CN114369678B - Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of beet - Google Patents
Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of beet 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 beet. The primer pair combination for detecting the common transgenic elements and transgenic lines of the beet comprises primer groups for amplifying 13 common beet transgenic elements, 2 specific sequences of the transgenic lines and 1 beet internal reference gene Bv_GluA3 (glutamine synthetase leaf isozyme gene). The invention also relates to a kit and a detection method for detecting the beet transgenic element and the transgenic strain. 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 beet and products thereof, and has better application prospect.
Description
Technical Field
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 beet.
Background
Beet is an important commercial crop, and more than 95% of the beet planted in the united states and canada are transgenic beet. The transgenic beet lines currently on the market have three total of GTSB77, T120-7 and H7-1, wherein only H7-1 is approved by the Ministry of agriculture for sugar, beet pulp and sugar beet pulp
The main characters of the processing raw materials are as follows: disease resistance, insect resistance, quality improvement, and the like, wherein only H7-1 is approved by the agricultural department of China for sugar, beet pulp and processing raw materials. With the increasing concern of the direct or indirect production of large amounts of transgenic sugar beets into food products 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 strengthened. 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 beet transgenic elements and strains, which are used for solving the technical problems of low flux based on quantitative PCR technology and high transgene detection cost based on a gene chip and a digital PCR technology.
According to the technical scheme of the surface detection beet transgenic element and strain, 13 commonly used beet transgenic elements p35S, t35S, pFMV35S, pNOS, tNOS, tOCS, GUS, GOX, PAT, bar, NPtII, E93, cp4epsps and 2 specific nucleotide sequences of transgenic strains H7-1 and GTSB77, namely target molecules screened by the invention, and an internal reference gene BV_GLUA3 are taken as detection targets, and nucleotide sequences of multiple PCR amplification primer pairs are designed; 17 pairs of primers were developed which did not affect each other and which can be amplified efficiently by multiplex PCR. The multiplex PCR primer combination can be used for developing beet transgenic elements and strain detection kits.
In a specific technical scheme, in a first aspect, the application provides a transgenic element and strain primer pair combination for detecting beet, wherein the primer pair combination comprises 15 pairs of primers with the numbers of BvGMO1, bvGMO2, bvGMO3, bvGMO4, bvGMO5, bvGMO6, bvGMO7, bvGMO8, bvGMO9, bvGMO10, bvGMO11, bvGMO12, bvGMO13, bvGMO14 and BvGMO15, each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequence of the specific primer pair combination is shown in SEQ ID NO.1-SEQ ID NO. 30.
Also provided is a combination of two pairs of primers BvGMO16 and BvGMO17 for amplifying the beet internal reference gene BV_GLUA3, 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.31-SEQ ID NO. 34.
These primers were used to amplify specific nucleotide sequences of the following beet transgenic elements p35S, t35S, pFMV35S, pNOS, tNOS, tOCS, GUS, GOX, PAT, bar, NPtII, E93, cp4epsps and 2 transgenic lines H7-1, GTSB77, respectively, i.e., the target molecules screened by the present invention. The specific nucleotide sequences of the primers and the beet transgenic elements and strains amplified by the primers, 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 beet transgenic elements and lines, which is characterized in that the kit comprises the primer pair combination for detecting beet transgenic elements and lines according to claim 1 and the primer pair combination for amplifying beet reference genes BV_GLUA3 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 detecting transgenic beet seeds and related products.
The invention also provides a method for detecting transgenic elements and strains of beet, which is characterized by comprising the following steps:
1) The beet transgenic element, strain and beet reference gene are used for reference to obtain a multiplex PCR primer;
2) Obtaining DNA of beet 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; and analyzing the gene sequences in the high-throughput library to realize the detection of transgenic elements and strains of beet.
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 purpose of detecting the transgenic elements or strains of the beet in the sample, when the transgenic elements or strains of the beet are selected, detection primers for the internal reference genes of the beet are added to realize the 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 beet element, the strain and the internal reference gene, each primer is premixed according to a ratio of 1:1, and the mixture of the primers is carried out 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 1-17 is appropriately adjusted according to the specific detection sample. 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 beets we collected 13 common beet transgenic elements and 2 transgenic lines covering the common transgenic elements of most transgenic beet lines on the market, the logarithmic range of the multiplex PCR primers was: 1-17 pairs, compared with the conventional 8-pair specific multiplex PCR, have the advantages of high detection flux and sensitivity.
In particular, the high throughput sequencing may be second generation sequencing or 3 generation sequencing, and the resulting high throughput library may analyze the transgenic components of sugar beets from multiple dimensions, including but not limited to sugar beet transgenic 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 beet lines and products thereof.
The kit provided by the invention can sensitively detect the beet 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 a plurality of beet transgenic strains 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 whole test object comprises the current common transgenic elements and 2 transgenic strains of beet, and a new detection target sequence can be conveniently added, so that single target amplification failure is avoided, and the specificity, accuracy and sensitivity of detection are improved;
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 beet and products thereof.
Therefore, the invention overcomes the defects of time and labor waste and high cost in the prior art, and the provided beet transgenic line detection kit is simple in operation, quick and sensitive, large in detection flux, good in repeatability of detection results, low in cost of multi-sample multi-target sequence detection, and has important application to detection of transgenic products in and out of ports of a seed station and a customs.
The technical scheme of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Drawings
FIG. 1; structural schematic of transgenic beet T120-7
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 sugar beet internal genes used in the embodiment and the transgenic elements and strains are mainly collected in a common transgenic database, a national standard, an industry standard or the existing literature, so that the specificity and accuracy of detection of people are ensured. 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. 44.
Example 2 detection of sugar beet samples for transgenic Components
1. Experimental materials: the structural schematic diagram of the herbicide-resistant beet variety T120-7 and the transgenic material T120-7 is shown in figure 1. The experimental material was transformed into p35S, t35S, PAT, NPtII, tNOS, tOCS, the transgene content was 10%, and was used as our study material.
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: the promoters and terminators commonly used in negative samples also detect several sequences in negative sugar beet species, in this case 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 our invented beet transgene kit 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 beet variety H7-1 and GTSB77 transgenic standards transgenic samples of different mass percentages were prepared to evaluate the accuracy, specificity and sensitivity of the developed technology. Specifically, the transgene content of each sample was diluted in mass percent, specifically transgenic beet H7-1 and GTSB77 were diluted with negative beet 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 H7-1 and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line GTSB77, 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 does not detect any transgenic component in a negative sample, so 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 351 sugar beet 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 351 test samples, only 2 samples are inconsistent, and the consistency of the detection result is as high as 99.43%, 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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Claims (5)
1. A multiplex PCR primer pair combination for detecting beet transgenic elements and transgenic lines is characterized in that the primer pair combination comprises 15 pairs of primers with the numbers of BvGMO1, bvGMO2, bvGMO3, bvGMO4, bvGMO5, bvGMO6, bvGMO7, bvGMO8, bvGMO9, bvGMO10, bvGMO11, bvGMO12, bvGMO13, bvGMO14 and BvGMO15, each pair of primers consists of a forward primer and a reverse primer, the specific primer pair combination nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO.30, and the beet transgenic lines are H7-1 and GTSB77.
2. The primer pair combination according to claim 1, further comprising two primer pair combinations numbered BvGMO16 and BvGMO17 for amplifying beet internal reference gene bv_glua3, wherein each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequences of the primers are shown in SEQ ID No.31-SEQ ID No. 34.
3. A kit for detecting a transgenic sugar beet element and transgenic line, comprising the primer pair combination for detecting a transgenic sugar beet element and transgenic line of claim 1 and the primer pair combination for amplifying sugar beet reference gene bv_glua3 of claim 2, wherein the transgenic sugar beet line is H7-1 and GTSB77.
4. The test kit of claim 3, further comprising a multiplex PCR premix.
5. Use of the primer pair combination of claim 1 or 2, the detection kit of claim 3 or 4 for detecting transgenic beet varieties, which are H7-1 and GTSB77, and related products.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103205499A (en) * | 2013-04-11 | 2013-07-17 | 中华人民共和国上海出入境检验检疫局 | Loop-mediated isothermal amplification (LAMP) on-site rapid detection method for transgenic beet H7-1 |
| CN107164514A (en) * | 2017-06-22 | 2017-09-15 | 中华人民共和国黄埔出入境检验检疫局 | Transgenic beet GTSB77 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103205499A (en) * | 2013-04-11 | 2013-07-17 | 中华人民共和国上海出入境检验检疫局 | Loop-mediated isothermal amplification (LAMP) on-site rapid detection method for transgenic beet H7-1 |
| CN107164514A (en) * | 2017-06-22 | 2017-09-15 | 中华人民共和国黄埔出入境检验检疫局 | Transgenic beet GTSB77 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
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