CN106995841B - Multiplex PCR (polymerase chain reaction) kit for detecting transgenic soybeans and detection method - Google Patents
Multiplex PCR (polymerase chain reaction) kit for detecting transgenic soybeans and detection method Download PDFInfo
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Abstract
The invention discloses a multiplex PCR kit and a detection method for detecting transgenic soybeans, which can quickly detect 12 transgenic soybeans approved to be imported by the Ministry of agriculture of China, including MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547, DP356043, DP305423, MON87769, MON87708, 305423 multiplied by 40-3-2, MON87701 multiplied by MON 89788; the multiple PCR kit disclosed by the invention is based on a public primer mediated multiple PCR method, and comprises the detection of a public primer, multiple primers and a multiple PCR system; the specificity and the sensitivity of a multiplex PCR system are improved by a two-step annealing temperature method, the multiplex PCR method has compatibility, detection targets can be continuously increased, and the improvement of the detection flux of the multiplex PCR is facilitated. The method has the advantages of high sensitivity, economy, rapidness, simple operation and the like, and can be applied to food detection.
Description
Technical Field
The invention belongs to the field of food/gene detection, and particularly relates to a multiple PCR (polymerase chain reaction) kit for detecting transgenic soybeans and a detection method.
Background
At present, the detection technologies of transgenic crops reported at home and abroad are mainly divided into three categories, one is a detection technology based on nucleic acid; secondly, protein-based enzymology and immunology detection technology; the third category is new transgenic detection technology, including biosensors and biochips developed to automation technology, near infrared spectroscopy and mass spectrometry based on modern analytical instruments, and the like. The second type of protein-based detection technology is to detect proteins expressed by foreign genes in transgenic plants, which are easily destroyed in the processing process to inactivate, decompose or disappear, and the detection result is easy to have false negative and the repeatability of the method is poor. The novel transgenic detection technology developed in recent years, such as a biosensor, a chromatography, a near infrared spectroscopy and the like, is still in a research stage at present, and has the defects that the technology cannot be effectively solved or objectively exists temporarily, for example, the chromatography is only suitable for qualitative detection and analysis when the components of transgenic plants and products thereof are changed greatly, and the detection technology cannot be effectively popularized and applied in a short period. Therefore, the standard of the transgenic component detection method released by the ministry of agriculture in China is mainly based on nucleic acid detection technology, and the detection technology comprises PCR technology, constant temperature amplification technology, southern hybridization, gene chip technology, fluorescent quantitative PCR and the like.
The multiplex PCR is a PCR technology for simultaneously amplifying multiple target sequences by using multiple pairs of primers in the same reaction system on the basis of common PCR. Since the report of multiplex PCR, it has been widely used in many fields of nucleic acid diagnosis, such as genetic disease diagnosis, mutation and polymorphism analysis, pathogen detection, transgene identification, etc. due to its high efficiency, sensitivity and economic simplicity; multiplex PCR systems, generally comprising the following reagents: PCR buffer, Taq enzyme, dNTP, MgCl2A multiplex primer, a template and deionized water.
The imported transgenic soybeans currently allowed in China are up to 12 (specifically including GTS-40-3-2, MON87701, MON89788, MON87701 XMON 89788, CV127-9, A2704-12, DP356043, DP305423, A5547, 305423X 40-3-2, MON87769 and MON87708, wherein the MON87701 XMON 89788 and 305423X 40-3-2 are transgenic soybeans with compound characters).
The transgenic crop is cultivated by transferring exogenous target gene into plant by means of gene engineering technology, and has excellent characteristics of resisting insect, resisting herbicide, resisting stress, improving nutrients, etc. The growing area of transgenic crops worldwide has increased from 0.17 billion hectares in 1996 to 1.797 billion hectares in 2015. With the continuous development of transgenic technology, the industrialization and commercialization degree of transgenic crops is continuously deepened, and China successively sets up relevant laws and regulations aiming at the transgenic crops, however, public worry about the safety of transgenic food and the right to know whether the edible food contains transgenic ingredients are still increased day by day, so that the rapid, accurate and sensitive transgenic food detection technology is one of the basis and the key of all laws and regulations such as transgenic safety evaluation and the like which really fall into the real places.
Disclosure of Invention
The invention aims to provide a multiple PCR kit for detecting transgenic soybeans, which is used for single or simultaneous multiple PCR detection of 12 transgenic soybeans which are currently approved to be imported (i.e. have issued safety certificates) in China.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a multiple PCR kit for detecting transgenic soybeans comprises a common primer and a chimeric primer aiming at the transgenic soybeans; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer aiming at the transgenic soybean is one or more of nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO. 18.
In the above technical scheme, the multiplex PCR kit for detecting transgenic soybean further comprises Taq polymerase, dNTP, MgCl2PCR buffer solution, deionized water and a positive control primer pair; wherein the sequences of the positive control primer pair are preferably SEQ ID NO.19 and SEQ ID NO. 20; can be used for detecting the quality of the DNA template and the amplification efficiency of a reaction system;
SEQ ID NO.19：GCCCTCTACTCCACCCCCATCC
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG 。
in the technical scheme, a two-step annealing temperature method is adopted during PCR reaction, and the first 10 circulating annealing temperatures are 61 ℃; the annealing temperature was 54 ℃ for the last 30 cycles. The annealing temperature of the first step is 5-10 ℃ higher than that of the second step; for example, the annealing temperature of the first 10 cycles is 7 ℃ higher than that of the second 30 cycles, so that the detection specificity can be effectively improved; when multiple PCR reactions are carried out, the final concentration of each chimeric primer aiming at the transgenic soybean is 8-80 nM, and the final concentration of the common primer is 1600 nM.
The invention also discloses a method for detecting the transgenic soybean component in the product, which comprises the steps of extracting the genomic DNA of the product to be detected as a template (for example, the connecting region of the insertion gene of the transgenic soybean and the soybean genome is a target sequence), carrying out PCR reaction by adopting a two-step annealing temperature method in the presence of the chimeric primer and the common primer, and carrying out electrophoretic analysis on the PCR product to finish the detection of the transgenic soybean component in the product; finally, performing electrophoretic analysis on the PCR product amplification to finish the detection of the transgenic soybean component in the product; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer is one or more of nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO. 18.
In the technical scheme, the annealing temperature of the first-step (namely, the first 10 cycles) multiple PCR amplification is 5-10 ℃ higher than that of the second-step (namely, the last 30 cycles) multiple PCR amplification; the final concentration of each chimeric primer is 8-80 nM, and the final concentration of the common primer is 1600 nM; the product is a food product comprising a soy component. The method specifically comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 10 cycles; extending for 2min at 72 ℃; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min. The first 10 cycles, at high annealing temperature, the specific primer in the low concentration chimeric primer amplifies the target sequence, producing a PCR product with a common primer end; in the last 30 cycles, the high concentration of the common primer is amplified in large quantities using the PCR product with the common primer end generated in the early stage as a template at the low annealing temperature. The method can effectively and simply detect whether the products, especially the food contain the transgenic soybean ingredients, and makes great contribution to national right of knowledge and food safety.
The method for detecting the nucleic acid of the transgenic soybean ingredients disclosed by the invention is accurate, can be used for simultaneously detecting the target gene in 12, has accurate detection results, and can solve the public worry about the safety of transgenic food and the right of knowing whether the edible food contains the transgenic ingredients.
The invention further discloses a primer for detecting the transgenic soybean, which comprises a public primer and a chimeric primer aiming at the transgenic soybean; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer aiming at the transgenic soybean is one or more of nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO. 18.
The invention also discloses application of the primer for detecting the transgenic soybean in preparing a multiplex PCR kit for detecting the transgenic soybean or application of the primer for detecting the transgenic soybean in claim 8 in detecting the transgenic soybean.
In the invention, the sequence SEQ ID NO.1 of the public primer and the nucleotide sequence of the chimeric primer are as follows:
the chimeric primer is a chimeric primer aiming at 12 transgenic soybeans; a2704 shares the upstream primer SEQ ID NO.11 with A5547, and MON89788, MON87769 and MON87708 shares the downstream primer SEQ ID NO. 18. 305423 × 40-3-2 and MON87701 × MON89788 are transgenic soybeans stacked by two genes, and if the transgenic soybeans DP305423 and GTS40-3-2 are detected simultaneously in a sample, the sample may contain the transgenic soybeans 305423 × 40-3-2; if transgenic soybean MON87701 and MON89788 are detected simultaneously in the sample, it is likely that the sample will contain transgenic soybean MON87701 xmon 89788.
In the technical scheme, the public primer has no homology with the related genome of the transgenic soybean, and when the transgenic soybean genome is used as a template for amplification, no specific product exists no matter what conditions are; when the amplification product of the chimeric primer is taken as a template, the common primer can amplify a specific product; and the Tm value of the common primer is 5-10 ℃ lower than that of the specific primer of the transgenic soybean target.
In the technical scheme, the final concentration of the common primer is 1600nM and the final concentration of the chimeric primer aiming at the transgenic soybean is 8-80 nM when PCR reaction is carried out, and the PCR reaction system is used for amplifying 12 transgenic soybeans MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547, DP356043, DP305423, MON87769, MON87708, 305423 × 40-3-2 and MON87701 × MON89788, such as SEQ ID NO.2/3:80nM, SEQ ID NO.4/5: 60nM, SEQ ID NO.6/7: 64nM, SEQ ID NO.8/9: 8nM, SEQ ID NO.10:32nM, SEQ ID NO.11: 48nM, SEQ ID NO.12: 16nM, SEQ ID NO.13/14: 24nM, SEQ ID NO.15/16: 8nM, SEQ ID NO.17: 16nM and SEQ ID NO.18: 32 nM; during PCR reaction, a two-step annealing temperature method is adopted, and the first-step annealing temperature is 5-10 ℃ higher than the second-step annealing temperature.
The kit disclosed by the invention can be used for detecting transgenic soybean components in products, particularly food, and the multiplex PCR disclosed by the invention is a public primer mediated multiplex PCR combined two-step annealing temperature method, specifically, a two-step annealing temperature method is adopted, a transgenic soybean inserted gene sequence and a soybean gene connecting region sequence are taken as target sequences, a chimeric primer is used for amplifying 10 cycles firstly, the annealing temperature is 61 ℃, the first-step multiplex PCR amplification is carried out, a PCR product at the tail end of the public primer can be generated, the public primer takes the PCR product with the tail end of the public primer amplified in the first step as a template, the second-step multiplex PCR amplification of the later 30 cycles is carried out, the annealing temperature is 54 ℃, the multiplex PCR amplification of the transgenic soybean is completed, the multiplex PCR amplification annealing temperature of the first 10 cycles is 5-10 ℃ higher than the multiplex PCR amplification annealing temperature of the later 30 cycles, the target sequences of the chimeric primer with low concentration are amplified at the early cycle, a PCR product with the tail end of the public primer is generated at high annealing temperature, the multiplex amplification of the chimeric primer with low concentration, the PCR product generated at the low annealing temperature is used for carrying out, the multiplex amplification of a large amount of the PCR product generated at the high-amplification temperature, the PCR amplification of the public primer, the multiple PCR system comprises a PCR system with the concentration of a PCR system of a2O4.5 μ L; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃Fire 30s, extension 30s at 72 ℃ for 10 cycles; extending for 2min at 72 ℃; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min. Finally, agarose gel electrophoresis analysis is carried out, and the result shows that the multiplex PCR kit for detecting the transgenic soybeans can detect the genomes of 10 transgenic soybeans simultaneously or independently and specifically; by utilizing the constructed plasmid template, the sensitivity of the multiplex PCR kit for detecting the transgenic soybean can reach 0.1 percent (w/w) of the content of the components of the transgenic soybean, namely 0.1ng/100ng of DNA sample.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
(1) the multiplex PCR kit for detecting the transgenic soybeans disclosed by the invention can simultaneously and independently detect 12 types of transgenic soybeans approved to be imported in China, namely MON87701, GTS40-3-2, MON89788, CV127, A2704, A5547, DP356043, DP305423, MON87769, MON87708, 305423 multiplied by 40-3-2 and MON87701 multiplied by MON89788, so that the use amount of reagents can be saved, the detection cost is reduced, the detection efficiency is improved, and the multiplex PCR kit has the advantages of high sensitivity, economy, quickness, simplicity in operation and the like;
(2) when the kit disclosed by the invention is used for detection, the concentration of the chimeric primers is reduced to 8nM per strip, and the concentration of the common primers is 1600 nM; the concentration of the chimeric primers is reduced, so that more pairs of chimeric primers can be added, and the multiple PCR detection flux is improved; the single common primer is adopted, so that the formation of dimer is reduced, the high concentration is favorable for amplifying the amount of specific products, and the detection sensitivity is improved;
(3) the kit disclosed by the invention adopts a two-step annealing temperature method in PCR reaction, and the first 10 cycles of amplification adopt high annealing temperature (61 ℃) to facilitate the specific binding of chimeric primers and reduce non-specific products; the low annealing temperature (54 ℃) of the next 30 cycles of amplification enables the common primer to play an amplification role; the extension time is increased by 72 ℃ for 2min additionally in the middle, which is beneficial to obtaining enough templates for the common primers;
(4) in the kit disclosed by the invention, a public primer is connected with the 5' end of each multiple PCR specific primer to form a chimeric primer; the concentration of a primer pair can be reduced by the common primer-mediated multiple PCR, and the formation of dimers is reduced, so that the detection flux of the multiple PCR is improved; the defects of non-specific amplification and easy dimer existing in the existing multiplex PCR system are overcome through creative design;
(5) the multiple PCR detection system has high specificity, and the sensitivity is up to 0.1% (w/w) (the content of transgenic soybean components), namely 0.1ng/100ng DNA sample; the method is carried out by a single tube, is simple to operate, has the advantages of high sensitivity, economy, quickness and the like, and has practical application value;
(6) the multiplex PCR (multiplex PCR) disclosed by the invention can be used for simultaneously detecting a plurality of target molecules in one PCR tube, is simpler, more convenient and quicker than southern hybridization and a gene chip method, is more economical than fluorescence quantitative PCR, and is not easy to pollute and has high flux compared with a constant temperature amplification technology (comprising a loop-mediated isothermal amplification method). This technique has received much attention in nucleic acid detection technology because of its advantages such as high efficiency, sensitivity, and economical simplicity.
Drawings
FIG. 1 is a specific detection electropherogram of a common primer of the invention;
FIG. 2 is a graph showing the results of sensitivity of the common primers of the present invention;
FIG. 3 is a diagram showing the results of electrophoresis in the detection of the specificity of the multiplex system of the present invention;
FIG. 4 is a graph showing the sequencing results of the transgenic soybean of the present invention;
FIG. 5 is a graph showing the results of sensitivity of the multiplex assay system of the present invention.
Detailed Description
The invention will be further described with reference to the following examples and the accompanying drawings
Examples
1. Screening of common primers and sensitivity of detection thereof
The quality of the common primer as a core technology in the whole technology system directly influences the success or failure of the technology. The common primer SEQ ID NO.1 adopted by the invention and the genomic DNA of the transgenic soybean and the non-transgenic soybean are subjected to PCR amplification, specific products cannot be amplified within the annealing temperature gradient range of 45-65 ℃, for example, as shown in figure 1, a-k are amplification results of the common primer and the genomic DNA of the transgenic soybean and the non-transgenic soybean, and the results show that the specific products cannot be amplified by the common primer, wherein a lane 0 is a blank control, a lane M is a 100bp DNA Marker, and lanes 1-8 are annealing temperature gradients of 45-65 ℃; the template a is non-transgenic soybean genomic DNA, the template b is GTS40-3-2 genomic DNA, the template c is A2704-12 genomic DNA, the template d is MON89788 genomic DNA, the template e is DP356043 genomic DNA, the template f is DP305423 genomic DNA, the template g is CV127-9 genomic DNA, the template h is MON87701 genomic DNA, the template i is A5547-127 genomic DNA, the template j is MON87708 genomic DNA, and the template k is MON87769 genomic DNA.
The sensitivity of the detection of the common primer SEQ ID NO.1 is further detected through the constructed recombinant plasmid template, the detection sensitivity reaches 100 copies, as shown in figure 2, a Lane M is a 100bp DNA marker, a Lane 0 is a blank control, and the plasmid copy numbers corresponding to the templates in the Lanes 1-8 are 10 in sequence1、102、103、104、105、106、107、108。
In the invention, when the genomic DNA of the transgenic soybean and the non-transgenic soybean is used as a template for amplification, no amplification product exists no matter how the amplification condition is optimized; when the multiple chimeric primers with the common primer have amplification products, specific products can be amplified by the common primer.
2. The multiplex PCR kit for detecting the transgenic soybeans comprises a conventional multiplex PCR component, and also comprises chimeric primers and common primers aiming at the 10 transgenic soybeans; the sequences of the chimeric and common primers are shown in Table 1, and the conventional multiplex PCR components include Taq polymerase, dNTP, MgCl2PCR buffer solution, deionized water and a positive control primer pair; the positive control primer pair can be used for detecting the quality of a DNA template and the amplification efficiency of a reaction system, and has the specific sequence as follows: SEQ ID NO. 19: GCCCTCTACTCCACCCCCATCC,
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG。
Table 1 shows chimeric primers and common primers for 12 types of transgenic soybeans imported in China
3. Specific detection of multiplex systems
A PCR method adopting multiple primers, a common primer and a single template is adopted to amplify various genomes or target genes for detecting the transgenic soybeans, wherein the DNA templates adopt 10 kinds of extracted corresponding transgenic soybean genome DNA, the concentration is about 25 ng/mu L, and a 25 mu L reaction system adopted by the multiple PCR comprises 4 mu L of 10 mu M common primers, 4 mu L of chimeric primer mixtures with different final concentrations (SEQ ID NO.2/3:80nM, SEQ ID NO.4/5: 60nM, SEQ ID NO.6/7: 64nM, SEQ ID NO.8/9: 8nM, SEQ ID NO.10:32nM, SEQ ID NO.11: 48nM, SEQ ID NO.12: 16nM, SEQ ID NO.13/14: 24nM, SEQ ID NO.15/16: 8nM, SEQ ID NO.17: 16nM and SEQ ID NO.18: 32 nM), 2 mu L of 2 × Dream Green Mix 12.5 mu L, 1 mu L of DNA template and ddH 12L of sterile primers2O5.5 μ L; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 10 cycles; extending for 2min at 72 ℃; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min. FIG. 3 is a diagram showing the results of electrophoresis of the specificity detection of the multiplex system, wherein 0 is blank control, N is negative control, lanes 1-10 are the results of agarose gel electrophoresis of PCR products, in this order, MON87701, DP356043, DP305423, CV127, MON87769, A2704, GTS40-3-2, MON87708, MON89788, and A5547, N is negative control, M is DNA standard molecular weight (band sizes from bottom to top are 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000bp in this order), PM is product marker, and the band sizes are shown on FIG. 3; the result shows that the specific PCR product with expected size can be amplified from the corresponding template by using the multiplex system of the invention to detect the target DNA, and the band is clear. The amplified product was further sequenced and proved to be the corresponding detection sequence of transgenic soybean (fig. 4). The experimental results in this section show that the multiplex system of the present invention has high specificity.
4. Sensitive detection of multiplex detection systems
The system sensitivity detection adopts 10 transgenic soybeans A2704-12, MON89788, CV127, MON87701, A5547-127, MON87708, MON87769, DP356043, DP305423 and GTS40-3-2 genomic DNA solutions to be mixed in equal volumes (wherein the initial concentration of DP356043, DP305423 and GTS40-3-2 is 2.5 ng/mu L, and the initial concentration of the rest 7 is 25 ng/mu L), then the mixed genomic DNA solution is subjected to 10-fold gradient dilution by using TE buffer solution to sequentially obtain 10-fold, 100-fold, 1000-fold and 10000-fold genomic DNA dilution mixed solutions, 4 mu L of each dilution solution is taken as a template (the concentration of each transgenic soybean genomic DNA template is 10ng, 1ng, 0.1ng and 0.01ng respectively, and the 3 corresponding concentrations of DP356043, DP305423 and GTS40-3-2 are 1ng, 0.1ng, 0.01ng and 0.001ng respectively), and the 3 gene target targets of PCR reaction is respectively carried out by gradient reaction, ddH2The multiplex PCR adopts 25 muL reaction system comprising 10 muM public primer 4 muL, 1 muM chimeric primer pair mixture for corresponding variety of transgenic soybean 2 muL, 2 × Dream Taq Green Mix 12.5 muL, DNA template 4 muL, sterilized ddH2O2.5 mu L; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 10 cycles; extending for 2min at 72 ℃; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min. The PCR reaction product was electrophoresed through 2% agarose gel, the analysis result of the gel imaging system is shown in FIG. 5, lane M is 100bpDNA Ladder, lane 0 is blank negative control using deionized water as template, and 1-4 are diluted mixed solution of 10000, 1000, 100 and 10 times of 10 kinds of transgenic soybean genomic DNA respectively as genomic DNA template. The result shows that the sensitivity of the public primer-mediated multiple PCR established by the invention can detect 10 kinds of transgenic soybean genomic DNA diluted mixed solution diluted by 1000 times, the system sensitivity exceeds 0.1 percent, and the detection is superior to the national requirements on transgenic food detection standards.
SEQUENCE LISTING
<110> Suzhou university
<120> multiplex PCR kit for transgenic soybean detection and detection method
<160>20
<210>1
<211>17
<212>DNA
<213> Artificial Synthesis
<400>1
CTCGTCAACTCCGCAAG 17
<210>2
<211>42
<212>DNA
<213> Artificial Synthesis
<400>2
CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG 42
<210>3
<211>40
<212>DNA
<213> Artificial Synthesis
<400>3
<210>4
<211>37
<212>DNA
<213> Artificial Synthesis
<400>4
CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG 37
<210>5
<211>39
<212>DNA
<213> Artificial Synthesis
<400>5
CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG 39
<210>6
<211>42
<212>DNA
<213> Artificial Synthesis
<400>6
CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA 42
<210>7
<211>42
<212>DNA
<213> Artificial Synthesis
<400>7
CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA 42
<210>8
<211>41
<212>DNA
<213> Artificial Synthesis
<400>8
CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG 41
<210>9
<211>39
<212>DNA
<213> Artificial Synthesis
<400>9
CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT 39
<210>10
<211>43
<212>DNA
<213> Artificial Synthesis
<400>10
CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG 43
<210>11
<211>40
<212>DNA
<213> Artificial Synthesis
<400>11
<210>12
<211>42
<212>DNA
<213> Artificial Synthesis
<400>12
CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG 42
<210>13
<211>39
<212>DNA
<213> Artificial Synthesis
<400>13
CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC 39
<210>14
<211>39
<212>DNA
<213> Artificial Synthesis
<400>14
CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT 39
<210>15
<211>42
<212>DNA
<213> Artificial Synthesis
<400>15
CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC 42
<210>16
<211>42
<212>DNA
<213> Artificial Synthesis
<400>16
CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT 42
<210>17
<211>43
<212>DNA
<213> Artificial Synthesis
<400>17
CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG 43
<210>18
<211>36
<212>DNA
<213> Artificial Synthesis
<400>18
CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG 36
<210>19
<211>22
<212>DNA
<213> Artificial Synthesis
<400>19
GCCCTCTACTCCACCCCCATCC 22
<210>20
<211>23
<212>DNA
<213> Artificial Synthesis
<400>20
GCCCATCTGCAAGCCTTTTTGTG 23
Claims (5)
1. A multiple PCR kit for detecting transgenic soybeans is characterized in that: the multiple PCR kit for detecting the transgenic soybeans comprises a common primer and a chimeric primer aiming at the transgenic soybeans; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer aiming at the transgenic soybean is a nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18:
SEQ ID NO.1：CTCGTCAACTCCGCAAG
SEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG
SEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT
SEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG
SEQ IDNO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG
SEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA
SEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA
SEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG
SEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT
SEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG
SEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC
SEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG
SEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC
SEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT
SEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC
SEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT
SEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG
SEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG。
2. the multiplex PCR kit for detecting transgenic soybean according to claim 1, wherein: the multiple PCR kit for detecting the transgenic soybean further comprises Taq polymerase, dNTP and MgCl2PCR buffer solution, deionized water and a positive control primer pair.
3. The multiplex PCR kit for detecting transgenic soybean according to claim 2, wherein: the positive control primer pair has sequences of SEQ ID NO.19 and SEQ ID NO. 20;
SEQ ID NO.19：GCCCTCTACTCCACCCCCATCC
SEQ ID NO.20：GCCCATCTGCAAGCCTTTTTGTG。
4. the primer for detecting the transgenic soybean is characterized in that: the primer for detecting the transgenic soybean comprises a common primer and a chimeric primer aiming at the transgenic soybean; the sequence of the public primer is SEQ ID NO. 1; the chimeric primer aiming at the transgenic soybean is a nucleotide sequence shown in SEQ ID NO.2 to SEQ ID NO.18:
SEQ ID NO.1：CTCGTCAACTCCGCAAG
SEQ ID NO.2：CTCGTCAACTCCGCAAGATATTGACCATCATACTCATTGCTG
SEQ ID NO.3：CTCGTCAACTCCGCAAGTCACTTTCTTGAATTAGCTTGCT
SEQ ID NO.4：CTCGTCAACTCCGCAAGCTTTTGCCCGAGGTCGTTAG
SEQ ID NO.5：CTCGTCAACTCCGCAAGGCCCTTTGGTCTTCTGAGACTG
SEQ ID NO.6：CTCGTCAACTCCGCAAGCGTCAGGAATAAAGGAAGTACAGTA
SEQ ID NO.7：CTCGTCAACTCCGCAAGATTTCTAACCTGGCTGCTATAGTTA
SEQ ID NO.8：CTCGTCAACTCCGCAAGTGTATAGGAAAGCGCAAACTGATG
SEQ ID NO.9：CTCGTCAACTCCGCAAGATTAGGGTTTCAGCAGGTTCGT
SEQ ID NO.10：CTCGTCAACTCCGCAAGAAAATACAAATTTAACACTTCATTGG
SEQ ID NO.11：CTCGTCAACTCCGCAAGTACCAATGCTTAATCAGTGAGGC
SEQ ID NO.12：CTCGTCAACTCCGCAAGGAATGCAACACACTGTAACAATTTG
SEQ ID NO.13：CTCGTCAACTCCGCAAGAACCCTTCAATTTAACCGATGC
SEQ ID NO.14：CTCGTCAACTCCGCAAGTTGCGAAGGATAGTGGGATTGT
SEQ ID NO.15：CTCGTCAACTCCGCAAGGGCAGTAACTTGAAAGACTATGAAC
SEQ ID NO.16：CTCGTCAACTCCGCAAGATGAGAAGATGGTTTTTTCCAAGGT
SEQ ID NO.17：CTCGTCAACTCCGCAAGGGTAATCTAAACATGCATGAGAAATG
SEQ ID NO.18：CTCGTCAACTCCGCAAGTGTCGTTTCCCGCCTTCAG。
5. use of the primer for detecting transgenic soybean of claim 4 for preparing a multiplex PCR kit for detecting transgenic soybean; the transgenic soybean is GTS-40-3-2, MON87701, MON89788, MON87701 XMON 89788, CV127-9, A2704-12, DP356043, DP305423, A5547, 305423X 40-3-2, MON87769 and MON 87708.
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| CN108866224B (en) * | 2018-05-25 | 2022-03-11 | 苏州市食品检验检测中心 | Multiplex PCR (polymerase chain reaction) kit for detecting thirteen transgenic soybeans and detection method |
| CN108676911B (en) * | 2018-07-18 | 2022-03-01 | 四川华汉三创生物科技有限公司 | Detection kit and method for common transgenic soybean strain |
| CN110373488A (en) * | 2019-06-12 | 2019-10-25 | 中国检验检疫科学研究院 | It is a kind of detect transgene component DNA standard sample and its application |
| CN110343779A (en) * | 2019-07-03 | 2019-10-18 | 中国农业科学院作物科学研究所 | Multiplex PCR detects primer combination, kit and the application of genetically engineered soybean |
| CN111197102B (en) * | 2020-01-10 | 2023-08-11 | 苏州市食品检验检测中心 | Nucleic acid composition for detecting 9 transgenic soybean lines and application thereof |
| CN112280886B (en) * | 2020-11-03 | 2022-04-05 | 大连海关技术中心 | PCR primer composition and method for detecting transgenic soybean strain MON87708 XMON 89788 |
| CN112760413A (en) * | 2021-03-22 | 2021-05-07 | 苏州大学 | Application of public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection |
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