than six times, otherwise, the interference between 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 simultaneously detect a plurality of transgenic crops; however, the cost is high, special instruments and equipment are needed, and operators are required to have high professional quality, and the factors limit the wide application of the technology in detection.

Therefore, the development of 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, and aims to solve 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 soybean transgenic components, the primer pair combination comprising:

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

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

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

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

the nucleotide sequence of the primer pair for specifically amplifying tPIII is shown in SEQ ID NO.9 to SEQ ID NO. 10;

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

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

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

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

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

a primer pair for specifically amplifying tsf1, wherein the nucleotide sequences of the primer pair are shown as SEQ ID NO.21 to SEQ ID NO. 22;

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

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

a primer pair for specifically amplifying Cry1A.105, wherein the nucleotide sequence of the primer pair 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, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.31 to SEQ ID NO. 32;

the nucleotide sequences of the primer pairs for specifically amplifying Cry2Ab are shown as SEQ ID NO.33 to SEQ ID NO. 34;

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

the nucleotide sequences of the primer pairs for specifically amplifying ATcsr1-2 are shown in SEQ ID NO.37 to SEQ ID NO. 38;

the nucleotide sequences of the primer pairs for specifically amplifying the tAASLL are shown as SEQ ID NO.39 to SEQ ID NO. 40;

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

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

the nucleotide sequence of the primer pair for specifically amplifying the AtRbcs-transit-peptide is shown in SEQ ID NO.45 to SEQ ID NO. 46;

the nucleotide sequence of the primer pair for specifically amplifying the AAD-12 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-SEQ ID NO. 50;

a primer pair for specifically amplifying pFMV35S, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.51 to SEQ ID NO. 52;

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

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

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

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

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

the nucleotide sequences of the primer pairs for specifically amplifying H3At-intron are shown in SEQ ID NO.63 to SEQ ID NO. 64;

the nucleotide sequence of the primer pair for specifically amplifying the pH4A748 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 in SEQ ID NO.67 to SEQ ID NO. 68;

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

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

and/or, a primer pair for specifically amplifying pCSVMV, wherein the nucleotide sequences are shown as 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.

Optionally, the nucleotide sequences of two pairs of primers of the primer pair for amplifying the soybean reference gene Gm _ Lectin _ control are shown as 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, tATHASL, DMO, tORF23, AtRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, Cry1F, CTP2, Tev-5UTR, hpppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, pAtUbitron, pAtUbititn 10 and pCSVtmV.

In a second aspect, a kit for detecting soybean transgenic components comprises the primer pair combination for detecting soybean transgenic components in the first aspect.

Optionally, the kit includes a first container, and the primer pair combination is contained in the first container.

Optionally, the kit further comprises a multiplex PCR master mix.

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

In a fourth aspect, a method for detecting transgenic components of soybean, said method comprising the steps of:

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

adding the primer pair combination into a reaction system by taking the DNA as a template to carry out amplification reaction to obtain an amplification product;

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

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

Optionally, the reaction system comprises: the total volume is 30-50 μ l; primer pair: 2-5 mul; 2 × buffer: 15-30 ul; multiplex amplification enzyme: 0.5-1 μ l; the balance being water.

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

in the primer pair combination provided by the embodiment of the application, the nucleotide sequence of the primer combination is shown as SEQ ID NO.1-SEQ ID NO. 78. The amplification product of the primer pair combination can be subjected to high-throughput sequencing and analysis once to obtain a plurality of detection results, including transgenic components, judgment of whether a sample to be detected contains target molecules or not, and determination of 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 avoids the problem that the traditional Real-time PCR technology can only realize one purpose at a time, multiple target transgenic components in a sample can be covered only by carrying out amplification and detection for multiple times, avoids the problems that more false positive and false negative results appear when multiple PCR is carried out in the prior art, can carry out more than 9 PCR reactions, can complete the simultaneous detection of multiple target molecules in the sample through one-time high-throughput sequencing and analysis, greatly improves the detection efficiency, and simultaneously considers the detection throughput and the cost.

Drawings

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

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

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

FIG. 2 is a schematic structural diagram of a transgenic line DAS-44406-6 provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings 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 invention belongs. If there is a conflict, the present specification will control. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. For example, the room temperature may be a temperature within a range of 10 to 35 ℃.

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

In order to solve the technical problems, the general idea of the embodiment of the application is as follows:

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

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

The specific correspondence between the primers and the amplified nucleotide sequences of the soybean transgenic elements, i.e., the target molecules, and the corresponding primer pairs and the nucleotide sequences of the primers is shown in table 1.

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

When the primers are designed, 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, and 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.

In some embodiments, the primer pair combination further comprises a primer pair for amplifying a soybean reference gene Gm _ Lectin _ control.

In order to realize the purpose of quantitative detection of the transgenic components of the soybeans in the samples, when the soybean transgenic elements are selected, detection primers of soybean internal reference genes are added, and further the quantitative detection of the content of the transgenic components in the samples is realized.

In some embodiments, two pairs of primers of the primer pair for amplifying the soybean reference gene Gm _ Lectin _ control have 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 to avoid that the DNA content cannot be detected efficiently 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, tATHASL, DMO, tORF23, AtRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, Cry1F, CTP2, Tev-5UTR, hpppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, pAtUbitron, pAtUbititn 10 and pCSVtmV.

In the embodiment of the application, a nucleotide sequence of a detection element of a common soybean transgenic product, namely a target molecule and an internal reference gene, is screened as a detection target. The method comprises 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, tAHASL, DMO, tORF23, AtRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, Cry1F, CTP2, Tev-5UTR, hpppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, pAtUbibitron 10 and pCSVMV, and sequences including soybean internal reference gene _ Letrol.

Next, the present invention developed multiplex PCR primer combinations for detecting the transgenic elements and soybean reference genes, wherein 37 pairs are directed to 37 transgenic elements and 2 pairs are directed to 1 reference gene. The primers do not conflict 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 combinations: 1-39, primer pairs for specifically amplifying soybean transgenic elements selected from the following group can be included, including but not limited to 39 pairs or more. The later stage can be carried out according to the newly collected transgenic element, the logarithm combination of multiple PCR primers is periodically increased, and the amplification effect is excellent after verification of 3000 pairs; compared with the conventional 8-pair specific multiplex PCR, the method has the advantages of high detection flux and high sensitivity.

Suitably adjusted according to the conditions of the specific test sample. In the later period, the number of the newly collected transgenic elements can be increased periodically, and 3000 pairs of primer combinations are tried, so that the amplification effect is still good. In order to realize the detection of transgenic components in soybean, 39 pairs of sequences of common soybean transgenic elements and reference genes are collected, and the logarithmic range of the multiple PCR primers is as follows: 1-39 pairs, compared with the conventional 8-pair specific multiplex PCR, has the advantages of high detection flux and high 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 the transgene elements in our embodiment.

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

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

Specifically, when the components of the multiplex PCR premix comprise the combination of the transgenic element of the soybean and the primers of the reference gene, each primer is premixed according to the proportion of 1:1, and the primers are mixed according to different experimental purposes, wherein in a specific implementation example, the concentration of each primer is 2 nM.

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

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

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

s3, performing high-throughput sequencing on the amplification product to obtain a high-throughput library;

and S4, analyzing the gene sequence in the high-throughput library to obtain a result for detecting the transgenic components of the soybean.

In some embodiments, the reaction system comprises: the total volume is 30-50 μ l; primer pair: 2-5 mul; 2 × buffer: 15-30 ul; multiplex amplification enzyme: 0.5-1 μ l; the balance being water.

Preferably, the reaction system comprises: total 30 μ l, primer pair: 2. mu.l, 2 XBuffer: 15ul, multiplex amplification enzyme: 0.5 mul; supplementing the rest water; the high-throughput library was found to be at a concentration of greater than 2 ng/ul.

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

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

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

In the reproducibility test of the invention, the detection result reproducibility r of each sample among different libraries and different library establishing batches is 100%, and the accuracy rate a is 99.2%.

The kit provided by the invention has high specificity in detecting various transgenic components in a complex template.

The process of the present invention will be described in detail below with reference to examples, comparative examples and experimental data.

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

The target transgenic components, namely mainly transgenic elements and reference genes, are comprehensively collected from a common transgenic database, national standards, industrial standards or existing documents as far as possible so as to ensure the specificity and the accuracy of detection. The names of the selected transgenic elements and the reference gene are shown in the above table 1.

The method comprises the steps of designing multiple PCR primers by using Primer3Plus, wherein the length of the primers is between 18 and 30bp, the primers are not interfered with each other, the primers are mainly used for evaluating dimers among the primers or hairpin structures inside the primers and non-specific amplification of non-target sequences, all the primers after evaluation can be combined into a Primer pool for multiple PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction. Specific primer sequences include: SEQ ID NO.1-SEQ ID NO. 78.

Example 2 detection of Soybean samples containing transgenic Components

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

Preparation of DNA template: the extraction of plant genome adopts CTAB or high-efficiency plant genome DNA extraction kit (DP350) of Tiangen Biochemical technology (Beijing) Co. In this example, DNA of a sample to be tested was extracted using a Tiangen DNA extraction kit, and each sample was subjected to three biological replicates.

PCR amplification, library construction and sequencing

Amplifying the genome DNA of the sample by 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 mixing to obtain 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. In the step, key parameters such as amplification cycle number and sequencing depth need to be researched and adjusted according to requirements such as detection accuracy and sensitivity; the step can also be connected with the step of the third-generation sequencing related topic so as to realize the advantage complementation between the second-generation sequencing and the third-generation sequencing.

4. Determination of the results

1) And judging whether the pollution is acceptable according to the signal index S of the transgenic component in the test sample and the signal index P of the transgenic component in the blank control, wherein: the blank noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequenced fragments of transgenic components and the total number of sequenced fragments in the blank, respectively. The signal index S of the test sample is Nt/Nt, where Nt and Nt represent the number of sequencing fragments of the transgenic component and the total number of sequencing fragments in the test sample, respectively. S/P ratio

2) Determination of transgenic results

And 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 the homologous alignment, wherein the targets comprise a transgenic element and an internal reference gene. Absolute quantification of the transgene components was achieved based on the number of sequenced sequences at each target position. When the sequence sequences on the internal reference gene and the transgenic element are compared to exceed a specified threshold, qualitatively judging that the sample contains transgenic components; when the sample contains the transgenic component, the content of the exogenous gene in the sample is quantitatively judged according to the ratio of the sequencing sequence of the transgenic element and the internal reference gene.

The calculation formula of the transgene content in this example is shown in (A):

ctest DNA-transgene content of test sample

tTi-number of sequencing sequences per transgenic element in a test sample

tRi-number of sequencing sequences of each reference Gene fragment detected in test sample

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

n-total number of detected transgenic element fragments in the Standard

According to the present example, we tested a total of 2 samples, each of which was replicated for three organisms, and the results are shown in Table 2: promoters and terminators commonly used in negative samples will also detect several sequences in negative soybean, in this case we require that sequences with a number of sequencing reads less than 5 be filtered out. The present invention provides that contamination in the detection system can be judged to be acceptable when the signal-to-noise ratio is greater than 10 times. When the signal-to-noise ratio of the transgenic component in the sample is greater than 10, the nucleic acid of the transgenic component detected in the sample is judged.

TABLE 2 transgenic test results of the test samples of this example 2

As can be seen from Table 2, all the transgenic elements in the sample of the 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 shown that the soybean transgene kit of our invention can be used to detect transgenic products.

Example 3 evaluation of accuracy, specificity and sensitivity

Transgenic soybean lines DAS-44406-6 and MON89788 transgenic standards different mass percentages of transgenic samples were prepared to assess the accuracy and sensitivity of the developed technology. Specifically, the transgene content of each sample is diluted by mass percent, specifically, transgenic soybeans DAS-44406-6 and MON89788 are respectively diluted by negative soybeans to 10%, 1%, 0.1%, 0.05%, 0.025% and 0.01% samples, which respectively correspond to the diluted sample numbers of transgenic lines DAS-44406-6 (A1, A2, A3, A4, A5, A6) and MON89788 (B1, B2, B3, B4, B5, B6). 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 between the average of multiple determinations and the true value, expressed as an error. The specificity is also called true negative rate, and the true negative detected by multiple detections accounts for the percentage of all negatives. Sensitivity refers to the lowest level of transgene content that can be detected at 95% confidence, i.e., the lower limit of detection. The assay was performed according to the method of example 2, with triplicates per sample, and the results are shown in Table 3. Meanwhile, a gene structure schematic diagram of the soybean line DAS-44406-6 is obtained.

Table 3 accuracy and sensitivity evaluation of the method of the invention.

Note: + represents detection, -represents no detection, a1 and B1 represent 10% transgene content, a2 and B2 represent 1% transgene content,

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

As can be seen from Table 3, the kit can stably detect each transgenic element in a sample with the transgenic content of 0.05%, but does not detect any transgenic component in a negative sample, so that the kit has strong specificity, can obviously distinguish the sample with the transgenic content of 0.05% from the negative sample, and has technical stability and detection sensitivity with the transgenic content of 0.05%.

Example 4 application of the method of our invention to the actual detection of samples

In order to verify the accuracy of the invention and the function of the batch sample in transgenic detection, 263 soybean leaf samples of unknown genotype of a certain company are selected for detection in a laboratory, the detection is carried out according to the method of the embodiment 2, the detection result is compared with the storage type of the company, and the consistency of the result is counted. Analysis results show that only 2 samples in 263 test samples have inconsistent results, and the consistency of the detection results is up to 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 invention at least have the following technical effects or advantages:

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

2) the detection objects are complete and comprise the current common transgenic element sequence and transgenic strain of the soybean, and a new detection sequence can be conveniently added, so that the amplification failure of a single target is avoided, and the specificity, the accuracy and the sensitivity of the detection are improved;

3) the kit is fused with a second-generation sequencing platform to sequence the amplification product, so that the detection flux and the repeatability of the system are improved, and the detection result can be directly digitalized and is suitable for large-scale detection of the transgenic soybean and the products thereof. Therefore, the invention overcomes the defects of time and labor waste and high cost in the prior art, and the provided soybean transgenic detection kit has the advantages of simple operation, rapidness, sensitivity, large detection flux, good repeatability of detection results and low detection cost of multi-sample multi-target sequences, and has important application to the detection of transgenic products at seed stations, agricultural institutions and customs entry and exit ports.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be 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. Also, 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 an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. 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 invention. Thus, the present invention 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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<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgaatcctgt tgccggtctt 20

<210> 8

<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 combination for detecting soybean transgenic components is characterized by comprising:

the nucleotide sequences of the primer pair for specifically amplifying pTsf1 are shown in SEQ ID NO.19 to SEQ ID NO. 20;

the nucleotide sequences of the primer pairs for specific amplification tsf1 are shown in SEQ ID NO.21 to SEQ ID NO. 22;

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

the nucleotide sequences of the primer pair for specific amplification of ATcsr1-2 are shown in SEQ ID NO.37 to SEQ ID NO. 38;

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

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

the nucleotide sequences of the primer pairs for specifically amplifying the cp4epsps are shown in SEQ ID NO. 69-SEQ ID NO. 70;

2. The primer pair combination according to claim 1, further comprising a primer pair for amplifying soybean reference gene Gm _ Lectin _ control.

3. The primer pair combination according to claim 1, wherein the nucleotide sequences of two pairs of primers of the primer pair for amplifying the soybean reference gene Gm _ Lectin _ control are shown as SEQ ID No.75-SEQ ID No. 78.

4. The primer pair combination of claim 1, further comprising a primer pair that specifically amplifies a soybean transgene 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, tATHASL, DMO, tORF23, AtRbcs-transit-peptide, AAD-12, tORF1, pFMV35S, Cry1F, CTP2, Tev-5UTR, hpppdPf, H4A-terminator, H3At-intron, pH4A748, 2mepsps, pAtUbitron, pAtUbititn 10 and pCSVtmV.

5. A kit for detecting soybean transgenic components, which is characterized by comprising the primer pair combination for detecting soybean transgenic components in any one of claims 1 to 4.

6. The kit of claim 5, wherein the kit comprises a first container containing the primer pair combination.

7. The kit of claim 5, wherein the kit further comprises a multiplex PCR premix.

8. Use of the primer pair combination of any one of claims 1 to 4 or the kit of any one of claims 5 to 7 for the detection of transgenic soybean and related products thereof.

9. A method for detecting transgenic components of soybean, comprising the steps of:

obtaining DNA of soybean to be detected and the primer pair combination of any one of claims 1-4;

10. The method of claim 9, wherein the reaction system comprises: the total volume is 30-50 μ l; primer pair: 2-5 mul; 2 × buffer: 15-30 ul; multiplex amplification enzyme: 0.5-1 μ l; the balance being water.