CN114774566B - Primer pair combination, kit and detection method for detecting cotton transgenic component - Google Patents

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 transgenic components of cotton. The primer pair combination comprises nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO. 66. The primer pair combined amplification product can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, so that the condition that the traditional Real-time PCR technology can only detect one target molecule at a time is avoided, the condition that more false positive and false negative results appear when multiple PCR is performed in the prior art is avoided, more than 9 PCR reactions can be performed through one-time high-throughput sequencing and analysis, the detection efficiency is improved, and meanwhile, the detection flux and cost are taken into consideration.

Description

Primer pair combination, kit and detection method for detecting genetically modified components of cotton

Technical Field

The 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 components of cotton.

Background Art

Cotton (Gossypium hirsutum L.) is one of the important economic crops in my country. Traditional breeding technology, mainly based on hybrid breeding, has made certain progress in improving cotton yield, fiber quality and stress resistance, and has achieved considerable social and economic benefits. The rapid development of modern biotechnology, especially plant genetic engineering, has provided new ideas and methods for the innovation of cotton germplasm resources. It can break the reproductive isolation between species and introduce excellent genes that control trait inheritance into cotton in a purposeful and planned manner through direct or indirect transfer methods. Without changing the original characteristics, it can break the unfavorable gene linkage and achieve directional changes in biological traits.

The use of genetic modification technology to increase cotton yield, improve fiber quality, and enhance insect resistance, disease resistance, and stress resistance is an important supplement to conventional breeding.

With the increasing attention paid by the society to the safety of genetically modified products, the detection of genetically modified ingredients in agricultural products has been included in the detection items of domestic and foreign inspection and quarantine departments and has been gradually strengthened. Therefore, it is crucial to develop efficient and convenient genetically modified food detection technology. The detection technology of genetically modified products mainly includes 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 ordinary qualitative PCR, nested PCR, loop-mediated isothermal amplification technology (LAMP), fluorescent quantitative PCR multiple PCR and other methods. Compared with the ordinary qualitative PCR method, nested PCR has high detection sensitivity and is prone to false positives. LAMP is simple to operate and has strong specificity, but the primer design is relatively complex, which is easy to cause DNA contamination and affect subsequent experiments. The fluorescent 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 ordinary multiplex PCR method can detect multiple genes simultaneously in one reaction, but generally not more than six times, otherwise the interference between primers is large, affecting the detection effect. Gene chips and digital PCR technology 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, they are expensive, require specialized instruments and equipment, and require operators to have high professional qualities. These factors limit the widespread application of this technology in detection.

Therefore, developing an efficient, sensitive and high-throughput method for detecting genetically modified products has become a key issue that needs to be urgently addressed.

Summary of the invention

The present application provides a primer pair combination, a kit and a detection method for detecting genetically modified components in cotton, so as to solve the technical problem of how to efficiently detect genetically modified components in cotton.

In a first aspect, the present application provides a primer pair combination for detecting genetically modified components in cotton, the primer pair combination comprising:

A primer pair for specific amplification of p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;

A primer pair for specific amplification of t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4;

A primer pair for specific amplification of pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;

A primer pair for specific amplification of pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;

A primer pair for specific amplification of tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

A primer pair for specific amplification of pZmUbi, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

A primer pair for specific amplification of tE9, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;

A primer pair for specific amplification of t7s, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16;

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

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

A primer pair for specific amplification of pS7S7, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22;

A primer pair for specific amplification of NPtII, the nucleotide sequences of which are shown in SEQ ID NO.23 to SEQ ID NO.24;

A primer pair for specifically amplifying tORF25, the nucleotide sequences of which are shown in SEQ ID NO.25 to SEQ ID NO.26;

A primer pair for specific amplification of pTsf1, the nucleotide sequences of which are shown in SEQ ID NO.27 to SEQ ID NO.28;

A primer pair for specific amplification of crylF, the nucleotide sequences of which are shown in SEQ ID NO.29 to SEQ ID NO.30;

A primer pair for specific amplification of CTP2, the nucleotide sequences of which are shown in SEQ ID NO.31 to SEQ ID NO.32;

A primer pair for specific amplification of Cry1Ac, the nucleotide sequences of which are shown in SEQ ID NO.33 to SEQ ID NO.34;

A primer pair for specific amplification of CryAb/Ac, the nucleotide sequences of which are shown in SEQ ID NO.35 to SEQ ID NO.36;

A primer pair for specific amplification of Cry2Ae, the nucleotide sequences of which are shown in SEQ ID NO.37 to SEQ ID NO.38;

The primer pair for specifically amplifying Cry2Ab shown in the figure has nucleotide sequences as SEQ ID NO.39 to SEQ ID NO.40;

A primer pair for specific amplification of CpTI, the nucleotide sequences of which are shown in SEQ ID NO.41 to SEQ ID NO.42;

A primer pair for specifically amplifying H4A-terminator, the nucleotide sequences of which are shown in SEQ ID NO.43 to SEQ ID NO.44;

A primer pair for specific amplification of H3At-intron, the nucleotide sequences of which are shown in SEQ ID NO.45 to SEQ ID NO.46;

A primer pair for specific amplification of pH4A748, the nucleotide sequences of which are shown in SEQ ID NO.47 to SEQ ID NO.48;

A primer pair for specifically amplifying 2mepsps, the nucleotide sequences of which are shown in SEQ ID NO.49 to SEQ ID NO.50;

A primer pair for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.51 to SEQ ID NO.52;

A primer pair for specifically amplifying tOCS, the nucleotide sequences of which are shown in SEQ ID NO.53 to SEQ ID NO.54;

A primer pair for specific amplification of pCSVMV, the nucleotide sequences of which are shown in SEQ ID NO.55 to SEQ ID NO.56;

A primer pair for specific amplification of TpSSuAT, the nucleotide sequences of which are shown in SEQ ID NO.57 to SEQ ID NO.58;

A primer pair for specific amplification of pCISV, the nucleotide sequences of which are shown in SEQ ID NO.59 to SEQ ID NO.60;

A primer pair for specific amplification of zmhsp, the nucleotide sequences of which are shown in SEQ ID NO.61 to SEQ ID NO.62;

A primer pair for specific amplification of Tev-5UTR, the nucleotide sequences of which are shown in SEQ ID NO.63 to SEQ ID NO.64;

And/or, a primer pair for specifically amplifying DMO, whose nucleotide sequences are shown in SEQ ID NO.65 to SEQ ID NO.66.

Optionally, the primer pair combination also includes a primer pair for amplifying cotton internal reference genes Gh_Sad1 and Gh_SAH7.

Optionally, the primer pairs for amplifying the cotton internal reference gene Gh_Sad1 include two pairs of primer pairs, and the nucleotide sequences thereof are shown in SEQ ID NO.67-SEQ ID NO.70.

Optionally, the primer pairs for amplifying the cotton internal reference gene Gh_SAH7 include two pairs of primer pairs, whose nucleotide sequences are shown in SEQ ID NO.71-SEQ ID NO.74.

Optionally, the primer pair combination includes a primer pair that specifically amplifies a cotton transgenic element selected from the following group: p35S, t35S, pNOS, pFMV35S, tNOS, pZmUbi, tE9, t7s, PAT, bar, pS7S7, NPtII, tORF25, pTsfl, cry1F, CTP2, Cry1Ac, CryAb/Ac, Cry2Ae, Cry2Ab, CpTI, H4A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, tOCS, pCSVMV, TpSSuAT, pCISV, zmhsp, Tev-5UTR and DMO.

In a second aspect, the present application provides a kit for detecting genetically modified components in cotton, the kit comprising the primer pair combination for detecting genetically modified components in cotton described in the first aspect.

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

Optionally, the kit further comprises a multiplex PCR premix.

In a third aspect, the present application provides the use of the primer pair combination described in the first aspect or the kit described in the second aspect in detecting genetically modified cotton and related products thereof.

In a fourth aspect, the present application provides a method for detecting genetically modified components in cotton, the method comprising the following steps:

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

Using the DNA as a template, adding the primer pair combination into a reaction system, performing an amplification reaction, and obtaining an amplified product;

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

The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the genetically modified components of cotton.

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

The primer pair combination provided in the embodiment of the present application, the nucleotide sequence of the primer pair combination is shown as SEQ ID NO.1 to SEQ ID NO.66; the amplification product of the primer pair combination avoids the traditional Real-time PCR technology that can only achieve one purpose at a time, and requires multiple amplifications and detections to cover multiple target transgenic components in the sample, avoiding the occurrence of more false positive and false negative results when performing multiple PCR in the prior art, and can perform more than 9 PCR reactions. It can complete the simultaneous detection of multiple target molecules in the sample through one high-throughput sequencing and analysis, greatly improving the detection efficiency, while taking into account the detection throughput and cost.

BRIEF DESCRIPTION OF THE 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 of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

FIG1 is a schematic diagram of a process for detecting genetically modified components in cotton provided in an embodiment of the present application;

FIG. 2 is a schematic diagram of the structure of the transgenic line T304-40 provided in the examples of the present application.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. If there is a conflict, the present specification takes precedence. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. For example, room temperature can refer to a temperature within the range of 10 to 35°C.

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.

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

According to a typical embodiment of the present invention, a primer pair combination for detecting genetically modified components of cotton is provided, the primer pair combination comprising: a primer pair for specifically amplifying p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2;

A primer pair for specific amplification of t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4;

A primer pair for specific amplification of pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6;

A primer pair for specific amplification of pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8;

A primer pair for specific amplification of tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10;

A primer pair for specific amplification of pZmUbi, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12;

A primer pair for specific amplification of tE9, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14;

A primer pair for specific amplification of t7s, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16;

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

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

A primer pair for specific amplification of pS7S7, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22;

A primer pair for specific amplification of NPtII, the nucleotide sequences of which are shown in SEQ ID NO.23 to SEQ ID NO.24;

A primer pair for specifically amplifying tORF25, the nucleotide sequences of which are shown in SEQ ID NO.25 to SEQ ID NO.26;

A primer pair for specific amplification of pTsf1, the nucleotide sequences of which are shown in SEQ ID NO.27 to SEQ ID NO.28;

A primer pair for specific amplification of cry1F, the nucleotide sequences of which are shown in SEQ ID NO.29 to SEQ ID NO.30;

A primer pair for specific amplification of CTP2, the nucleotide sequences of which are shown in SEQ ID NO.31 to SEQ ID NO.32;

A primer pair for specific amplification of CrylAc, the nucleotide sequences of which are shown in SEQ ID NO.33 to SEQ ID NO.34;

A primer pair for specific amplification of CryAb/Ac, the nucleotide sequences of which are shown in SEQ ID NO.35 to SEQ ID NO.36;

A primer pair for specific amplification of Cry2Ae, the nucleotide sequences of which are shown in SEQ ID NO.37 to SEQ ID NO.38;

The primer pair for specifically amplifying Cry2Ab shown in the figure has nucleotide sequences as SEQ ID NO.39 to SEQ ID NO.40;

A primer pair for specific amplification of CpTI, the nucleotide sequences of which are shown in SEQ ID NO.41 to SEQ ID NO.42;

A primer pair for specifically amplifying H4A-terminator, the nucleotide sequences of which are shown in SEQ ID NO.43 to SEQ ID NO.44;

A primer pair for specific amplification of H3At-intron, the nucleotide sequences of which are shown in SEQ ID NO.45 to SEQ ID NO.46;

A primer pair for specific amplification of pH4A748, the nucleotide sequences of which are shown in SEQ ID NO.47 to SEQ ID NO.48;

A primer pair for specifically amplifying 2mepsps, the nucleotide sequences of which are shown in SEQ ID NO.49 to SEQ ID NO.50;

A primer pair for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.51 to SEQ ID NO.52;

A primer pair for specifically amplifying tOCS, the nucleotide sequences of which are shown in SEQ ID NO.53 to SEQ ID NO.54;

A primer pair for specific amplification of pCSVMV, the nucleotide sequences of which are shown in SEQ ID NO.55 to SEQ ID NO.56;

A primer pair for specific amplification of TpSSuAT, the nucleotide sequences of which are shown in SEQ ID NO.57 to SEQ ID NO.58;

A primer pair for specific amplification of pCISV, the nucleotide sequences of which are shown in SEQ ID NO.59 to SEQ ID NO.60;

A primer pair for specific amplification of zmhsp, the nucleotide sequences of which are shown in SEQ ID NO.61 to SEQ ID NO.62;

A primer pair for specific amplification of Tev-5UTR, the nucleotide sequences of which are shown in SEQ ID NO.63 to SEQ ID NO.64;

And/or, a primer pair for specifically amplifying DMO, whose nucleotide sequences are shown in SEQ ID NO.65 to SEQ ID NO.66.

In the present application, the commonly used cotton transgenic product detection element nucleotide sequences, i.e., target molecules and internal reference genes, were screened as detection targets. 33 commonly used transgenic elements were included: p35S, t35S, pNOS, pFMV35S, tNOS, pZmUbi, tE9, t7s, PAT, bar, pS7S7, NPtII, tORF25, pTsfl, crylF, CTP2, CrylAc, CryAb/Ac, Cry2Ae, Cry2Ab, CpTI, H4A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, tOCS, pCSVMV, TpSSuAT, pCISV, zmhsp, Tev-5UTR, DMO and sequences including 2 cotton internal reference genes Gh_Sadl and Gh_SAH7; a high-throughput sequencing and analysis can be performed to obtain multiple test results, including genetically modified components, determine whether the sample to be tested contains exogenous genes, determine the copy numbers of internal reference genes and exogenous genes in the sample to be tested, so as to determine the content of exogenous genes.

Next, the present invention develops a multiplex PCR primer combination for detecting the transgenic elements and cotton internal reference genes, wherein 33 pairs are for 33 transgenic elements and 4 pairs are for 2 internal reference genes. The primers do not conflict with each other and can be amplified efficiently by multiplex PCR. The multiplex PCR primer combination can be used to develop a transgenic element detection kit.

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

In order to achieve the purpose of quantitative detection of genetically modified components in cotton samples, the above-mentioned cotton genetically modified elements were selected and primers for detecting cotton internal reference genes were introduced, thereby achieving quantitative detection of genetically modified components in samples.

In some embodiments, the primer pairs for amplifying the cotton internal reference gene Gh_Sad1 include two pairs of primer pairs, and the nucleotide sequences thereof are shown in SEQ ID NO.67-SEQ ID NO.70.

The reason for selecting two pairs of primers is to avoid the 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.

In some embodiments, the primer pairs for amplifying the cotton internal reference gene Gh_SAH7 include two pairs of primer pairs, whose nucleotide sequences are shown in SEQ ID NO.71-SEQ ID NO.74.

The specific correspondence between the primers and the nucleotide sequences of the cotton transgenic elements amplified by them, that is, 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 and primer sequences screened by the present invention.

When designing primers, in order to enhance the applicability and sensitivity of the primers, the length of the primers is set between 18-30bp, the primers do not interfere with each other, and all primers can be combined into a primer pool for multiple 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.

In some embodiments, the primer pair combination also includes a primer pair that specifically amplifies a cotton transgenic element selected from the following group: p35S, t35S, pNOS, pFMV35S, tNOS, pZmUbi, tE9, t7s, PAT, bar, pS7S7, NPtII, tORF25, pTsfl, cry1F, CTP2, Cry1Ac, CryAb/Ac, Cry2Ae, Cry2Ab, Cp于I, H4A-terminator, H3At-intron, pH4A748, 2mepsps, cp4epsps, tOCS, pCSVMV, TpSSuAT, pCISV, zmhsp, Tev-5UTR and DMO.

In the embodiment of the present application, on the basis of the existing primer pair combination, it can also include other primer pair combinations that specifically amplify the same cotton transgenic element, and can also include a pair array of multiple PCR primers that can be regularly added according to newly collected transgenic elements in the later stage, which has been verified to be up to 3,000 pairs.

In a second aspect, the present application provides a kit for detecting genetically modified components in cotton, the kit comprising the primer pair combination for detecting genetically modified components in cotton described in the first aspect.

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

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

Specifically, when the components of the multiplex PCR premix include the transgenic elements of the cotton and the primer sets of the internal reference gene, each primer is premixed at a ratio of 1:1, and the primers are mixed according to different experimental purposes. In the specific implementation example, the concentration of each primer is 2nM.

In a third aspect, the present application provides the use of the primer pair combination described in the first aspect or the kit described in the second aspect in detecting genetically modified cotton and related products thereof.

In a fourth aspect, the present application provides a method for detecting genetically modified components in cotton, as shown in FIG1 , the method comprising the following steps:

Obtaining the DNA of cotton to be tested and the combination of primer pairs;

Using the DNA as a template, adding the primer pair combination into a reaction system, performing an amplification reaction, and obtaining an amplified product;

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

The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the genetically modified components of cotton.

In the kit of this application, the primer pair number range is: 1-37 pairs, which are appropriately adjusted according to the specific conditions of the test sample. 37 pairs of commonly used cotton transgenic elements and internal reference gene sequences were collected, with a pair number range of: 1-37 pairs, which has the advantages of high detection throughput and sensitivity compared to the conventional 8 pairs of specific multiplex PCR.

Specifically, high-throughput sequencing can be second-generation sequencing or third-generation sequencing, and the resulting high-throughput library can analyze the components of the transgenic from multiple dimensions, including but not limited to the transgenic elements in the implementation case.

Preferably, the environment/procedure of the amplification reaction in the method includes: pre-denaturation at 94°C for 15 minutes; the first step of 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 step of amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 26 cycles.

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 qualified if it is greater than 2 ng/ul.

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

In the reproducibility test of the present invention, the reproducibility rate of the detection results between different libraries of each sample and between different library construction batches is r=100%, and the accuracy rate is a=98.8%.

The kit of the present invention has high specificity in detecting multiple transgenic components in a complex template.

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

Example 1 Screening of target transgenic components and design of multiplex PCR amplification primers

The target transgenic components, i.e., commonly used cotton transgenic elements, and the target internal reference genes used are collected comprehensively from commonly used transgenic databases, national standards, industry standards or existing literature as much as possible to ensure the specificity and accuracy of the detection. The names of the transgenic elements and internal reference genes screened are shown in Table 1 above.

Primer3Plus was used to design multiple PCR primers. The length of the primers was between 18-30 bp. The primers did not interfere with each other. This mainly evaluated the dimers between primers, the hairpin structure inside the primers, and the non-specific amplification of non-target sequences. All primers after evaluation can be combined into a primer pool for multiple PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction. The specific primer sequences include: SEQ ID NO.1 to SEQ ID NO.74.

Example 2 Detecting whether cotton samples contain genetically modified ingredients

1. Experimental materials: Transgenic cotton line T304-40 (Figure 2). The experimental material was transformed with pS7S7, bar, CrylAb/Ac, p35S, and tNOS, and its transgenic content was 10%. It was used as research material.

2. Preparation of DNA template: Plant genomes were extracted using CTAB or the high-efficiency plant genome DNA extraction kit (DP350) of 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.

3. PCR amplification, library construction and sequencing

The genomic DNA of the sample is amplified using 37 pairs of multiplex PCR amplification primers; the amplified products of each sample are connected to the sequencing adapter and the specific sample DNA barcode and then mixed to form a high-throughput sequencing library; the high-throughput sequencing library is detected using a high-throughput sequencing platform and the high-throughput sequencing data is quality controlled. This step requires the adjustment of key parameters such as the number of amplification cycles and sequencing depth according to the requirements of detection accuracy and sensitivity; this step can also be connected to the third-generation sequencing step to achieve complementary advantages between the second-generation sequencing and the third-generation sequencing.

4. Determination of results

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

2) Determination of transgenic results

Using the DNA barcode and homology alignment of the sample to be tested, each sequencing fragment is assigned to each target position of each target species, and the targets include transgenic elements and internal reference genes. According to the number of sequencing sequences at each target position, the absolute quantification of transgenic components is achieved. When the sequencing sequences on the internal reference gene and transgenic element exceed the specified threshold, it is qualitatively determined that the sample contains transgenic components; when the sample contains transgenic components, the content of the exogenous gene in the sample is quantitatively determined based on the ratio of the sequencing sequences of the transgenic element and the internal reference gene.

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

CtestDNA - Testing samples for GMO content

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

tRi——The number of sequencing sequences of each reference gene fragment detected in the test sample

m——The total number of internal reference gene fragments detected in the test sample

n——The total number of transgenic element fragments detected in the standard

According to this example, a total of 2 samples were tested, and three biological replicates were made for each sample. The results are shown in Table 2: Several sequences of the promoters and terminators commonly used in negative samples will also be detected in negative cotton, and sequences with less than 5 sequencing reads are required to be filtered out. The present invention stipulates 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. When the signal-to-noise ratio of the genetically modified component in the sample is greater than 10, it is determined that the nucleic acid of the genetically modified component is detected in the sample.

Table 2 Genetically modified test results of the samples tested in Example 2

As can be seen from the table, all transgenic elements in the T304-40 sample were effectively detected in three repeated experiments, and the content was close to 10%; this table shows that the cotton transgenic kit can be used to detect transgenic products.

Example 3 Accuracy, specificity and sensitivity evaluation

The transgenic samples of different mass percentages of herbicide-resistant cotton varieties T304-40 and GHB119 transgenic standards were prepared to evaluate the accuracy and sensitivity of the developed technology. Specifically, the transgenic content of each sample was diluted by mass percentage. Specifically, transgenic cotton T304-40 and GHB119 were diluted with negative cotton into 10%, 1%, 0.1%, 0.05%, 0.025% and 0.01% samples, respectively, corresponding to the dilution sample numbers (A1, A2, A3, A4, A5, A6) of transgenic line T304-40 and the dilution sample numbers (B1, B2, B3, B4, B5, B6) of GHB119. The accuracy of qualitative detection refers to the ratio of true positives to true negatives, and the quantitative accuracy refers to the degree of consistency between the average value of multiple measurements and the true value, expressed as an error. Specificity is also called the true negative rate, which is the percentage of true negatives detected by multiple tests to all negatives. Sensitivity refers to the lowest content of genetically modified components that can be detected at a confidence level of 95%, 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.

Table 3 Accuracy and sensitivity evaluation of the method of the present invention

Note: + represents detected, - represents undetected, A1 and B1 represent 10% GMO content, A2 and B2 represent 1% GMO content, A3 and B3 represent 0.1% GMO content, A4 and B4 represent 0.05% GMO content, A5 and B5 represent 0.025% GMO content, A6 and B6 represent 0.01% GMO content.

As can be seen from the above table, the kit can stably detect each transgenic element in a sample with a transgenic content of 0.05%. The kit can clearly distinguish samples with a transgenic content of 0.05% from negative samples, and has technical stability and detection sensitivity of a transgenic content of 0.05%.

Example 4

In order to verify the accuracy of the present invention and its role in the detection of transgenics in batch samples, the laboratory selected 164 cotton leaf samples of unknown genotypes from a company for detection, and performed the detection according to the method of Example 2, and compared the detection results with the company's storage type and the consistency of the statistical results. The analysis results showed that among the 164 test samples, only 2 samples had inconsistent results, and the consistency of the detection results was as high as 98.8%, thus well proving the accuracy and good application prospects.

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

1) The operation is simple. After one sample pretreatment, single-tube PCR amplification, library construction and sequencing, multiple samples or multiple transgenic components in one sample can be detected simultaneously. It has the characteristics of parallel analysis and multiple judgments, which greatly improves the detection efficiency of transgenic products;

2) The test objects are complete, including the common transgenic element sequences of cotton, and new detection sequences can be easily added to avoid the failure of single target amplification, thus improving the specificity, accuracy and sensitivity of the detection;

3) The kit integrates the second generation sequencing platform to sequence the amplified products, which improves the detection throughput and repeatability of the system, and the detection results can be directly digitized, which is suitable for large-scale detection of genetically modified cotton and its products. Therefore, the present invention overcomes the defects of the prior art that it is time-consuming, labor-intensive and costly. The cotton transgenic detection kit provided is simple to operate, rapid and sensitive, has a large detection throughput, good repeatability of detection results, and low cost for multi-sample multi-target sequence detection. It has important applications for the detection of genetically modified products at seed stations, agricultural science institutes and customs import and export ports. It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants are intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Without more constraints, an element defined by the phrase "comprising a..." does not exclude the existence of other identical elements in the process, method, article or apparatus comprising the element.

The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Sequence Listing

<110> Jianghan University

<120> Primer pair combination, kit and detection method for detecting genetically modified components in cotton

<160> 74

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 1

aaagagactcctttgccccg 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

tgtcgtcacc ttcaccttcg 20

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 3

agggtttcgc tcatgtgttg a 21

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 4

agctcggtacccctggattt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

gcaacgattg aaggagccac 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 6

tggaacgtca gtggagcatt 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

tagtgagtgg gctgtcagga 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

tccagcacat gcatcatggt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 9

ttgaatcctg ttgccggtct 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 10

aacatagatg acaccgcgcg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 11

ttagatccgt gctgctagcg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 12

gacccgacaa acaagtgcac 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 13

cattgcgcac acaccagaat 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 14

agaggccacg atttgacaca 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 15

gagcgaggaa agtatcgggc 20

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 16

tgccaagtgc caaccatatc a 21

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 17

gtctatggag gcgcaaggtt 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 18

catgccatcc acatgcttg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 19

catcgagaca agcacggtca 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 20

gagacgtaca cggtcgactc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 21

gtgccgggat cagagacatt 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 22

agcatcacag atcctgagcg 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 23

aagccggtct tgtcgatcag 20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 24

aaaagcggcc attttccacc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 25

tgcgtcgcac agtgaaaatc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 26

tttcgcgagc tcgagatacc 20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 27

tagcgttgaa tccgaaacca 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 28

cacggctaaa caggggcata 20

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 29

atcctgtctt cgtccgagga 20

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 30

agagagtcaa tggtcccgga 20

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 31

agcatccacg agctttatccg 20

<210> 32

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 32

ggacgaagct cagagccaat 20

<210> 33

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 33

gaacaacaac gtgccacctc 20

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 34

atctgggtga tggagtcgga 20

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 35

tttctgctca gcgagttcgt 20

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 36

agtaggatcg gcttcccact 20

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 37

ctgcgatgca tacaacgtgg 20

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 38

ggatcctctt gccgatcagg 20

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 39

tggacatcaa cgtgaccctg 20

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 40

atcagtacag cggcgagatg 20

<210> 41

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 41

tcactcggct tgcaaatcct 20

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 42

catccctggacttgcaaggt 20

<210> 43

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 43

tgaggataac gaaagggcgg 20

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 44

tcgtgtccgc gtttcagtag 20

<210> 45

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 45

tgttactgtc tccgcgatct g 21

<210> 46

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 46

ccctagttct cgatacacgg c 21

<210> 47

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 47

cctgcaggtc gaggagaaat 20

<210> 48

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 48

taccaaccgg cactcacaaaa 20

<210> 49

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 49

ggagatgatg ggagcgaagg 20

<210> 50

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 50

gcccttcctc aacagatgct 20

<210> 51

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 51

gcctccgatg atcgacgaat 20

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 52

atccacgccattgagcttga 20

<210> 53

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 53

ggcagaaccg gtcaaaccta 20

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 54

ggtgcccgta actttcggta 20

<210> 55

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 55

tgtcgtcagt gcttacgtct 20

<210> 56

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 56

gcagatcaat atgcggcaca 20

<210> 57

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 57

cacgtggcattattccagcg 20

<210> 58

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 58

ttggagtgat cggagggtct 20

<210> 59

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 59

aaggctgatg tgacctgtcg 20

<210> 60

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 60

ctcgttgggt cacatcgtca 20

<210> 61

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 61

ctgcctttgt tactgccacg 20

<210> 62

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 62

agcacaggcagaacacgatt 20

<210> 63

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 63

ggctatcgtt cgtaaatggt gaaa 24

<210> 64

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 64

acgaatctca agcaatcaag ca 22

<210> 65

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 65

cgatccagag atggacggtg 20

<210> 66

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 66

tcacgccgct tctagttgtt 20

<210> 67

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 67

ggcaaactca gcggaaactg 20

<210> 68

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 68

tggatggggg tggagtagag 20

<210> 69

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 69

ggcaaactca gcggaaactg 20

<210> 70

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 70

tggatggggg tggagtagag 20

<210> 71

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 71

actccccatg catcacagtg 20

<210> 72

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 72

gcacgaactt gttccagctg 20

<210> 73

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 73

actccccatg catcacagtg 20

<210> 74

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 74

gcacgaactt gttccagctg 20

Claims (8)

1. a kind of primer pair composition that detects cotton genetically modified composition, it is characterized in that, described primer pair composition comprises: A pair of primers for specifically amplifying p35S, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2; A pair of primers for specifically amplifying t35S, the nucleotide sequences of which are shown in SEQ ID NO.3 to SEQ ID NO.4; A pair of primers for specifically amplifying pNOS, the nucleotide sequences of which are shown in SEQ ID NO.5 to SEQ ID NO.6; A pair of primers for specifically amplifying pFMV35S, the nucleotide sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.8; A pair of primers for specifically amplifying tNOS, the nucleotide sequences of which are shown in SEQ ID NO.9 to SEQ ID NO.10; A pair of primers for specifically amplifying pZmUbi, the nucleotide sequences of which are shown in SEQ ID NO.11 to SEQ ID NO.12; A pair of primers for specifically amplifying tE9, the nucleotide sequences of which are shown in SEQ ID NO.13 to SEQ ID NO.14; A pair of primers for specifically amplifying t7s, the nucleotide sequences of which are shown in SEQ ID NO.15 to SEQ ID NO.16; A pair of primers for specifically amplifying PAT, the nucleotide sequences of which are shown in SEQ ID NO.17 to SEQ ID NO.18; A pair of primers for specifically amplifying bar, the nucleotide sequences of which are shown in SEQ ID NO.19 to SEQ ID NO.20; A pair of primers for specifically amplifying pS7S7, the nucleotide sequences of which are shown in SEQ ID NO.21 to SEQ ID NO.22; A pair of primers for specifically amplifying NPtII, the nucleotide sequences of which are shown in SEQ ID NO.23 to SEQ ID NO.24; A pair of primers for specifically amplifying tORF25, the nucleotide sequences of which are shown in SEQ ID NO.25 to SEQ ID NO.26; A pair of primers for specifically amplifying pTsf1, the nucleotide sequences of which are shown in SEQ ID NO.27 to SEQ ID NO.28; A pair of primers for specifically amplifying cry1F, the nucleotide sequences of which are shown in SEQ ID NO.29 to SEQ ID NO.30; A pair of primers for specifically amplifying CTP2, the nucleotide sequences of which are shown in SEQ ID NO.31 to SEQ ID NO.32; A pair of primers for specifically amplifying Cry1Ac, the nucleotide sequences of which are shown in SEQ ID NO.33 to SEQ ID NO.34; A pair of primers for specifically amplifying CryAb/Ac, the nucleotide sequences of which are shown in SEQ ID NO.35 to SEQ ID NO.36; A pair of primers for specifically amplifying Cry2Ae, the nucleotide sequences of which are shown in SEQ ID NO.37 to SEQ ID NO.38; The primer pair for specifically amplifying Cry2Ab is shown, and its nucleotide sequence is as shown in SEQ ID NO.39 to SEQ ID NO.40; A pair of primers for specifically amplifying CpTI, the nucleotide sequences of which are shown in SEQ ID NO.41 to SEQ ID NO.42; A pair of primers for specifically amplifying H4A-terminator, the nucleotide sequences of which are shown in SEQ ID NO.43 to SEQ ID NO.44; A pair of primers for specifically amplifying H3At-intron, the nucleotide sequences of which are shown in SEQ ID NO.45 to SEQ ID NO.46; A pair of primers for specifically amplifying pH4A748, the nucleotide sequences of which are shown in SEQ ID NO.47 to SEQ ID NO.48; A pair of primers for specifically amplifying 2mepsps, the nucleotide sequences of which are shown in SEQ ID NO.49 to SEQ ID NO.50; A pair of primers for specifically amplifying cp4epsps, the nucleotide sequences of which are shown in SEQ ID NO.51 to SEQ ID NO.52; A pair of primers for specifically amplifying tOCS, the nucleotide sequences of which are shown in SEQ ID NO.53 to SEQ ID NO.54; A pair of primers for specifically amplifying pCSVMV, the nucleotide sequences of which are shown in SEQ ID NO.55 to SEQ ID NO.56; A pair of primers for specifically amplifying TpSSuAT, the nucleotide sequences of which are shown in SEQ ID NO.57 to SEQ ID NO.58; A pair of primers for specifically amplifying pCISV, the nucleotide sequences of which are shown in SEQ ID NO.59 to SEQ ID NO.60; A pair of primers for specifically amplifying zmhsp, the nucleotide sequences of which are shown in SEQ ID NO.61 to SEQ ID NO.62; A pair of primers for specifically amplifying Tev-5UTR, the nucleotide sequences of which are shown in SEQ ID NO.63 to SEQ ID NO.64; And, a pair of primers for specifically amplifying DMO, the nucleotide sequences of which are shown in SEQ ID NO.65 to SEQ ID NO.66. 2. primer pair composition according to claim 1, is characterized in that, described primer pair composition also comprises the primer pair for amplifying cotton internal reference gene Gh_Sad1. 3. primer pair composition according to claim 2, is characterized in that, the primer pair of amplification cotton internal reference gene Gh_Sad1 comprises two pairs of primer pairs, and its nucleotide sequence is as SEQ ID NO.67-SEQ ID NO.70 shown. 4. A test kit for detecting genetically modified components of cotton, characterized in that the kit comprises the primer pair composition for detecting genetically modified components of cotton according to any one of claims 1-3. 5. The kit according to claim 4, wherein the kit comprises a first container containing the primer pair composition. 6. The kit according to claim 4, characterized in that, the kit also includes multiplex PCR master mixes. 7. Application of the primer pair composition according to any one of claims 1-3 or the kit according to any one of claims 4-6 in detecting transgenic cotton and related products thereof. 8. A method for detecting genetically modified cotton ingredients, characterized in that the method comprises the following steps: Obtain the DNA of the cotton to be tested and the primer pair composition described in any one of claims 1-3; Using the DNA as a template, adding the primer pair composition into the reaction system, performing an amplification reaction, and obtaining an amplification product; performing high-throughput sequencing on the amplified product to obtain a high-throughput library; The gene sequence in the high-throughput library is analyzed to obtain the result of detecting the genetically modified components of cotton.

Images