CN114807407B

CN114807407B - Primer pair combination, kit and detection method for detecting soybean transgenic strain

Info

Publication number: CN114807407B
Application number: CN202210224821.8A
Authority: CN
Inventors: 李论; 彭海; 陈利红; 高利芬; 李甜甜; 周俊飞; 方治伟; 万人静; 肖华锋
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: CN114807407A

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 a soybean transgenic strain. The primer pair combination comprises nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO. 40; judging the transgenic strain in the sample to be tested; the high cost of amplification and detection by the traditional gene chip and digital PCR technology is avoided, so that the primer can obtain a plurality of genes in transgenic crops or detect a plurality of transgenic crop detection results simultaneously by one-time high-throughput sequencing and analysis during detection, thereby saving the cost and facilitating popularization.

Description

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

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 a soybean transgenic strain.

Background

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 in 1 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.

It is therefore important to develop a method for the efficient, sensitive and identification of transgenic lines.

Disclosure of Invention

The application provides a primer pair combination, a kit and a detection method for detecting a soybean transgenic strain, which are used for solving the technical problem of how to rapidly identify the transgenic strain with low cost.

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

a primer pair for specifically amplifying MON87769, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 4;

a primer pair for specifically amplifying GTS40-3-2, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 8;

a primer pair for specifically amplifying MON89788, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 12;

a primer pair for specifically amplifying MON87705, the nucleotide sequence of which is shown in SEQ ID NO.13 to SEQ ID NO. 14;

a primer pair for specific amplification 356043, the nucleotide sequence of which is shown in SEQ ID NO.15 to SEQ ID NO. 16;

the nucleotide sequence of the primer pair of the specific amplification 305523 is shown as SEQ ID NO.17 to SEQ ID NO. 18;

a primer pair for specifically amplifying CV127, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;

a primer pair for specifically amplifying MON87708, the nucleotide sequence of which is shown in SEQ ID NO.21 to SEQ ID NO. 22;

a primer pair for specifically amplifying MON87701, the nucleotide sequence of which is shown in SEQ ID NO.23 to SEQ ID NO. 24;

a primer pair for specifically amplifying FG72, the nucleotide sequence of which is shown as SEQ ID NO.25 to SEQ ID NO. 26;

the nucleotide sequence of the primer pair for specifically amplifying A2704-12 is shown in SEQ ID NO.27 to SEQ ID NO. 28;

a primer pair for specifically amplifying A5547-127, the nucleotide sequence of which is shown in SEQ ID NO.29 to SEQ ID NO. 30;

a primer pair for specifically amplifying DAS-68416-4, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.31 to SEQ ID NO. 32;

a primer pair for specifically amplifying DAS-81419-2, 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 DAS-44406-6, the nucleotide sequence of which is shown in SEQ ID NO.35 to SEQ ID NO. 36;

a primer pair for specifically amplifying MON87751, the nucleotide sequence of which is shown in SEQ ID NO.37 to SEQ ID NO. 38;

and/or a primer pair for specifically amplifying SYHT0H2, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO.39 to SEQ ID NO. 40.

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.41-SEQ ID NO. 44.

Optionally, the primer pair combination further includes:

primer pairs that specifically amplify soybean transgenic line specific sequences selected from the group consisting of: MON87769, GTS40-3-2, MON89788, MON87705, 356043, 305523, CV127, MON87708, MON87701, FG72, a2704-12, a5547-127, DAS-68416-4, DAS-81419-2, DAS-44406-6, MON87751, SYHT0H2, and ATcsr1-2.

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

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, the application provides a primer pair combination according to the first aspect and application of the detection kit according to the second aspect in detection of soybean transgenic lines and related products thereof.

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

obtaining DNA and primer pair combination of soybean to be tested;

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 sequence in the high-throughput library to obtain the result of detecting the soybean transgenic line.

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 primer pair combination provided by the embodiment of the application comprises nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO. 40; judging the transgenic strain in the sample to be tested; the high cost of amplification and detection by a gene chip and a digital PCR technology is avoided, so that the primer can obtain a plurality of genes in transgenic crops or detect a plurality of transgenic crop detection results simultaneously by one-time high-throughput sequencing and analysis during detection, thereby saving the cost and facilitating popularization.

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 a transgenic soybean strain according to an embodiment of the present 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 transgenic soybean line, the primer pair combination comprising:

In the embodiment of the application, the length of each primer in the primer pair is 18 bp-30 bp, and because the dimer between the primers or the hairpin structure inside the primers can be controlled by the length, the amplification reaction between the primers can not be affected within the length range, and further the normal amplification of the detection primer pair in one amplification reaction can be realized; when the length is larger or smaller than the end value of the range, the amplification reaction of the primer is affected, and the amplified product structures are mutually agglomerated and interfered, so that the amplified product is impure and the detection accuracy is affected.

Specifically, each pair of primers consists of a forward primer and a reverse primer. The specific correspondence between the above primers and the nucleotide sequences of the soybean transgenic lines 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 target molecules selected according to the application and primer sequences thereof

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 purpose of detecting the soybean transgenic strain in the sample, when the soybean transgenic strain is selected, a detection primer for the soybean internal reference gene is added, and the content of the transgenic strain is quantitatively detected.

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.41-SEQ ID NO. 44.

The reason for selecting two primer pairs is that: the method can avoid the problem that a pair of reference genes cannot be effectively detected due to unstable detection results caused by the reference genes or low DNA content in a sample with detection.

The primer logarithmic range is as follows: the pairs 20-22 are appropriately adjusted according to the specific detection sample. The later period can be increased periodically according to the newly collected transgenic line, 3000 pairs of primer combinations are tried, and the amplification effect is still good. To achieve the detection of transgenic lines in soybean, we collected 22 pairs of sequences of commonly used soybean transgenic lines and internal reference genes, the logarithmic range of the multiplex PCR primers was: 20-22 pairs, compared with the conventional 8-pair specific multiplex PCR, have the advantages of high detection flux and sensitivity.

In some embodiments, the primer pair combination comprises: primer pairs that specifically amplify soybean transgenic line specific sequences selected from the group consisting of: MON87769, GTS40-3-2, MON89788, MON87705, 356043, 305523, CV127, MON87708, MON87701, FG72, a2704-12, a5547-127, DAS-68416-4, DAS-81419-2, DAS-44406-6, MON87751, SYHT0H2, and ATcsr1-2.

The nucleotide sequence of the common soybean transgenic line, namely the target molecule and the internal reference gene, are screened as detection targets. The primer pair combination comprises: 20 pairs for 17 transgenic lines and 2 pairs for 1 reference gene. Other primer pairs can be added for the soybean transgenic strain specific sequence, the primer pairs do not conflict with each other, and efficient amplification can be performed through multiplex PCR. The multiplex PCR primer composition can be used for developing a transgenic line detection kit.

The logarithmic range of primer pair combinations includes, but is not limited to: 18-20 pairs, preferably 19 or 20 pairs, wherein the transgene element 13 pairs and the internal reference gene 2 pairs, and the logarithmic combination of the multiplex PCR primers can be increased periodically according to the newly collected transgene elements in the later period, and the amplification effect is excellent, wherein the logarithmic combination can reach 3000 pairs; compared with the conventional 8-pair specific multiplex PCR, the method has the advantages of high detection flux and high sensitivity.

Notably, are: 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.

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

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

All primer combinations are added into the same first container, so that one-time detection is realized, and each primer group is not required to be used for independent detection.

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

Specifically, when the components of the multiplex PCR premix include the combination of each primer group of the transgenic strain of soybean and 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, the concentration of each primer is 2nM.

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

obtaining DNA and primer pair combination of soybean to be tested;

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

By using the method, all target transgenic lines of multiple samples can be detected at one time, and the method has the advantages of high flux, high sensitivity, accuracy, rapidness and the like, and can be applied to qualitative and quantitative detection of transgenic lines of soybeans and products thereof.

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 environment/procedure of the amplification reaction of the method 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 TouchDown 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.

Still preferably, the reaction system of the method 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.

The kit provided by the application can sensitively detect the transgenic line 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=98.8%.

The kit has high specificity for detecting various transgenic lines 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 lines and design of multiplex PCR amplification primers

The target transgenic strain is mainly a transgenic strain and an internal reference gene, and is comprehensively collected from a common transgenic 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 transgenic lines and reference genes selected are as described 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. 44.

Example 2 detection of the Soybean sample containing transgenic lines

1. Experimental materials: transgenic materials GTS40-3-2, 356043, 305523, DAS-81419-2 and DAS-44406-6 with transgene content of 1%, 10% and 10% respectively were used as the research 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 22 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 transgenic line in the test sample and the signal index P of the control transgenic line, 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, respectively, of the transgenic line in the control. The signal index of the test sample s=nt/Nt, where, and Nt represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of the transgenic line 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 lines and internal reference genes. Absolute quantification of transgenic lines was achieved based on the number of sequenced sequences at each target position. When the sequencing sequences on the reference gene and the transgenic lines are compared and exceed a specified threshold value, qualitatively judging that the sample contains the transgenic lines; when the sample contains the transgenic strain, the content of the exogenous gene in the sample is quantitatively determined according to the ratio of the sequence of the transgenic strain to the sequence of the internal reference gene.

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

CtestDNA	testing the transgene content of the sample
		tTi	Number of sequenced sequences for each transgenic line in the test sample
tRi	-number of sequenced sequences of each reference gene fragment detected in the test sample
		m	-total number of reference gene fragments detected in the test sample
n	Total number of transgene line fragments detected in standard

According to this example, we examined 6 samples in total, 5 transgenic lines and one negative sample, three biological replicates per sample, and the results are shown in Table 2, which we require sequence filtering out with a number of sequencing reads less than 5. 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 strain in the sample is greater than 10, judging that the nucleic acid of the transgenic strain is detected in the sample.

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

Note that: + represents detection

As can be seen from table 2, each corresponding transgenic line in the sample was effectively detected in three repeated experiments, and the content was close to that of the transgenic line; 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 GTS40-3-2 and 305523 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 GTS40-3-2 and 305523 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) of transgenic line GTS40-3-2 and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line 305523, 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 content of transgenic lines 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.

TABLE 3 evaluation of accuracy and sensitivity of the methods of the application

Note that: + represents detected, -represents undetected, A1 and B1 represent transgene content of 10%, A2 and B2 represent transgene content of 1%, 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 line in the sample with the transgenic content of 0.05%, and detect none of the transgenic lines in the negative sample, which shows that the specificity is strong, and the kit 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 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 174 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 174 test samples, only 2 samples are inconsistent in result, and the consistency of the detection result is as high as 98.8%, 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 lines 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 current common transgenic strain sequence and the current common transgenic strain of soybean, 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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<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

aaaaacgtcc gcaatgtgtt 20

<210> 32

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

tgaacttgct aacatatttc aatgga 26

<210> 33

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

gctatattta gcacttgata ttca 24

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

atcagtacag cggcgagatg 20

<210> 35

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

ttgttcttgt tgtttcctct tt 22

<210> 36

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

cctcaattgc gagctttcta at 22

<210> 37

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

tttattttgt agatatttcc cctc 24

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

taacttttgg tgtgatgatg ct 22

<210> 39

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

aattgggtac catgcccg 18

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

tgtgccattg gtttagggtt 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

ggcaaactca gcggaaactg 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

tggatggggg tggagtagag 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

actccccatg catcacagtg 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

gcacgaactt gttccagctg 20

Claims

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

and a primer pair for specifically amplifying SYHT0H2, wherein the nucleotide sequences of the primer pair are shown in SEQ ID NO.39 to SEQ ID NO. 40.

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.41 to SEQ ID No. 44.

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

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

6. The kit of claim 4, 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 detecting transgenic lines of soybean.

8. A method of detecting a transgenic line of soybean, 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;

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

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.