Primer combination, kit, detection method and application for detecting transgenic components of tobaccoTechnical 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 of tobacco.
Background
Tobacco (Nicotiana tabacum l.) belongs to the family solanaceae, genus nicotiana. Some islands originating in the oceangoing, america and the south pacific. Tobacco is an important commercial crop, and is also the first sharp industry in countries around the world, and the planting area is very large. How to improve the quality and yield of tobacco and stress resistance is a matter of concern to researchers. The above problems can be ameliorated by obtaining tobacco plants and their progeny by transgenic techniques, since tobacco is readily tissue-cultured and transformed. However, the safety problem of transgenic products has been a major concern. Therefore, it is important to develop efficient and convenient transgene detection technology.
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 reproducibility, 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 number of the genes is not more than six, otherwise, the interference among primers is large, and the detection effect is influenced. The gene chip and the digital PCR technology are also common transgenic product detection technologies, and the method has 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 technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
the nucleotide sequence of the common tobacco transgene product detection element, namely the target molecule and the internal reference gene, are screened as detection targets. Including 15 transgenic elements commonly used for detection: p35S, t35S, pFMV35S, tNOS, PAT, bar, NPtII, hpt, CP epsps, E93, TMV-CP, pVY_CP, CMV_CP, cry1Ab and GUS, and sequence NT_UBI comprising the 1 tobacco reference gene.
Next, the present invention developed multiplex PCR primer compositions for detecting the transgenic elements and tobacco reference genes, wherein 15 pairs for 15 transgenic elements and 1 pair for 1 reference gene. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR. The multiplex PCR primer composition can be used for developing a transgenic element detection kit.
In a specific technical scheme, in a first aspect, the application provides a primer pair combination for detecting transgenic components of tobacco, wherein the primer pair combination comprises 15 pairs of primers with the numbers of NtGMO1, ntGMO2, ntGMO3, ntGMO4, ntGMO5, ntGMO6, ntGMO7, ntGMO8, ntGMO9, ntGMO10, ntGMO11, ntGMO12, ntGMO13, ntGMO14 and NtGMO15, and each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequence of the specific primer pair combination is shown as SEQ ID No.1-SEQ ID No. 30.
Also provided is a primer pair combination with the number of NtGMO16 for amplifying the tobacco reference gene NT_UBI, and the nucleotide sequence of the primer pair combination is shown as SEQ ID NO.31-SEQ ID NO. 32.
These above primers were used to amplify the following tobacco transgenic elements, respectively: specific correspondence of the above primers to the tobacco transgene element nucleotide sequences amplified by the primers, i.e., the numbers of the target molecules and corresponding primer pairs and the nucleotide sequences of the primers, are shown in Table 1, p35S, t35S, pFMV S, tNOS, PAT, bar, NPtII, hpt, CP epsps, E93, TMV-CP, pVY_CP, CMV_CP, cry1Ab and GUS.
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 primers have high sensitivity and strong applicability.
In another aspect, the application provides a kit for detecting a tobacco transgenic component, which is characterized in that the kit comprises the primer pair combination for detecting the tobacco transgenic component according to claim 1 and the primer pair combination for amplifying the tobacco internal reference gene NT_UBI according to claim 2.
Preferably, the detection kit further comprises a multiplex PCR premix.
Also provided is the use of the primer pair combination of claim 1 or 2 and the detection kit of claim 3 or 4 for detecting transgenic tobacco seeds and related products.
Also provided is a method of detecting a transgenic component of tobacco, the method comprising the steps of:
1) The tobacco transgenic element and the tobacco internal reference gene are used for reference to obtain a multiplex PCR primer;
2) Obtaining DNA of tobacco to be tested; adding the multiplex PCR primer into a reaction system by taking the DNA as a template, and performing an amplification reaction to obtain an amplification product; carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library; the gene sequences in the high throughput library are analyzed to effect detection of tobacco transgene components.
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 concentration of the high-throughput library is greater than 2 ng/ul.
In order to realize the purpose of detecting the transgenic components of the tobacco in the sample, when the transgenic components of the tobacco are selected, a detection primer for the internal reference genes of the tobacco is added, and the content of the transgenic components is quantitatively detected.
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 primers have high sensitivity and strong applicability.
Specifically, when the components of the multiplex PCR premix include the combination of the transgene element of the tobacco and each primer group of the reference gene, each primer is premixed according to a ratio of 1:1, and the mixture of each primer 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 1-16 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, 3000 pairs of primer combinations are tried, and the amplification effect is still good. To achieve detection of transgenic components in tobacco, we collected 16 pairs of commonly used tobacco transgenic elements, the log ranges of the multiplex PCR primers were: 1-16 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 third generation sequencing, and the resulting high throughput library can analyze the components of the transgene from multiple dimensions, including but not limited to transgenic elements in our embodiment.
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 tobacco and products thereof.
The kit provided by the invention can sensitively detect the transgenic component with the 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 has high specificity for detecting various transgenic components in complex templates.
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 the transgenic products is greatly improved;
2) The test object is complete, contains the transgene element sequence and the transgene strain which are common at present in tobacco, can conveniently add a new detection sequence, avoids single target amplification failure, and improves the specificity, accuracy and sensitivity of detection;
3) The kit fuses with a second generation sequencing platform to sequence the amplified product, so that the detection flux and repeatability of the system are improved, the detection result can be directly digitized, and the kit is suitable for large-scale detection of transgenic tobacco and products thereof. Therefore, the invention overcomes the defects of time and labor waste and high cost in the prior art, and the provided tobacco transgene detection kit is simple to operate, quick and sensitive, large in detection flux, good in detection result repeatability and low in detection cost of multiple-sample multi-target sequences, and has important application to the detection of transgenic products in seed stations, agricultural institutions and customs access ports.
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 diagram of transgenic tobacco
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. The drawings illustrate preferred embodiments of the invention. 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 screening of target transgenic Components and design of multiplex PCR amplification primers
S1, screening of target transgenic components
In the embodiment of the application, the target transgene components are mainly transgene elements and internal reference genes, and are comprehensively collected from a common transgene database, a national standard, an industry standard or existing literature as much as possible so as to ensure the specificity and the accuracy of detection. Wherein the transgenic elements and reference genes selected are as described in Table 1 above:
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 non-target sequences, 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. 32.
Example 2 detection of whether tobacco samples contain transgenic Components
1. Experimental materials: over-expressing tobacco OE-GUS-7, OE-CBF1-3 transgenic material was stored in this experiment.
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 30 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 the step of the third generation sequencing related task so as to realize the complementary advantages between the second generation sequencing and the third generation sequencing.
4. Determination of results
1) Determining whether the contamination is acceptable based on the signal index S of the transgene component in the test sample and the signal index P of the transgene component in the blank, wherein: the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total number of sequenced fragments of the transgenic component, respectively, in the control. The signal index s=nt/Nt of the test sample, where, and Nt represent the number of sequenced fragments of the transgenic component and the total number of sequenced fragments, respectively, in the test sample. Signal to noise ratio = S/P
2) Determination of transgene outcome
And (3) distributing each sequencing fragment to each target position of each target species by utilizing the DNA bar code of the sample to be tested and homology comparison, wherein the targets comprise transgenic elements and internal reference genes. Absolute quantification of the transgene component is achieved based on the number of sequenced sequences at each target location. 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 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 sequenced sequences of each transgenic element in test sample
tRi number of sequenced sequences of each reference gene fragment detected in the test sample
m-total number of internal Gene fragments detected in test sample
n-total number of transgene element fragments detected in standard
According to this example we detected a total of 2 samples, each with three biological replicates, the results are shown in table 2 and figure 1: promoters and terminators commonly used in negative samples also detect several sequences in negative tobacco species, i have in this example required that sequences with a number of sequencing reads of 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 component in the sample is greater than 10, judging the nucleic acid of the detected transgenic component in the sample. Specifically, all transgenic elements in OE-GUS-7 samples were efficiently detected in three replicates and were present at approximately 10%; from this table it is demonstrated that the tobacco transgene 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
Herbicide resistant tobacco varieties OE-GUS-7 and OE-CBF1-3 transgenic standards transgenic samples of different mass percentages were prepared to evaluate the accuracy and sensitivity of the developed technology. Specifically, the transgene content of each sample was diluted in mass percent, specifically, transgenic tobacco OE-GUS-7 and OE-CBF1-3 were diluted with negative tobacco 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) and OE-CBF1-3 (B1, B2, B3, B4, B5, B6) of transgenic line RT-73, 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 amount of transgene component 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 can detect 1 transgenic component at most 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
To verify the accuracy of the invention and the role in the transgene testing of bulk samples, the laboratory selected 108 tobacco leaf samples of unknown genotypes from a company for testing, tested according to the method of example 2, and compared the test results with the preservation type of the company, and the consistency of the statistical results. The analysis result shows that in 108 test samples, only 2 samples are inconsistent in result, and the consistency of the detection result is up to 98.1%, so that the accuracy and the good application prospect of the method are better proved.
It should be noted that in this document, relational terms such as "first" and "second" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
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