Primer pair combination, kit and detection method for detecting transgenic cotton lines

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a primer pair combination, a kit, and a detection method for detecting genetically modified cotton lines. The nucleotide sequences of the primer pair combination are shown as SEQ ID NO. 1 to SEQ ID NO. 24. This method avoids the problem that traditional real-time PCR technology can only achieve one goal at a time, requiring multiple amplification and detection to cover multiple target genetically modified components in a sample. It also avoids the high number of false positive and false negative results that occur in existing multiplex PCR techniques. Nine or more multiplex PCR reactions can be performed through a single high-throughput sequencing and analysis, eliminating the need for separate testing of each primer set, thereby greatly improving detection efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a primer pair combination, a kit and a detection method for detecting transgenic cotton lines. Background Art

[0002] Traditional breeding techniques, primarily based on hybridization, have long made progress in improving cotton yield, fiber quality, and stress resistance, yielding considerable social and economic benefits. The rapid development of modern biotechnology, particularly plant genetic engineering, has provided new approaches and methods for the innovation of cotton germplasm resources. These approaches can overcome reproductive isolation between species and, through direct or indirect transfer, purposefully and systematically introduce superior genes that control trait inheritance into cotton. This, without altering existing characteristics, can break undesirable gene linkage and achieve targeted changes in biological traits. Using genetic engineering to increase cotton yield, improve fiber quality, and enhance insect, disease, and stress resistance is an important supplement to conventional breeding. With growing public concern about the safety of genetically modified products, testing for genetically modified ingredients in agricultural products has been incorporated into the repertoire of domestic and international inspection and quarantine authorities and is being gradually strengthened. Therefore, the development of efficient and convenient testing technologies for genetically modified foods is crucial.

[0003] The detection technologies for genetically modified products mainly include protein-based detection methods and nucleic acid-based detection methods. At present, the nucleic acid-based PCR detection method is still the most common and accurate genetically modified detection technology, which mainly includes conventional qualitative PCR, nested PCR, loop-mediated isothermal amplification technology (LAMP), fluorescence quantitative PCR multiplex PCR and other methods. Compared with the conventional qualitative PCR method, nested PCR has higher detection sensitivity and is prone to false positives. LAMP is simple to operate and has strong specificity, but the primer design is relatively complex and is prone to DNA contamination, affecting subsequent experiments. The fluorescence quantitative PCR method has the advantages of good repeatability, high sensitivity, and less nucleic acid cross-contamination, but it is expensive and requires special detection instruments. The conventional multiplex PCR method can detect multiple genes simultaneously in one reaction, but generally does not exceed six times, otherwise the interference between primers is large, affecting the detection effect. Gene chips and digital PCR technologies are also commonly used technologies for detecting genetically modified products. They both have the advantages of high throughput, high sensitivity, and strong specificity, and can detect multiple genes in one genetically modified crop in parallel or detect multiple genetically modified crops at the same time. However, their high cost, the need for specialized instruments and equipment, and the requirement for operators to have high professional qualities have limited the widespread application of this technology in detection.

[0004] Therefore, it is very important to develop an efficient, sensitive and authentic method for detecting genetically modified products. Summary of the Invention

[0005] The present application provides a primer pair combination, a kit and a detection method for detecting transgenic cotton lines, in order to solve the technical problem of how to efficiently and quickly identify transgenic cotton lines.

[0006] In the first aspect, the present application provides a primer pair combination for detecting transgenic cotton lines, the primer pair combination comprising

[0007] A primer pair for specifically amplifying MON15985a, the nucleotide sequences of which are shown in SEQ ID NO. 1 to SEQ ID NO. 2;

[0008] A primer pair for specifically amplifying MON1445, comprising two pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO. 3 to SEQ ID NO. 6;

[0009] A primer pair for specifically amplifying MON88913, the nucleotide sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 8;

[0010] A primer pair for specifically amplifying GHB614, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

[0011] A primer pair specifically amplifying LLCotton25, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

[0012] A primer pair for specifically amplifying DAS-24236-5, the nucleotide sequences of which are shown in SEQ ID NO. 13 to SEQ ID NO. 14;

[0013] A primer pair for specifically amplifying DAS-21023-5, the nucleotide sequences of which are shown in SEQ ID NO. 15 to SEQ ID NO. 16;

[0014] A primer pair for specifically amplifying MON531, the nucleotide sequences of which are shown in SEQ ID NO. 17 to SEQ ID NO. 18;

[0015] A primer pair for specifically amplifying GHB119, the nucleotide sequences of which are shown in SEQ ID NO. 19 to SEQ ID NO. 20;

[0016] A primer pair for specifically amplifying T304-40, the nucleotide sequences of which are shown in SEQ ID NO. 21 to SEQ ID NO. 22;

[0017] And / or, a primer pair for specifically amplifying MON88701, the nucleotide sequences of which are shown in SEQ ID NO.22 to SEQ ID NO.24.

[0018] Optionally, the primer pair combination includes: a primer pair that specifically amplifies a cotton transgenic line-specific sequence selected from the following group: MON15985a, MON1445, MON88913, GHB614, LLCotton25, DAS-24236-5, DAS-21023-5, MON531, GHB119, T304-40 and MON88701.

[0019] Optionally, the primer pair combination further includes a primer pair for amplifying cotton internal reference genes Gh_Sad1 and Gh_SAH7.

[0020] Optionally, two pairs of primers for amplifying the cotton internal reference gene Gh_Sad1 have nucleotide sequences as shown in SEQ ID NO. 25 to SEQ ID NO. 28.

[0021] Optionally, two pairs of primers for amplifying the cotton internal reference gene Gh_SAH7 have nucleotide sequences as shown in SEQ ID NO.29 to SEQ ID NO.32.

[0022] In a second aspect, the present application provides a kit for detecting transgenic cotton lines, the kit comprising the primer pair combination described in the first aspect.

[0023] Optionally, the kit includes a first container containing the primer pair combination.

[0024] Optionally, the kit further comprises a multiplex PCR premix.

[0025] In a third aspect, the present application provides the use of the primer pair combination described in the first aspect and the detection kit described in the second aspect in detecting transgenic cotton lines and related products.

[0026] In a fourth aspect, the present application provides a method for detecting transgenic cotton lines, the method comprising the following steps:

[0027] Obtaining the DNA of cotton to be tested and the primer pair combination described in the first aspect;

[0028] Using the DNA as a template, adding the primer pair combination into the reaction system to perform an amplification reaction to obtain an amplified product;

[0029] Performing high-throughput sequencing on the amplified product to obtain a high-throughput library;

[0030] The gene sequences in the high-throughput library are analyzed to obtain the results of detecting the cotton transgenic lines.

[0031] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0032] The nucleotide sequences of the primer pair combinations provided in the embodiments of the present application are shown in SEQ ID NO. 1 to SEQ ID NO. 24; they avoid the problem that traditional real-time PCR technology can only achieve one purpose at a time, requiring multiple amplifications and detections to cover multiple target transgenic components in a sample, and avoid the high number of false positive and false negative results that occur in existing technologies when performing multiplex PCR. More than 9 PCR reactions can be performed, and high-throughput sequencing and analysis can be performed in one go without the need for separate detection of each primer set. This allows for rapid and efficient simultaneous detection of multiple target molecules in a sample at one time, greatly improving detection efficiency while balancing detection throughput and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0035] Figure 1 This is a schematic diagram of the process of detecting transgenic cotton lines provided in the embodiments of the present application. DETAILED DESCRIPTION

[0036] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0037] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of a conflict, this specification takes precedence. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. For example, room temperature may refer to a temperature within the range of 10 to 35°C.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0039] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0040] According to a typical embodiment of the present invention, a primer pair combination for detecting transgenic cotton lines is provided, the primer pair combination comprising: a primer pair for specifically amplifying MON15985a, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;

[0041] A primer pair for specifically amplifying MON1445, comprising two pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO. 3 to SEQ ID NO. 6;

[0042] A primer pair for specifically amplifying MON88913, the nucleotide sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 8;

[0043] A primer pair for specifically amplifying GHB614, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

[0044] A primer pair specifically amplifying LLCotton25, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

[0045] A primer pair for specifically amplifying DAS-24236-5, the nucleotide sequences of which are shown in SEQ ID NO. 13 to SEQ ID NO. 14;

[0046] A primer pair for specifically amplifying DAS-21023-5, the nucleotide sequences of which are shown in SEQ ID NO. 15 to SEQ ID NO. 16;

[0047] A primer pair for specifically amplifying MON531, the nucleotide sequences of which are shown in SEQ ID NO. 17 to SEQ ID NO. 18;

[0048] A primer pair for specifically amplifying GHB119, the nucleotide sequences of which are shown in SEQ ID NO. 19 to SEQ ID NO. 20;

[0049] A primer pair for specifically amplifying T304-40, the nucleotide sequences of which are shown in SEQ ID NO. 21 to SEQ ID NO. 22;

[0050] And / or, a primer pair for specifically amplifying MON88701, the nucleotide sequences of which are shown in SEQ ID NO.22 to SEQ ID NO.24.

[0051] During primer design, in order to enhance the applicability and sensitivity of the primers, the primer lengths are set between 18-30 bp, the primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction. It has been confirmed by use that they have high sensitivity and strong applicability.

[0052] Nucleotide sequences of commonly used transgenic cotton lines, i.e., target molecules and internal reference genes, were screened for detection. Twelve commonly used transgenic lines were included: MON15985a, MON1445, MON88913, GHB614, LLCotton25, DAS-24236-5, DAS-21023-5, MON531, GHB119, T304-40, and MON88701, as well as sequences of two cotton internal reference genes, Gh_Sad1 and Gh_SAH7.

[0053] Next, the present invention developed a multiplex PCR primer combination for detecting the transgenic lines and cotton internal reference genes. The combination includes 12 primer pairs specific for 11 transgenic lines and 4 primer pairs specific for two internal reference genes. These primers are independent of each other and enable efficient amplification via multiplex PCR. This multiplex PCR primer combination can be used to develop a transgenic line detection kit.

[0054] It is worth noting that the amplification products of the primer pair combination can be subjected to high-throughput sequencing and analysis to obtain multiple test results, including transgenic components, determining whether the test sample contains exogenous genes, and determining the copy numbers of internal reference genes and exogenous genes in the test sample to determine the content of exogenous genes.

[0055] In some embodiments, the primer pair combination includes: a primer pair that specifically amplifies a cotton transgenic line-specific sequence selected from the group consisting of MON15985a, MON1445, MON88913, GHB614, LLCotton25, DAS-24236-5, DAS-21023-5, MON531, GHB119, T304-40, and MON88701.

[0056] In the embodiment of the present application, in addition to the existing primer pair combinations, other primer pair combinations that specifically amplify the same cotton transgenic elements may also be included. The number of pairs of multiple PCR primers that can be regularly added based on newly collected transgenic elements may also be included. It has been verified that the number of pairs can reach 3,000.

[0057] In some embodiments, the primer pair combination further comprises a primer pair for amplifying cotton internal reference genes Gh_Sad1 and Gh_SAH7.

[0058] In order to achieve the purpose of quantitative detection of cotton transgenic lines in samples, while selecting the above-mentioned cotton transgenic line primers, detection primers for cotton internal reference genes were added, thereby achieving quantitative detection of the transgenic line content in the sample.

[0059] In some embodiments, the two pairs of primers for amplifying the cotton internal reference gene Gh_Sad1 have nucleotide sequences as shown in SEQ ID NO. 25 to SEQ ID NO. 28.

[0060] In some embodiments, the two pairs of primers for amplifying the cotton internal reference gene Gh_SAH7 have nucleotide sequences as shown in SEQ ID NO. 29 to SEQ ID NO. 32.

[0061] The reason for selecting two pairs of primers for each internal reference gene is to avoid instability of the test results caused by the internal reference gene or the inability to effectively detect a pair of internal reference genes when the DNA content in the sample to be tested is low.

[0062] In the examples of the present application, the number of primer pairs ranges from 1 to 16 pairs; it is adjusted appropriately according to the specific conditions of the test sample. In the later stage, it can be regularly increased according to the newly collected transgenic lines. The primer combination has been tried for more than 3,000 pairs, and the amplification effect is still very good. In order to detect transgenic lines in cotton, 16 pairs of commonly used transgenic cotton lines and sequences of internal reference genes were collected. The number of pairs of multiplex PCR primers ranges from 1 to 16 pairs, preferably 12 to 16 pairs; compared with the conventional 8-pair specific multiplex PCR, it has the advantages of high detection throughput and sensitivity.

[0063] In a second aspect, the present application provides a kit for detecting transgenic cotton lines, the kit comprising the primer pair combination described in the first aspect.

[0064] In some embodiments, the kit includes a first container containing the primer pair combination.

[0065] In some embodiments, the kit further comprises a multiplex PCR premix.

[0066] Specifically, when the components of the multiplex PCR premix include the transgenic cotton line and primer sets for the internal reference gene, each primer is premixed at a 1:1 ratio. The primers are mixed according to different experimental purposes. In a specific embodiment, the concentration of each primer is 1.8-2.2 nM. The concentrations of each primer include but are not limited to: 1.9, 2.0, and 2.2 nM.

[0067] In a third aspect, the present application provides the use of the primer pair combination described in the first aspect and the detection kit described in the second aspect in detecting transgenic cotton lines and related products.

[0068] In a fourth aspect, the present application provides a method for detecting transgenic cotton lines, the method comprising the following steps:

[0069] Obtaining DNA of cotton to be tested and the primer pair combination according to any one of claims 1 to 5;

[0070] Using the DNA as a template, adding the primer pair combination into the reaction system to perform an amplification reaction to obtain an amplified product;

[0071] Performing high-throughput sequencing on the amplified product to obtain a high-throughput library;

[0072] The gene sequences in the high-throughput library are analyzed to obtain the results of detecting the cotton transgenic lines.

[0073] Specifically, high-throughput sequencing can be second-generation sequencing or third-generation sequencing. The resulting high-throughput library can analyze the transgenic lines in the sample from multiple dimensions, including but not limited to the transgenic lines in the implementation case.

[0074] Using the above method, all target transgenic lines of multiple samples can be detected at one time. It has the advantages of high throughput, high sensitivity, accuracy and speed, and can be applied to the qualitative and quantitative detection of transgenic lines of cotton and its products.

[0075] In the embodiment of the present application, the environment / procedure of the amplification reaction includes: pre-denaturation at 94°C for 15 minutes; the first amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 65°C to 57°C for 60 seconds, 10 Touch Down cycles (the temperature of annealing and extension is reduced by 0.8°C in each cycle); the second amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 26 cycles.

[0076] In the embodiment of the present application, the reaction system includes: a total system of 30 μl, a primer pair: 2 μl, a 2× buffer: 15 ul, and a multiplex amplification enzyme: 0.5 μl; the remainder is supplemented with water; the concentration of the high-throughput library is greater than 2 ng / ul to be qualified.

[0077] Preferably, the environment / procedure of the amplification reaction in the method includes: pre-denaturation at 94°C for 15 minutes; a first amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 65°C to 57°C for 60 seconds, and 10 touch-down cycles (the annealing and extension temperature is reduced by 0.8°C in each cycle); a second amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, and 26 cycles.

[0078] More preferably, the reaction system of the method comprises: a total system of 30 μl, primer pairs: 2 μl, 2× buffer: 15 ul, multiplex amplification enzyme: 0.5 μl; the remainder is supplemented with water; the concentration of the high-throughput library is greater than 2 ng / ul to be qualified.

[0079] The kit provided by the present invention can sensitively detect transgenic strains with a content of 0.05% in a sample.

[0080] In the reproducibility test of the present invention, the reproducibility rate r of the detection results between different libraries of each sample and between different library construction batches is 100%, and the accuracy rate a is 98.7%.

[0081] The kit of the present invention has high specificity in detecting multiple transgenic lines in a complex template.

[0082] The kit of the present invention has high specificity in detecting multiple transgenic lines in a complex template. The method of the present invention will be described in detail below with reference to examples, comparative examples and experimental data.

[0083] Example 1 Screening of target transgenic lines and design of multiplex PCR amplification primers

[0084] Target transgenic lines, i.e., transgenic lines, and target internal reference genes are collected as comprehensively as possible from commonly used transgenic databases, national standards, industry standards, or existing literature to ensure the specificity and accuracy of the test. The names of the transgenic lines and internal reference genes screened are shown in Table 1 above:

[0085] Multiplex PCR primers were designed using Primer3Plus. Primers were designed to be between 18 and 30 bp in length and to avoid interference between primers. This approach primarily evaluated primer dimers, hairpin structures within primers, and nonspecific amplification of non-target sequences. All evaluated primers were combined into a primer pool for multiplex PCR amplification, ensuring that all designed primers could be amplified in a single reaction. Specific primer sequences include: SEQ ID NO. 1 to SEQ ID NO. 32. These primers were used to amplify the following transgenic cotton lines: MON15985a, MON1445, MON88913, GHB614, LLCotton25, DAS-24236-5, DAS-21023-5, MON531, GHB119, T304-40, and MON88701. The specific correspondence between these primers and the nucleotide sequences of the transgenic cotton lines amplified by them, 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.

[0086] Table 1 Target molecules and primer sequences screened by the present invention.

[0087]

[0088]

[0089]

[0090] Example 2 Detecting whether cotton samples contain transgenic lines

[0091] 1. Experimental materials: Transgenic materials MON1445, LLCotton25, MON531, GHB119, T304-40, with transgenic contents of 1%, 1%, 10%, 10% and 10% respectively, were used as research materials.

[0092] 2. Preparation of DNA template: Plant genomes were extracted using CTAB or the high-efficiency plant genomic DNA extraction kit (DP350) from Tiangen Biochemical Technology (Beijing) Co., Ltd. In this example, the DNA of the test samples was extracted using the Tiangen DNA extraction kit, and three biological replicates were performed for each sample.

[0093] 3. PCR amplification, library construction and sequencing

[0094] The genomic DNA of each sample is amplified using 16 pairs of multiplex PCR primers. The amplified products of each sample are ligated to sequencing adapters and sample-specific DNA barcodes before being mixed to create a high-throughput sequencing library. This library is then analyzed using a high-throughput sequencing platform, and the data quality control is performed. This step requires adjustments to key parameters such as the number of amplification cycles and sequencing depth based on accuracy and sensitivity requirements. This step can also be combined with third-generation sequencing to complement the advantages of second- and third-generation sequencing.

[0095] 4. Determination of results

[0096] 1) Determine whether the contamination is acceptable based on the signal index S of the transgenic strain in the test sample and the signal index P of the transgenic strain in the blank control, where: the blank control noise index P = nc / Nc, where nc and Nc represent the number of sequenced fragments and the total number of sequenced fragments in the blank control, respectively. The signal index S of the test sample = nt / Nt, where nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments in the test sample, respectively. Signal-to-noise ratio = S / P

[0097] 2) Determination of transgenic results

[0098] Using DNA barcodes and homology alignments from the test sample, each sequenced fragment is assigned to each target position for each target species, including transgenic strains and internal reference genes. Absolute quantification of transgenic strains is achieved based on the number of sequenced reads at each target position. When the sequenced reads from the internal reference gene and transgenic strain exceed a specified threshold, the sample is qualitatively confirmed to contain a transgenic strain. If a transgenic strain is present in the sample, the exogenous gene content is quantitatively determined based on the ratio of the sequenced reads from the transgenic strain to the internal reference gene.

[0099] The calculation formula for the transgenic content in this example is shown in (A):

[0100]

[0101] CtestDNA——Test samples for GMO content

[0102] tTi - the number of sequenced reads for each transgenic line in the test sample

[0103] tRi - the number of sequencing reads for each reference gene fragment detected in the test sample

[0104] m ——the total number of internal reference gene fragments detected in the test sample

[0105] n ——The total number of transgenic line fragments detected in the standard

[0106] In this example, six samples were tested: five transgenic lines and one negative sample. Three biological replicates were performed for each sample. The results are shown in Table 2. Sequences with fewer than five sequencing reads were filtered out. The present invention stipulates that contamination in the detection system is acceptable when the signal-to-noise ratio is greater than 10. When the signal-to-noise ratio of the transgenic line in the sample is greater than 10, the nucleic acid of the transgenic line is determined to be detected in the sample.

[0107]

[0108] Table 2 Genetically modified test results of the samples tested in Example 2.

[0109] Note: + represents detection

[0110] As shown in Table 2, each corresponding transgenic line in the sample was effectively detected in three repeated experiments, and the content was close to its content; this table shows that the cotton transgenic kit can be used to detect transgenic products.

[0111] Example 3 Accuracy, specificity and sensitivity evaluation

[0112] The accuracy and sensitivity of the developed technology were evaluated by preparing transgenic cotton standards containing GHB119 and T304-40 with varying mass percentages of transgenic content. Specifically, the transgenic content of each sample was diluted using negative cotton to 10%, 1%, 0.1%, 0.05%, 0.025%, and 0.01% concentrations, respectively. These samples correspond to the dilution numbers (A1, A2, A3, A4, A5, A6) for GHB119 and (B1, B2, B3, B4, B5, B6) for T304-40. Qualitative accuracy refers to the ratio of true positives to true negatives, while quantitative accuracy refers to the degree of agreement between the average of multiple measurements and the true value, expressed as error. Specificity, also known as the true negative rate, is the percentage of true negatives detected in multiple tests compared to all negatives. Sensitivity refers to the lowest level of transgenic lines that can be detected at a 95% confidence level, i.e., the detection limit. The test was performed according to the method of Example 2, with three biological replicates for each sample. The results are shown in Table 3.

[0113] Table 3 Accuracy and sensitivity evaluation of the method of the present invention.

[0114]

[0115]

[0116]

[0117] Note: + represents detected, - represents not detected, A1 and B1 represent a GM content of 10%, A2 and B2 represent a GM content of 1%, A3 and B3 represent a GM content of 0.1%, A4 and B4 represent a GM content of 0.05%, A5 and B5 represent a GM content of 0.025%, and A6 and B6 represent a GM content of 0.01%.

[0118] As shown in Table 3, the kit consistently detected each transgenic line in samples with a transgenic content of 0.05%. In negative samples, a single positive sign indicated that a specific sequence of a transgenic line was detected in the negative control group. This demonstrates strong specificity, with the kit clearly distinguishing between samples with a transgenic content of 0.05% and negative samples, demonstrating technical stability and sensitivity at a transgenic content of 0.05%.

[0119] Example 4

[0120] To validate the accuracy of the present invention and its utility in batch GMO detection, the laboratory selected 237 cotton leaf samples of unknown genotype from a company for testing according to the method in Example 2. The test results were compared with the company's storage type and statistically analyzed for consistency. The analysis revealed that only three of the 237 test samples showed discrepancies, resulting in a high consistency of 98.7%, demonstrating the accuracy and promising application prospects of the method.

[0121] One or more technical solutions in the embodiments of the present invention may have at least the following technical effects or advantages:

[0122] 1) Simple operation: After one-time sample pretreatment, single-tube PCR amplification, library construction and sequencing, multiple samples or multiple transgenic lines in one sample can be detected simultaneously. It has the characteristics of parallel analysis and multiple judgments, greatly improving the detection efficiency of transgenic products;

[0123] 2) The test targets are comprehensive, including the sequences of the most common transgenic cotton lines, and new detection sequences can be easily added to avoid single target amplification failure, thereby improving the specificity, accuracy and sensitivity of the test;

[0124] 3) The kit integrates a second-generation sequencing platform to sequence amplified products, improving the system's detection throughput and reproducibility. Furthermore, the test results can be directly digitized, making it suitable for large-scale testing of genetically modified cotton and its products. Therefore, the present invention overcomes the time-consuming, labor-intensive, and costly shortcomings of existing technologies. The provided cotton GMO detection kit offers simple operation, rapid and sensitive detection, high detection throughput, and highly reproducible test results. It also offers low-cost detection of multiple samples and multiple target sequences, making it a valuable tool for testing genetically modified products at seed stations, agricultural science institutes, and customs import and export ports.

[0125] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations are intended to cover non-exclusive inclusion, so that a process, method, article, or device that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or device that includes the element.

[0126] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein. Sequence Listing <110> Jianghan University; Wuhan Qingfa Hesheng Agricultural Development Co., Ltd. <120> Primer pair combination, kit and detection method for detecting transgenic cotton lines <140> 2022102142961 <141> 2022-03-04 <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 ctggtcaagg ctatttgatg gc 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tttgccgggg atcctctaga 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 acgctgcgga catctacatt 20 <210> 4 <211> twenty one <212> DNA <213> Artificial Sequence <400> 4 tggcccatta tgaaggcttg t 21 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <400> 5 acccttcaat ttaaccgatg ct 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 atctttggga ccactgtcgg 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 tgcttctccc agaatgatcg g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 cgcattcgag cttcttcacg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 gcccctcgaa acttgttcct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 cagaagcttg ataacgcggc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 ctaagccccc atttggacgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 ggggaagagg ctgaaatgct 20 <210> 13 <211> twenty four <212> DNA <213> Artificial Sequence <400> 13 atgtagacac gtcgaaataa agat 24 <210> 14 <211> twenty three <212> DNA <213> Artificial Sequence <400> 14 cagtgataac aacaccctga gtc 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 ttttgcccga ggtcgttagg 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 agtgtcgtgc tccaccatg 19 <210> 18 <211> twenty four <212> DNA <213> Artificial Sequence <400> 18 cgtcaggaat aaaggaagta cagt 24 <210> 18 <211> twenty three <212> DNA <213> Artificial Sequence <400> 18 tctgaatttc taacctggct gct 23 <210> 19 <211> twenty two <212> DNA <213> Artificial Sequence <400> 19 gccctcctta tttatcccct ta 22 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <400> 20 agcaggttcg tttaaggatg aa 22 <210> twenty one <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty one ccatcatact cattgctgat cca 23 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty two tcaatctcag aactgtccgc a 21 <210> twenty three <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty three gcacgcttag tgtgtgtgtc aa 22 <210> twenty four <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty four tctagaacta gtggatcccc cg 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <400> 25 ggcaaactca gcggaaactg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <400> 26 tggatggggg tggagtagag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 ggcaaactca gcggaaactg 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 ggcaaactca gcggaaactg 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <400> 29 actccccatg catcacagtg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 actccccatg catcacagtg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 actccccatg catcacagtg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <400> 32 gcacgaactt gttccagctg 20

Claims

1. A primer pair combination for detecting transgenic cotton lines, characterized in that: The primer pair combination includes: a primer pair for specifically amplifying MON15985a, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2; A primer pair for specifically amplifying MON1445, comprising two pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO. 3 to SEQ ID NO. 6; A primer pair for specifically amplifying MON88913, the nucleotide sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 8; A primer pair for specifically amplifying GHB614, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10; A primer pair specifically amplifying LLCotton25, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12; A primer pair for specifically amplifying DAS-24236-5, the nucleotide sequences of which are shown in SEQ ID NO. 13 to SEQ ID NO. 14; A primer pair for specifically amplifying DAS-21023-5, the nucleotide sequences of which are shown in SEQ ID NO. 15 to SEQ ID NO. 16; A primer pair for specifically amplifying MON531, the nucleotide sequences of which are shown in SEQ ID NO. 17 to SEQ ID NO. 18; A primer pair for specifically amplifying GHB119, the nucleotide sequences of which are shown in SEQ ID NO. 19 to SEQ ID NO. 20; A primer pair for specifically amplifying T304-40, the nucleotide sequences of which are shown in SEQ ID NO. 21 to SEQ ID NO. 22; and a primer pair for specifically amplifying MON88701, the nucleotide sequences of which are shown in SEQ ID NO.23 to SEQ ID NO.

24.

2. The primer pair combination according to claim 1, characterized in that The primer pair combination includes a primer pair that specifically amplifies a cotton transgenic line-specific sequence selected from the group consisting of MON15985a, MON1445, MON88913, GHB614, LLCotton25, DAS-24236-5, DAS-21023-5, MON531, GHB119, T304-40, and MON88701.

3. The primer pair combination according to claim 1, characterized in that The primer pair combination also includes a primer pair for amplifying the cotton internal reference gene Gh_SAH7.

4. The primer pair combination according to claim 3, characterized in that The nucleotide sequences of the two pairs of primers for amplifying the cotton internal reference gene Gh_SAH7 are shown in SEQ ID NO.29 to SEQ ID NO.

32.

5. A kit for detecting transgenic cotton lines, characterized in that: The kit comprises the primer pair combination according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that The kit includes a first container containing the primer pair combination.

7. The kit according to claim 5, characterized in that The kit also includes a multiplex PCR premix.

8. Use of the primer pair combination according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 7 in detecting transgenic cotton lines.

9. A method for detecting transgenic cotton lines, characterized in that: The method comprises the following steps: Obtaining DNA of cotton to be tested and the primer pair combination according to any one of claims 1 to 4; Using the DNA as a template, adding the primer pair combination into the reaction system to perform an amplification reaction to obtain an amplified product; Performing high-throughput sequencing on the amplified product to obtain a high-throughput library; The gene sequences in the high-throughput library are analyzed to obtain the results of detecting the cotton transgenic lines.

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