CN114369675B - Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of potatoes - Google Patents
Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of potatoes Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a primer combination, a kit, a detection method and application for detecting transgenic components and transgenic strains of potatoes. The primer pair combination for detecting the common transgenic elements and transgenic lines of potatoes comprises primer groups for amplifying 15 common transgenic elements of potatoes, 2 specific sequences of the transgenic lines and 1 reference gene ST-LS1 (light-induced specific gene) of potatoes. The invention also relates to a kit and a detection method for detecting the transgenic elements and transgenic lines of potatoes. The technical scheme of the invention has simple operation and high detection efficiency; the test object is comprehensive, and has higher detection specificity, accuracy and sensitivity; can be used for large-scale detection of transgenic potatoes and products thereof, and has better application prospect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a primer combination, a kit, a detection method and application for detecting transgenic components and transgenic strains of potatoes.
Background
Potatoes are the fourth largest world food crop next to rice, corn, wheat. Potatoes are one of important crops in the world, have higher economic value and play a role in international agricultural product trade. Currently, more than 40 transgenic potatoes are co-approved for commercial planting worldwide, and the main characters are as follows: disease resistance, insect resistance, quality improvement, etc., the European Union has approved the production and use of transgenic potatoes, some of which are about to apply for transgenic biosafety certificates in China. With the increasing concern of the direct or indirect production of food from a large number of transgenic potatoes and the safety problems of transgenic products by the international society, the detection of transgenic components in agricultural products has been brought into the detection projects of inspection and quarantine departments at home and abroad and gradually enhanced. Therefore, development of efficient and convenient transgenic food detection technology is very important.
The detection technology of the transgenic products mainly comprises a protein-based detection method and a nucleic acid-based detection method. The current PCR detection method based on nucleic acid is still the most common and accurate transgene detection technology at present, and mainly comprises the methods of common qualitative PCR, nested PCR, loop-mediated isothermal amplification (LAMP), fluorescent quantitative PCR multiplex PCR and the like. Compared with the common qualitative PCR method, the nested PCR has higher detection sensitivity and is easy to cause false positive. LAMP is simple to operate and high in specificity, however, primer design is complex, DNA pollution is easy to cause, and subsequent experiments are affected. The fluorescent quantitative PCR method has the advantages of good repeatability, high sensitivity and less nucleic acid cross contamination, but has high cost and needs a special detection instrument. The common multiplex PCR method can detect a plurality of genes simultaneously in one reaction, but the weight is generally not more than 6, otherwise, the interference among primers is larger, and the detection effect is influenced. The gene chip and the digital PCR technology are also common transgenic product detection technologies, have the advantages of high flux, high sensitivity, strong specificity and the like, and can detect a plurality of genes in 1 transgenic crop in parallel or detect a plurality of transgenic crops simultaneously; however, the cost is high, special instruments and equipment are required, operators are required to have high professional quality, and the factors limit the wide application of the technology in detection.
Therefore, developing a high-efficiency, sensitive and high-flux transgenic product detection method becomes a key problem to be solved urgently.
Disclosure of Invention
The invention provides a primer pair combination, a kit and a detection method for surface detection of potato transgenic elements and strains, which are used for solving the technical problems of low flux of a quantitative PCR technology and high transgene detection cost of a gene chip and a digital PCR technology.
According to the technical scheme of the invention, 15 common potato transgenic elements p35S, t35S, pNOS, pFMV35S, tNOS, PAT, bar, NPtII, hpt, cp epsps, E93 and pVYCP, RLRV, pSSuAra, cryIIIa and 3 specific nucleotide sequences of transgenic lines EH92-527-1, RH149N09 and AV43-6-G7, namely target molecules screened by the invention, and an internal reference gene ST-LS1 are taken as detection targets, and nucleotide sequences of multiple PCR amplification primer pairs are designed; 21 pairs of primers were developed which did not affect each other and which allowed efficient amplification by multiplex PCR. The multiplex PCR primer combination can be used for developing potato transgenic elements and strain detection kits.
According to the specific technical scheme, in the first aspect, the application provides a pair combination of primers for detecting potato transgenic elements and strains, namely 19 pairs of primers comprising a number of StGMO1, stGMO2, stGMO3, stGMO4, stGMO5, stGMO6, stGMO7, stGMO8, stGMO9, stGMO10, stGMO11, stGMO12, stGMO13, stGMO14, stGMO15, stGMO16, stGMO17, stGMO18 and StGMO19, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of the pair combination is shown as SEQ ID NO.1-SEQ ID NO. 38.
Also provided is a combination of two pairs of primers, namely StGMO20 and StGMO21, for amplifying the potato reference gene ST-LS1, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of the primers is shown as SEQ ID NO.39-SEQ ID NO. 42.
These primers were used to amplify the specific nucleotide sequences of the following potato transgenic elements p35S, t35S, pNOS, pFMV S, tNOS, PAT, bar, NPtII, hpt, cp epsps, E93, pVYCP, RLRV, pSSuAra, cryIIIa and transgenic lines EH92-527-1, RH149N09, AV43-6-G7, respectively, i.e., the target molecules screened by the present invention, wherein the specific nucleotide sequences of transgenic line RH149N09 were amplified by two pairs of primers StGMO17 and StGMO 18. The specific nucleotide sequences of the above primers and the amplified potato transgenic elements and lines, the numbers of the corresponding primer pairs and the nucleotide sequences of the primer pairs are shown in table 1.
TABLE 1 target molecules selected according to the invention and primer sequences thereof
In the process of primer design, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be normally amplified in one amplification reaction, and the use proves that the sensitivity is high and the applicability is strong.
In another aspect, the application provides a kit for detecting transgenic elements and lines of potatoes, which is characterized by comprising the primer pair combination for detecting transgenic elements and lines of potatoes according to claim 1 and the primer pair combination for amplifying the reference gene ST-LS1 of potatoes according to claim 2.
Preferably, the detection kit further comprises a multiplex PCR premix.
The invention also provides application of the primer pair combination of claim 1 or 2 and the detection kit of claim 3 or 4 in detection of transgenic potatoes and related products.
The invention also provides a method for detecting transgenic elements and strains of potatoes, which is characterized by comprising the following steps of:
1) The potato transgenic element, strain and potato internal reference gene are used for reference to obtain a multiplex PCR primer;
2) Obtaining DNA of the potato to be detected; adding the multiplex PCR primer 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; the gene sequences in the high throughput library were analyzed to effect detection of transgenic elements and lines of potato.
Preferably, the environment/procedure of the amplification reaction of the method comprises: pre-denaturation at 94 ℃ for 15 min; the first amplification step, denaturation at 94℃for 20 seconds, annealing at 65℃to 57℃and extension for 60 seconds, 10 Touch Down cycles, (annealing and extension temperatures for each cycle reduced by 0.8 ℃); the second amplification step was performed by denaturation at 94℃for 20 seconds, annealing at 57℃and extension for 60 seconds, 26 cycles.
Still preferably, the reaction system of the method comprises: 30 μl of the total system, primer pair: 2 μl, 2 Xbuffer: 15ul, multiplex amplification enzyme: 0.5 μl; the rest water is used for supplementing; the high throughput library is qualified at a concentration greater than 2 ng/ul.
In order to realize the detection of the transgenic elements and strains of the potatoes in the samples, when the transgenic elements or strains of the potatoes are selected, detection primers for reference genes of the potatoes are added to realize quantitative detection of the content of transgenic components.
In the process of primer design, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be normally amplified in one amplification reaction, and the use proves that the sensitivity is high and the applicability is strong.
Specifically, when the components of the multiplex PCR premix include the primer sets for amplifying the transgenic potato elements, lines and reference genes, each primer is premixed in a ratio of 1:1, and the mixture of the primers is performed according to different experimental purposes, and in a specific implementation example, the concentration of each primer is 2nM.
In some embodiments, the primer pair number ranges are: the number of pairs 1-22 is appropriately adjusted according to the specific sample to be tested. The later period can be increased periodically according to the newly collected transgenic elements or strains, and the amplification effect is still good after 3000 pairs of primer combinations are tried. To achieve the detection of transgenic potatoes we collected 15 common potato transgenic elements and 3 transgenic lines covering the common transgenic elements of most transgenic potato lines on the market, the log ranges of the multiplex PCR primers were: 1-21 pairs, compared with the conventional 8-pair specific multiplex PCR, have the advantages of high detection flux and sensitivity.
In particular, high throughput sequencing can be second generation sequencing or 3 generation sequencing, and the resulting high throughput library can analyze potato transgene components from multiple dimensions, including but not limited to potato transgene elements or lines in our embodiments.
In some embodiments, the method can be used for detecting all target transgenic components of multiple samples at one time, has the advantages of high flux, high sensitivity, accuracy, rapidness and the like, and can be applied to qualitative and quantitative detection of the transgenic components of transgenic potato strains and products thereof.
The kit provided by the invention can sensitively detect the potato transgenic product with the transgenic content of 0.05% in the sample.
In the reproducibility test of the invention, the reproducibility r=100% of detection results between different libraries and different library-building batches of each sample and the accuracy a=100% are obtained.
The kit provided by the invention detects various potato transgenic lines in a complex template, and has high specificity.
The beneficial effects of the invention are as follows:
1) The method is simple to operate, multiple transgenic components in multiple samples or one sample can be synchronously detected by single-tube PCR amplification, library construction and sequencing through primary sample pretreatment, and the method has the characteristics of parallel analysis and multiple judgment, so that the detection efficiency of transgenic products is greatly improved;
2) The test object is complete, comprises the current common transgenic elements and 2 transgenic strains of the potato, can conveniently add a new detection target sequence, avoids single target amplification failure, and improves the specificity, accuracy and sensitivity of detection;
3) The kit fuses a second generation sequencing platform to sequence the amplified product, so that the flux and the repeatability of a detection system are improved, the detection result can be directly digitized, and the kit is suitable for large-scale detection of transgenic potatoes and products thereof.
Therefore, the invention overcomes the defects of time and labor waste and high cost in the prior art, and the provided kit for detecting the potato transgenic line is simple in operation, quick and sensitive, large in detection flux, good in repeatability of detection results, low in cost for detecting multiple-sample multi-target sequences, and has important application to detecting transgenic products in and out of ports of a seed station and a customs in an agricultural sciences.
The technical scheme of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Drawings
FIG. 1; schematic structural diagram of transgenic material ATBT04-30
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
Example 1 selection of target transgenic elements, lines and design of multiplex PCR amplification primers
S1, screening of target transgenic elements and strains
In the embodiment of the application, the target transgenic component mainly refers to transgenic elements and strains, and potato internal genes used in the application and the embodiment are mainly collected in a common transgenic database, a national standard, an industry standard or the existing literature, so as to ensure the specificity and accuracy of detection. The names of the transgenic elements, lines and internal reference genes screened are shown in Table 1.
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. Specific primer sequences include: SEQ ID NO.1-SEQ ID NO. 42.
Example 2 detection of whether Potato samples contain transgenic Components
1. Experimental materials: the insect-resistant potato variety ATBT04-30, and the structural schematic diagram of the transgenic material ATBT04-30 is shown in figure 1. The experimental materials were transformed into pRBCS4 and p35S, cryIIIa, NPtII, tNOS, tE, the transgene content was 10%, and the experimental materials were used as the research materials.
Preparation of DNA templates: the extraction of plant genome adopts a high-efficiency plant genome DNA extraction kit (DP 350) of CTAB or Tiangen biochemical technology (Beijing) limited company. In this example, three biological replicates were performed for each sample of sample DNA extracted using the root DNA extraction kit.
PCR amplification, library construction and sequencing
Amplifying genomic DNA of the sample using 22 pairs of multiplex PCR amplification primers; connecting the amplified product of each sample with a sequencing joint and a specific sample DNA bar code, and then mixing to form a high-throughput sequencing library; and detecting the high-throughput sequencing library by using a high-throughput sequencing platform and performing quality control on the high-throughput sequencing data. The step is to research and adjust key parameters such as amplification cycle number, sequencing depth and the like according to the requirements of detection accuracy, sensitivity and the like; the step can also be connected with third generation sequencing to realize the complementary advantages between second generation sequencing and third generation sequencing.
4. Determination of results
1) Determining whether the contamination is acceptable based on the signal index S of the transgenic element, line, and the signal index P of the transgenic element, line in the test sample and the blank, wherein:
the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of the transgenic element, line, respectively, in the control.
The signal index s=nt/Nt of the test sample, where, and Nt represent the number of sequenced fragments and total sequenced fragment number of the transgenic element, line, 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 utilizing the DNA bar code of the sample to be tested and homology comparison, wherein the targets comprise transgenic elements, strains and internal reference genes. Absolute quantification of transgenic elements or lines is achieved based on the number of sequenced sequences at each target position. Qualitatively judging that the sample contains transgenic components when the sequencing sequences on the reference gene and the transgenic element are compared to exceed a specified threshold value; when the sample contains the transgenic component, the content of the exogenous gene in the sample is quantitatively determined according to the ratio of the sequence of the transgenic component and the strain to the sequence of the internal reference gene. The calculation formula of the transgene content in this embodiment is shown in (a):
CtestDNA-transgenic content of test sample
tTi-number of sequencing sequences for each transgenic element and line in the test sample
tRi order of sequencing sequence of each internal reference gene fragment detected in the test sample
m-total number of internal Gene fragments detected in test sample
n-total number of transgenic elements and line fragments detected in Standard substance
According to this example we examined 2 samples in total, 1 transgenic line and one negative sample, three biological replicates per sample, the results are shown in table 2 and figure 1: promoters and terminators commonly used in negative samples also detect several sequences in negative potato species, in this example we require that sequences with a number of sequencing reads less than 5 be filtered out. The invention provides that when the signal to noise ratio is greater than 10 times, it can be determined that the contamination in the detection system is acceptable. And when the signal to noise ratio of the transgenic strain in the sample is greater than 10, judging that the nucleic acid of the transgenic strain is detected in the sample. Specifically, each corresponding transgenic element in the positive sample is effectively detected in three repeated experiments in China, and the content is close to that of the transgenic element; from this table it is demonstrated that the potato transgenic kit of our invention can be used to detect transgenic products.
TABLE 2 transgene test results for the test sample of example 2
Example 3 accuracy, specificity and sensitivity assessment
Transgenic potato varieties ATBT04-30 and EH92-527-1 transgenic standards transgenic samples of different mass percentages were prepared to evaluate the accuracy, specificity and sensitivity of the developed techniques. Specifically, the transgene content of each sample was diluted in mass percent, specifically transgenic potatoes ATBT04-30 and EH92-527-1 were diluted with negative potatoes 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 ATBT04-30 and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line EH92-527-1, respectively. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree of coincidence of the average value of multiple determinations with a true value, and is expressed by errors. The specificity is also called true negative rate, and the percentage of true negative detected by multiple detection is the percentage of all negative. Sensitivity refers to the lowest content of transgenic lines that can be detected at 95% confidence, i.e., the lower detection limit. The assay was performed as in example 2, with three biological replicates per sample, and the results are shown in table 3: the kit can stably detect each transgenic element in a sample with the transgenic content of 0.05%, and detects at most 1 transgenic component in a negative sample, which indicates that the kit has strong specificity, can obviously distinguish the sample with the transgenic content of 0.05% from the negative sample, and has technical stability and detection sensitivity with the transgenic content of 0.05%.
TABLE 3 evaluation of accuracy and sensitivity of the methods of the 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%.
Example 4 application of our inventive method to practical detection of samples
In order to verify the accuracy of the invention and the role in transgene detection of batch samples, a laboratory selects 101 potato leaf samples of unknown genotypes of a company for detection, adopts the detection method of the embodiment 2, compares the detection result with the preservation type of the company, and counts the consistency of the result. The analysis result shows that in 101 test samples, the results of only 1 sample are inconsistent, and the consistency of the detection results is as high as 99.0%, so that the accuracy of the method disclosed by the invention is better demonstrated.
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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<213> Artificial sequence ()
<400> 15
gatggattgc acgcaggttc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 16
ttcagtgaca acgtcgagca 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 17
ctcggagggc gaagaatctc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 18
gcgggagatg caataggtca 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 19
aggacgtcat caatacgggc 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 20
atcgatgatc caggtgtcgc 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 21
cattgcgcac acaccagaat 20
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 22
agaggccacg atttgacaca t 21
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 23
ccaaaagcaa gggagcaacc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 24
tgacatttgg cgaggttcca 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 25
ttcaacttcc gaggcacctc 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 26
tgtccccaac cgtttgacaa 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 27
cacgtggcat tattccagcg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 28
ttggagtgat cggagggtct 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 29
cttcaactac tggagcggca 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 30
gtacaccttc tcgccgttga 20
<210> 31
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 31
gtgtcaaaac acaatttaca gca 23
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 32
tcccttaatt ctccgctcat ga 22
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 33
agttgtcgag ggtcctatcc 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 34
tgagctatga gaaagcgcca 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 35
gcgtcagacc ccgtagaaaa 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 36
ccacgtccag taacgcgtta 20
<210> 37
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 37
gtggtcaatg aaagcgctgg 20
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 38
cctggggtgc ctaatgagtg 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 39
agatccattg ataggccgcg 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 40
agtccactcg tgcaaggaag 20
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 41
cttccttgca cgagtggact 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 42
caccccttct gtggattgct 20
Claims (8)
1. A primer composition for detecting potato transgenic elements and transgenic lines is characterized by comprising 19 pairs of primers numbered StGMO1, stGMO2, stGMO3, stGMO4, stGMO5, stGMO6, stGMO7, stGMO8, stGMO9, stGMO10, stGMO11, stGMO12, stGMO13, stGMO14, stGMO15, stGMO16, stGMO17, stGMO18 and StGMO19, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of the specific primer composition is shown in SEQ ID No.1-SEQ ID No. 38.
2. The primer composition of claim 1, further comprising two pairs of primers numbered StGMO21 and StGMO22 for amplifying the potato reference gene ST-LS1, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of each pair of primers is shown as SEQ ID NO.39-SEQ ID NO. 42.
3. A kit for detecting a transgenic element and a transgenic line of potato, comprising the primer composition for detecting a transgenic element and a transgenic line of potato of claim 1 and two pairs of primers for amplifying the reference gene ST-LS1 of potato of claim 2.
4. The test kit of claim 3, further comprising a multiplex PCR premix.
5. Use of the primer composition according to claim 1 or 2, the detection kit according to claim 3 or 4 for detecting transgenic potatoes and related products.
6. A method of detecting a transgenic line of potato, comprising the steps of:
1) The method comprises the steps of (1) carrying out reference by using a potato transgenic element, a characteristic nucleotide sequence of a transgenic strain and a potato reference gene to obtain a multiplex PCR primer composition, wherein the multiplex PCR primer composition is shown as SEQ ID NO.1-SEQ ID NO. 42;
2) Obtaining DNA of the potato to be detected; adding the multiplex PCR primer composition into a reaction system by taking the DNA as a template, and performing amplification reaction to obtain an amplification product; carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library; the gene sequences in the high-throughput library are analyzed to realize detection of common transgenic elements and transgenic lines of potatoes.
7. The method of claim 6, wherein the environment/procedure of the amplification reaction comprises: pre-denaturation at 94 ℃ for 5 min; the first step of amplification reaction, denaturation at 94 ℃ for 15s, annealing at 62-56 ℃ for 30s,12 Touch Down cycles, and the temperature of annealing and extension in each cycle is reduced by 0.5 ℃; the second amplification step was 15s denatured at 94℃and 30S annealed at 57℃for 22 cycles.
8. The method of claim 7, wherein the reaction system comprises: 40. Mu.l of the total system, 2.5. Mu.l of primer premix, 2 Xbuffer: 20 μl, multiplex PCR amplification enzyme: 0.5 μl; the rest is supplemented with water; the rest water is used for supplementing; the high throughput library is qualified at a concentration greater than 2 ng/ul.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420182A (en) * | 2002-10-11 | 2003-05-28 | 覃文 | Probe sequence and kit for real time fluorescence PCR detection of transgenic potato |
| CN101646782A (en) * | 2007-01-29 | 2010-02-10 | 科学公共卫生研究所（Iph） | The transgenic plant event detection |
| NZ595357A (en) * | 2007-01-29 | 2012-04-27 | Scient Inst Of Public Health Iph | A device to detect the presence or absence of events in a sample using prime numbers |
| AU2013201571B2 (en) * | 2007-01-29 | 2015-06-18 | Sciensano | Transgenic plant event detection |
| EP1950311A1 (en) * | 2007-01-29 | 2008-07-30 | Scientific Institute of Public Health (IPH) | Transgenic plant event detection |
| CN104328188B (en) * | 2014-11-05 | 2016-09-28 | 中国农业科学院生物技术研究所 | A kind of positive criteria plasmid for transgenic Rhizoma Solani tuber osi detection |
| CN105349693B (en) * | 2015-12-18 | 2018-08-14 | 山东出入境检验检疫局检验检疫技术中心 | Primer, probe and the method for transgenic potato AV43-6-G7 ore grade indexes |
| CN105586413A (en) * | 2016-01-29 | 2016-05-18 | 江汉大学 | Detection method of transgenic components in potatoes |
| CN107475431A (en) * | 2017-09-29 | 2017-12-15 | 山东世通检测评价技术服务有限公司 | A kind of method for differentiating transgenic potato |
| CN109680098A (en) * | 2019-03-07 | 2019-04-26 | 广东出入境检验检疫局检验检疫技术中心 | A kind of kit and its application based on droplet digital pcr quantitative detection potato ingredient |
| CN110004244A (en) * | 2019-03-18 | 2019-07-12 | 中国检验检疫科学研究院 | Marker group, composition and the application of comprehensive screening transgene component |
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