CN114657276B - Primer pair combination, kit and detection method for detecting rice transgenic line - Google Patents

Abstract

The application relates to the field of biotechnology, in particular to a primer pair combination, a kit and a detection method for detecting a rice transgenic line. The nucleotide sequence of the primer pair combination is shown as SEQ ID NO.1 to SEQ ID NO. 26; the amplification products combined by the primer pairs can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, so that more false positive or false negative results are avoided when multiple PCR is performed in the prior art; meanwhile, when the primer is used for detecting samples, multiplex PCR amplification of more than 9 target molecules in one sample or multiplex PCR amplification of multiple targets in multiple samples can be realized, and then a detection result of multiple targets in transgenic crops or multiple target molecules in multiple transgenic crops is obtained through one-time high-throughput sequencing and analysis, so that the detection efficiency and sensitivity are greatly improved, and the application and popularization are facilitated.

Description

Primer pair combination, kit and detection method for detecting rice transgenic line

Technical Field

The application relates to the field of biotechnology, in particular to a primer pair combination, a kit and a detection method for detecting a rice transgenic line.

Background

Rice is the most important ration crop in China, and has important significance in guaranteeing the grain safety in China. The application of the transgenic technology in different rice lines improves the comprehensive properties of the rice varieties, such as yield, resistance, adaptability and the like.

As a large number of transgenic products are introduced into the market, development of a method for identifying transgenic lines has been on the way, and detection techniques for transgenic products mainly include protein-based detection methods and nucleic acid-based detection methods. Western blotting, enzyme-linked immunosorbent assay (ELISA), immune test paper strips, protein chips and other methods are the main protein-based detection methods at present.

The current PCR detection method based on nucleic acid is mainly a multiplex PCR method, and can detect a plurality of genes simultaneously in one reaction, but the conventional electrophoresis detection result is difficult to judge, and false positive or false negative results are easy to appear; the conventional Real-time method is also limited in the number of exogenous genes detected in one PCR reaction and high in cost, requires special instruments and equipment, requires operators to have higher professional quality, and limits the wide application of the technology in detection.

Therefore, developing a product and a detection method of a transgenic line with high efficiency, sensitivity and flux becomes a key problem to be solved urgently.

Disclosure of Invention

The application provides a primer pair composition, a kit and a detection method for detecting a rice transgenic line, which are used for solving the technical problem that the conventional commonly used kit for detecting rice based on multiple PCR reactions cannot realize PCR primer amplification of more than 8 weight.

In a first aspect, the present application provides a primer pair composition for detecting a transgenic line of rice, wherein:

primer pairs for specifically amplifying Bt63, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.3 to SEQ ID NO. 4;

a primer pair for specifically amplifying G6H1, the nucleotide sequence of which is shown as SEQ ID NO.5 to SEQ ID NO. 6;

a primer pair for specifically amplifying LLRICE62, the nucleotide sequence of which is shown as SEQ ID NO.7 to SEQ ID NO. 8;

a primer pair for specifically amplifying LLRICE601, the nucleotide sequence of which is shown as SEQ ID NO.9 to SEQ ID NO. 10;

a primer pair for specifically amplifying KF2, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;

a primer pair for specifically amplifying KF8, the nucleotide sequence of which is shown in SEQ ID NO.13 to SEQ ID NO. 14;

a primer pair for specifically amplifying KMDF1, the nucleotide sequence of which is shown as SEQ ID NO.15 to SEQ ID NO. 16;

a primer pair for specifically amplifying T1C-9, the nucleotide sequence of which is shown as SEQ ID NO.17 to SEQ ID NO. 18;

a primer pair for specifically amplifying M12, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;

a primer pair for specifically amplifying T2A-1, the nucleotide sequence of which is shown as SEQ ID NO.21 to SEQ ID NO. 22; and, a step of, in the first embodiment,

the nucleotide sequences of the primer pair for specifically amplifying Kefeng6 are shown as SEQ ID NO. 23-24 and SEQ ID NO. 25-26.

Optionally, the primer pair composition further comprises a primer pair for specifically amplifying a rice transgenic line specific sequence selected from the group consisting of: bt63, G6H1, LLRICE62, LLRICE601, KF2, KF8, KMDF1, T1C-9, M12, T2A-1 and Kefeng6; the primer pair composition further comprises a primer pair for specifically amplifying the reference gene PLD.

Optionally, the primer pair for specifically amplifying the reference gene PLD comprises: the first pair has a nucleotide sequence shown in SEQ ID NO.27 to SEQ ID NO. 28; the second pair has the nucleotide sequences shown in SEQ ID NO.29 to SEQ ID NO. 30.

In a second aspect, the present application provides a kit for detecting a transgenic line of rice, the kit comprising a primer pair composition according to the first aspect.

Optionally, the kit further comprises a multiplex PCR premix.

Alternatively, the primer pair composition has a logarithmic range of 1-13 pairs.

Optionally, the kit comprises a first container containing the primer pair composition therein.

In a third aspect, the present application provides a method for detecting a transgenic line of rice using the primer pair composition of the first aspect, the method comprising the steps of:

obtaining a DNA and primer pair composition of rice to be detected;

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

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

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

Optionally, the concentration of the high-throughput library is more than or equal to 2ng/ul.

In a fourth aspect, the present application provides the use of a primer pair composition for the preparation of a detection kit for detecting a line of a transgenic rice;

the primer pair composition comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 26.

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

the primer provided by the embodiment of the application comprises a primer pair composition, wherein the nucleotide sequence of the primer pair composition is shown in SEQ ID NO.1 to SEQ ID NO. 26; the primer pair composition can perform one-time high-throughput sequencing and analysis on yield-increasing substances to obtain a plurality of detection results, wherein the detection results comprise transgenic strain determination, whether a sample to be detected contains the transgenic strain determination, and determination of copy numbers of reference genes and the transgenic strain in the sample to be detected so as to determine the content of the transgenic strain; the method avoids that the traditional Real-time PCR technology can only achieve one purpose at a time, and can cover a plurality of target transgenes in a sample only by carrying out amplification and detection for a plurality of times, thereby avoiding false positive or false negative results in the process of carrying out multiplex PCR in the prior art. When the primer is used for detection, more than 9 multiple PCR reactions can be performed, and the detection results of multiple target molecules in transgenic crops or multiple target molecules of the transgenic crops at the same time are obtained through one-time high-throughput sequencing and analysis, so that the detection efficiency and sensitivity are greatly improved, and the application and popularization are facilitated.

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

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

FIG. 2 is a schematic diagram of the gene structure of Bt63 samples provided in the examples of the present application;

FIG. 3 is a schematic diagram of the gene structure of LLRICE62 samples according to an embodiment of the present application.

Detailed Description

For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present application based on the embodiments herein.

In a first aspect, the present application provides a primer pair composition for detecting a transgenic line of rice, wherein:

primer pairs for specifically amplifying Bt63, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.3 to SEQ ID NO. 4;

a primer pair for specifically amplifying G6H1, the nucleotide sequence of which is shown as SEQ ID NO.7 to SEQ ID NO. 8;

a primer pair for specifically amplifying LLRICE62, the nucleotide sequence of which is shown as SEQ ID NO.7 to SEQ ID NO. 8;

a primer pair for specifically amplifying LLRICE601, the nucleotide sequence of which is shown as SEQ ID NO.9 to SEQ ID NO. 10;

a primer pair for specifically amplifying KF2, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;

a primer pair for specifically amplifying KF8, the nucleotide sequence of which is shown in SEQ ID NO.13 to SEQ ID NO. 14;

a primer pair for specifically amplifying KMDF1, the nucleotide sequence of which is shown as SEQ ID NO.15 to SEQ ID NO. 16;

a primer pair for specifically amplifying T1C-9, the nucleotide sequence of which is shown as SEQ ID NO.17 to SEQ ID NO. 18;

a primer pair for specifically amplifying M12, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;

a primer pair for specifically amplifying T2A-1, the nucleotide sequence of which is shown as SEQ ID NO.21 to SEQ ID NO. 22; and/or the number of the groups of groups,

the nucleotide sequences of the primer pair for specifically amplifying Kefeng6 are shown as SEQ ID NO. 23-24 and SEQ ID NO. 25-26.

In order to enhance the applicability of the primers to rice strain detection, at least 9 groups of primers are selected for detection, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can normally amplify target molecules in one amplification reaction, and experiments prove that the sensitivity is high and the applicability is strong. Meanwhile, the primer pair composition consists of a forward primer and a reverse primer, wherein one primer is an upstream primer and the other primer is a downstream primer.

In some embodiments, the primer pair composition further comprises a primer pair that specifically amplifies a rice transgenic line specific sequence selected from the group consisting of: bt63, G6H1, LLRICE62, LLRICE601, KF2, KF8, KMDF1, T1C-9, M12, T2A-1 and Kefeng6; the primer pair composition further comprises a primer pair for specifically amplifying the reference gene PLD.

In order to realize the effective amplification and detection of multiple groups of primers in one PCR reaction, when the primers are designed by utilizing the Primer, the Primer dimer, GC content, tm value, hairpin structure and the like are evaluated, then the amplified products of the multiple PCR primers are subjected to high-throughput sequencing, a high-throughput sequencing library can be obtained, and finally the comprehensive analysis of the high-throughput sequencing data is realized. Based on the existing primer pair composition, the primer pair for specifically amplifying other target molecules can be further included, and the primer pair combination can reach 3000 pairs through verification.

In addition, the nucleotide sequences of the target transgenic lines, i.e., as well as the target reference genes, including but not limited to, are comprehensively collected from transgenic databases, national standards, industry standards, or existing literature to ensure specificity and accuracy of the detection.

In some embodiments, the primer pair for specifically amplifying the reference gene PLD comprises: the first pair has a nucleotide sequence shown in SEQ ID NO.27 to SEQ ID NO. 28; the second pair has the nucleotide sequences shown in SEQ ID NO.29 to SEQ ID NO. 30.

In order to realize the quantitative detection of the transgenic rice strain, a rice internal reference gene PLD is introduced. The reason for using 2 primer pairs for the reference gene PLD is to avoid the condition that 1 pair of reference genes cannot be detected due to the influence of amplification efficiency.

In a second aspect, the present application provides a kit for detecting a transgenic line of rice, the kit comprising a primer pair composition according to the first aspect.

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

Specifically, the components of the multiplex PCR premix include the transgene element of the rape and each primer combination of the internal reference gene. A kind of electronic device. In combination, each primer was premixed in a ratio of 1:1, and each primer was mixed for different experimental purposes, and in the specific embodiment, each primer was 2 nM.

In some embodiments, the primer pair composition has a logarithmic range of 1-13 pairs.

The logarithmic range of multiplex PCR primers includes, but is not limited to: 11-13 pairs, preferably 13 pairs, wherein the number of pairs of transgenic elements 13 and the number of pairs of internal reference genes 1 can be increased periodically according to the newly collected transgenic elements, and the number of pairs of multiplex PCR primers can reach 3000 pairs after verification, so that the amplification effect is excellent; compared with the conventional 8-pair specific multiplex PCR, the method has the advantages of high detection flux and high sensitivity.

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

In a third aspect, the present application provides a method for detecting a kit according to the first aspect, as shown in fig. 1, the method comprising the steps of:

s1, obtaining a DNA and primer pair composition of rice to be detected;

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

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

s4, analyzing the gene sequences in the high-throughput library to obtain a result of detecting the rice transgenic line.

In particular, high throughput sequencing can be second generation sequencing or 3 generation sequencing, and the resulting high throughput library can analyze the composition of transgenes from multiple dimensions, including but not limited to transgenic lines in our embodiments. The method is applied to qualitative and quantitative detection of transgenic lines of rice and products thereof.

In the method of the embodiment of the present application, the amplification reaction comprises a general PCR amplification reaction or a real-time fluorescent PCR amplification reaction; the environment/procedure of the amplification reaction includes: pre-denaturation at 94 ℃ for 15 min; the first amplification step, denaturation at 94℃for 20 seconds, annealing at 65℃to 57℃and extension for 60 seconds, 10 Touch Down cycles, (annealing and extension temperatures for each cycle reduced by 0.8 ℃); a second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles; in addition, the reaction system includes, but is not limited to: 30 μl of the total system, primer pair: 2. mu.l, 2 Xbuffer: 15ul, multiplex amplification enzyme: 0.5 Mu.l; the remainder was replenished with water.

In some embodiments, the high throughput library is at a concentration of ≡2ng/ul.

The concentration of the high-flux library is controlled, the systematic deviation caused by cloning is avoided, the experimental operation is simplified, and the sequencing efficiency is improved.

In a fourth aspect, the present application provides the use of a primer pair composition for the preparation of a detection kit for detecting a line of a transgenic rice;

the primer pair composition comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 26.

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

Example 1 screening of target transgenes and design of multiplex PCR amplification primers

In the embodiment of the application, the target molecules, namely the specificity nucleotide sequences of the main transgenic strain and the internal reference gene, are mainly collected in a common transgenic database, a national standard, an industry standard or an existing literature, so that the specificity and the accuracy of detection are ensured. The names and sequences of the selected transgenic lines and reference genes are shown in Table 1.

Table 1 the target molecules screened according to the invention and their primer sequences.

S2, design of multiplex PCR amplification primer

In the embodiment of the application, primer3Plus is utilized to design multiple PCR primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, the main evaluation is to evaluate the dimer among the primers, or the hairpin structure inside the primers, and the nonspecific amplification of a non-target sequence, all the evaluated primers can be combined into a Primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

Example 1 detection of whether Rice samples contain specific sequences by the method of the invention

1. Experimental materials: a schematic diagram of the insect-resistant rice variety (Bt 63 or TT 51-1) and herbicide-resistant rice LLRICE62, and the transgenic material Bt63 is shown in FIG. 2. The experimental material is transferred into a self promoter, and the transgenic content of the specific sequence containing Bt63 strain is 10%; the structural diagram of herbicide-resistant rice LLRICE62 is shown in FIG. 3, and contains a strain-specific sequence; it was used as a material for our study.

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

PCR amplification, library construction and sequencing

Amplifying genomic DNA of the sample using 30 pairs of multiplex PCR amplification primers; wherein each sample is subjected to PCR amplification, and a sequencing joint is connected with a DNA bar code of a specific sample to form a high-throughput sequencing library; and detecting the high-throughput sequencing library by using a high-throughput sequencing platform, and performing quality control on the high-throughput sequencing data. The step is to research and adjust key parameters such as amplification cycle number, primer concentration, sequencing depth and the like according to the requirements of detection accuracy, sensitivity and the like; the step can also be connected with the step related to the third generation sequencing so as to realize the complementary advantages between the second generation sequencing and the third generation sequencing.

The environment/procedure of the amplification reaction includes: pre-denaturation at 94 ℃ for 15 min; the first amplification step, denaturation at 94℃for 20 seconds, annealing at 65℃to 57℃and extension for 60 seconds, 10 Touch Down cycles, (annealing and extension temperatures for each cycle reduced by 0.8 ℃); a second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles; in addition, the reaction system includes, but is not limited to: 30 μl of the total system, primer pair: 2. mu.l, 2 Xbuffer: 15ul, multiplex amplification enzyme: 0.5 Mu.l; the remainder was replenished with water.

4. Determination of results

1) Determining whether the contamination is acceptable based on the signal index S of the specific sequence in the test sample and the signal index P of the specific sequence in the blank, wherein:

the blank noise index p=nc/Nc, where Nc and Nc represent the number of sequenced fragments of the specific sequence and the total number of sequenced fragments, respectively, in the blank.

The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments of the specific sequence and the total number of sequenced fragments, respectively, in the test sample.

Signal to noise ratio = S/P,

2) Determination of transgene outcome

And (3) distributing each sequencing fragment to each target position of each target species by using the DNA bar code of the sample to be tested and homology comparison, wherein the targets comprise transgenic lines and internal reference genes. Based on the number of sequenced sequences at each target position, absolute quantification of specific sequences is achieved. When the sequencing sequences on the reference gene and the lines are compared to exceed a specified threshold, qualitatively judging that the sample contains specific transgenic line sequences; when the sample contains the specific transgene strain sequence, the content of the transgene in the sample is quantitatively judged according to the ratio of the specific sequence of the transgene strain to the sequencing sequence of the internal reference gene. The calculation formula of the transgene content in this embodiment is shown in (a):

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

According to this example we detected a total of 2 samples, each with three biological replicates, in this example we required sequence filtering out with a number of sequencing reads less than 5. The results are shown in Table 2: no sequence of any transgenic line was detected in the negative sample, and the invention provides that when the signal to noise ratio is greater than 10 times, it can be judged that the contamination in the detection system is acceptable. And when the signal to noise ratio of the specific sequence in the sample is greater than 10, judging that the nucleic acid of the specific sequence is detected in the sample.

Table 2 the transgene test results of the test sample of example 2.

From the above table, 2 strain-specific sequences in Bt63 samples were all detected, and three repeats all detected the target sequence, with a content close to 10%; the strain-specific sequences in the same LLRICE62 sample were also detected and the content was also close to 1%, indicating that the rice transgenic kit of our invention can be used to detect transgenic products.

Example 3 evaluation of accuracy and sensitivity of the method of the invention

Transgenic samples of different mass percentages were prepared using rice Bt63 and LLRICE62 transgenic standards to evaluate the accuracy and sensitivity of the developed techniques. Specifically, the transgene content of each sample was diluted with mass percent, specifically, transgenic rice Bt63 and LLRICE62 were diluted with negative rice to 10%,1%,0.1%,0.05%,0.025% and 0.01% samples, respectively, corresponding to diluted sample numbers (A1, A2, A3, A4, A5, A6) of transgenic line Bt63 and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line LLRICE62, respectively. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree of coincidence of the average value of multiple determinations with a true value, and is expressed by errors. Sensitivity refers to the lowest content of a specific sequence that can be detected at 95% confidence, i.e., the lower detection limit. The assay was performed as in example 2, with three replicates per sample, and the results are shown in table 3.

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

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

As can be seen from the above table, the kit can stably detect each transgenic line in a sample with the transgenic content of 0.05%, and can obviously distinguish a sample with the transgenic content of 0.05% from a negative sample without detecting a specific sequence in a negative sample, and has technical stability and detection sensitivity with the transgenic content of 0.05%.

Examples

Specificity analysis

Specificity, consistency and stability have been important criteria in determining whether a test system is viable. In example 4, we performed cross amplification of transgenic rice line test products with different exogenous genes to verify their specificity, consistency and stability.

In the embodiment, 12 pairs of transgenic rice strain specific primers in our kit are utilized to detect high specificity Bt63 (A1), 6H1 (A2), LLRICE62 (A3), LLRICE601 (A4), KF2 (A5), KF8 (A6), KMDF1 (A7), T1C-9 (A8), M12 (A9), T2A-1 (A10) and kefeng6 (A11) of target microorganisms in 11 transgenic rice strain hybrid templates, and the samples are mixed in equal proportion, diluted 10 times after mixing, and negative samples are taken as controls during each transgenic detection. The test procedure was carried out as in example 2 and the results are shown in Table 4: each of the transgenic lines tested was amplified using 13 pairs of primers, but we amplified only the target specific sequence, did not amplify the non-target molecule, and three replicates of each test sample gave consistent results, thus demonstrating the specificity, consistency and stability of our method.

TABLE 4 specificity analysis of the method of the invention

Example 5 application of the invention in the detection of batch transgenic products

In order to verify the accuracy of the invention and the role in transgene detection of batch samples, a laboratory selects 2864 rice leaf samples with unknown genotypes of a company to detect, the detection result is compared with the preservation type of the company, and the consistency of the result is counted. The analysis result shows that in 2864 test samples, only 17 samples have inconsistent results, and the consistency of the detection results is up to 99.4%, so that the accuracy and the good application prospect of the method are better proved.

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

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

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Claims (9)

1. A primer pair composition for detecting a transgenic rice line, wherein the primer pair composition comprises:

primer pairs for specifically amplifying Bt63, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.2 and SEQ ID NO.3 to SEQ ID NO. 4;

a primer pair for specifically amplifying G6H1, the nucleotide sequence of which is shown as SEQ ID NO.5 to SEQ ID NO. 6;

a primer pair for specifically amplifying LLRICE62, the nucleotide sequence of which is shown as SEQ ID NO.7 to SEQ ID NO. 8;

a primer pair for specifically amplifying LLRICE601, the nucleotide sequence of which is shown as SEQ ID NO.9 to SEQ ID NO. 10;

a primer pair for specifically amplifying KF2, the nucleotide sequence of which is shown as SEQ ID NO.11 to SEQ ID NO. 12;

a primer pair for specifically amplifying KF8, the nucleotide sequence of which is shown in SEQ ID NO.13 to SEQ ID NO. 14;

a primer pair for specifically amplifying KMDF1, the nucleotide sequence of which is shown as SEQ ID NO.15 to SEQ ID NO. 16;

a primer pair for specifically amplifying T1C-9, the nucleotide sequence of which is shown as SEQ ID NO.17 to SEQ ID NO. 18;

a primer pair for specifically amplifying M12, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;

a primer pair for specifically amplifying T2A-1, the nucleotide sequence of which is shown as SEQ ID NO.21 to SEQ ID NO. 22; and, a step of, in the first embodiment,

the nucleotide sequences of the primer pair for specifically amplifying Kefeng6 are shown as SEQ ID NO. 23-24 and SEQ ID NO. 25-26.

2. The primer pair composition of claim 1, further comprising a primer pair that specifically amplifies a reference gene PLD.

3. The primer pair composition according to claim 2, wherein the primer pair for specifically amplifying the reference gene PLD comprises: the first pair has a nucleotide sequence shown in SEQ ID NO.27 to SEQ ID NO. 28; the second pair has the nucleotide sequences shown in SEQ ID NO.29 to SEQ ID NO. 30.

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

5. The kit of claim 4, further comprising a multiplex PCR premix.

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

7. A method of using the primer pair composition of any one of claims 1-3 for detecting a transgenic line of rice, comprising the steps of:

obtaining a DNA and primer pair composition of rice to be detected;

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

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

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

8. The method of claim 7, wherein the high throughput library is at a concentration of ≡2ng/ul.

9. The primer pair composition is used for preparing a detection kit, and the detection kit is used for detecting rice transgenic lines;

the primer pair composition comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 26.

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