CN114657275B - Primer pair combination, kit and detection method for detecting transgenic alfalfa - Google Patents
Primer pair combination, kit and detection method for detecting transgenic alfalfa Download PDFInfo
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Abstract
The application relates to the field of biotechnology, in particular to a primer pair combination, a kit and a detection method for detecting transgenic alfalfa. The nucleotide sequence of the primer pair combination is shown in SEQ ID NO.1 to SEQ ID NO. 18; through the primer combination, the method is simple to operate, multiple transgenic components in multiple samples or one sample can be synchronously detected through one-time sample pretreatment, single-tube PCR amplification, library construction and sequencing, and the method has the characteristics of parallel analysis and multiple judgment, so that the detection efficiency of transgenic products is greatly improved, and the detection cost is saved.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a primer pair combination, a kit and a detection method for detecting transgenic alfalfa.
Background
Alfalfa is a perennial pasture of leguminous family, and has the characteristics of high nutrition, high yield, strong adaptability and the like. Simultaneously, alfalfa is a high protein, and has important functions in agriculture and animal husbandry production due to the advantages of high protein content, rich mineral elements, vitamins and carbohydrates, capability of carrying out biological nitrogen fixation and the like, and has high economic and ecological values.
The traditional cultivation method has the limitations of long time, low yield and high cost, and cannot meet the requirement of the current society on breeding diversification. Along with the continuous development of transgenic technology, the plant transgenic technology has extremely high application value in genetic improvement of alfalfa, fundamentally increases the breeding process and improves the production yield and quality. However, transgenic alfalfa is directly or indirectly manufactured into food, and the safety problem of transgenic products is increasingly concerned by the international society, and the detection of transgenic components in agricultural products is brought into the detection projects of inspection and quarantine departments at home and abroad and is 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 application provides a primer pair combination, a kit and a detection method for detecting transgenic alfalfa, which are used for solving the technical problems of low flux and high detection cost of detecting the transgenic alfalfa based on a quantitative PCR technology.
In a first aspect, the present application provides a primer pair combination for detecting transgenic alfalfa, the primer pair combination comprising:
a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying pFMV35S, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PAT, the nucleotide sequence of which is shown in SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying bar, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.15 to SEQ ID NO. 16;
and/or a primer pair for specifically amplifying cp4epsps, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.17 to SEQ ID NO. 18.
Optionally, the primer pair combination further comprises a primer pair that specifically amplifies an alfalfa transgenic line selected from the group consisting of: ms_j163 and/or ms_j101.
Optionally, the primer pair combination further includes:
a primer pair for specifically amplifying Ms_J163, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;
and/or a primer pair for specifically amplifying Ms_J101, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22.
Optionally, the nucleotide sequence of the primer pair for specifically amplifying Ms_Acc is shown as SEQ ID NO.23 to SEQ ID NO. 24.
Optionally, the primer pair combination further comprises a primer pair that specifically amplifies an alfalfa transgenic element selected from the group consisting of: p35S, t35S, pNOS, tNOS, pFMV S, PAT, bar, NPtII and cp4epsps.
In a second aspect, the present application provides a kit for detecting a primer pair combination of transgenic alfalfa, the kit comprising: the primer pair combination of the first aspect.
Optionally, the kit further comprises a multiplex PCR premix.
Optionally, the kit further comprises a first container, wherein the first container contains the primer pair combination.
In a third aspect, the present application provides a method for detecting transgenic alfalfa using the primer pair combination of the first aspect, the method comprising the steps of:
obtaining the DNA of alfalfa to be detected and the primer pair combination;
taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing amplification reaction to obtain an amplification product;
carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;
and analyzing the gene sequence in the high-flux library to obtain a result of detecting the transgenic alfalfa.
In a fourth aspect, the use of a alfalfa primer pair combination for the preparation and detection of alfalfa transgenic ingredients and transgenic lines;
the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 24.
Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:
the primer pair combination has a nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO. 18; through the primer combination, the method is simple to operate, multiple transgenic components in multiple samples or one sample can be synchronously detected through one-time sample pretreatment, single-tube PCR amplification, library construction and sequencing, and the method has the characteristics of parallel analysis and multiple judgment, so that the detection efficiency of transgenic products is greatly improved, and the detection cost is saved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
Fig. 1 is a schematic flow chart of a detection method of a kit for comprehensively detecting transgenic alfalfa according to an embodiment of the present application.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present application based on the embodiments herein.
There are many factors that affect multiplex PCR reactions, such as: the specificity of the primer, if the primer has stronger binding force with other non-target gene fragments in the system, the capability of the target gene to bind the primer is competed, so that the amplification efficiency is reduced; through the primer pair combination designed by the application, the detection efficiency and the accuracy of the amplified product obtained by the PCR reaction can be effectively improved.
In a first aspect, the present application provides a primer pair combination for detecting transgenic alfalfa, the primer pair combination comprising:
a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying pFMV35S, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown in SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PAT, the nucleotide sequence of which is shown in SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying bar, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.15 to SEQ ID NO. 16;
and/or a primer pair for specifically amplifying cp4epsps, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.17 to SEQ ID NO. 18.
The length of the primer is between 18 and 30bp, and the primers are not interfered with each other; the logarithmic range of multiplex PCR primers includes, but is not limited to: 1-12 pairs, preferably 9-12 pairs, wherein the 1-12 pairs are suitably adjusted according to the specific sample to be tested. To achieve detection of transgenic alfalfa, 9 common alfalfa transgenic elements and 2 transgenic lines were collected, covering the common transgenic elements for most transgenic alfalfa lines on the market, the log ranges of the multiplex PCR primers were: 1-12 pairs, compared with the conventional 8-pair specific multiplex PCR, have the advantages of high detection flux and sensitivity.
In some embodiments, the primer pair combination further comprises a primer pair that specifically amplifies a reference within an alfalfa transgenic line selected from the group consisting of: ms_j163 and/or ms_j101.
The PCR amplification primer pair is designed for the target alfalfa transgenic strain or transgenic element, and the multi-angle information of the genes to be detected can be rapidly and comprehensively obtained through high-throughput sequencing analysis, so that time and labor are saved; in order to realize that multiple groups of primers can be added simultaneously in one experimental flow, instead of multiple independent parallel experiments, the amplified products of multiple PCR primers can be subjected to high-throughput sequencing, so that the aims of saving labor and cost can be achieved.
In addition, the amplification product of the primer combination can be subjected to one-time high-throughput sequencing and analysis to obtain a plurality of detection results, wherein the detection results comprise transgenic components, judging whether a sample to be detected contains target molecules or not, and determining the copy numbers of reference genes and target molecules in the sample to be detected so as to determine the content of exogenous genes; the method has the advantages that the traditional Real-time PCR technology is avoided to realize only one purpose at a time, multiple times of amplification and detection are needed to cover multiple target transgenic components in a sample, and when the primer is used for detection, multiple genes in transgenic crops or detection results of multiple transgenic crops can be obtained through one-time high-throughput sequencing and analysis, so that the detection efficiency and sensitivity are greatly improved, the cost is saved, and the application and popularization are facilitated.
In some embodiments, the primer pair combination is:
a primer pair for specifically amplifying Ms_J163, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;
and/or a primer pair for specifically amplifying Ms_J101, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22.
In summary, in order to enhance the applicability and sensitivity of the primer pair, the designed primers have the length of 18-30bp, the primers do not interfere 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 primer pair has high sensitivity and strong applicability; meanwhile, the PCR amplification primer pair group consists of a forward primer and a reverse primer, wherein one primer is an upstream primer and the other primer is a downstream primer.
In some embodiments, the primer pair specifically amplifying ms_acc has a nucleotide sequence as set forth in SEQ ID No.23 to SEQ ID No. 24.
In order to achieve the purpose of detecting the transgenic components and transgenic strains of alfalfa, alfalfa internal reference genes are selected, and the effect of quantifying the content of the transgenic components in the sample can be achieved.
Preferably, the primer pair combination comprises primer pairs for amplifying 9 common alfalfa transgenic elements, 2 transgenic line-specific sequences, and 1 alfalfa internal reference gene ms_acc.
In some embodiments, the primer pair combination further comprises a primer pair that specifically amplifies an alfalfa transgenic element selected from the group consisting of: p35S, t35S, pNOS, tNOS, pFMV S, PAT, bar, NPtII and cp4epsps.
Based on the existing primer pair combination, the primer combination can also comprise other primer pair primer combinations for specifically amplifying the same alfalfa transgenic elements, and can also comprise a logarithmic group for periodically increasing multiple PCR primers according to the newly collected transgenic elements in the later period, and the amplification effect is still good as proved by 3000 pairs.
The specific nucleotide sequences of the primers and the alfalfa transgenic elements and lines amplified by the primers, the numbers of the corresponding primer pairs and the nucleotide sequences of the primer pairs are shown in table 1.
In a second aspect, the present application provides a kit for detecting a primer pair combination of transgenic alfalfa, the kit comprising: the primer pair combination of the first aspect.
In some embodiments, the kit further comprises a multiplex PCR premix.
Specifically, the components of the multiplex PCR premix include the transgene element of the rape and each primer combination of the internal reference gene. A kind of electronic device. In combination, each primer was premixed in a ratio of 1:1, and each primer was mixed for different experimental purposes, and in the specific embodiment, each primer was 2 nM.
In some embodiments, the kit further comprises a first container containing the primer pair combination therein.
In a third aspect, the present application provides a method for detecting a primer pair combination according to the first aspect, as shown in fig. 1, the method comprising the steps of:
s1, obtaining DNA of alfalfa to be detected and the primer pair combination;
s2, taking the DNA as a template, combining and adding the primer pair into a reaction system, and performing an amplification reaction to obtain an amplification product;
s3, carrying out high-flux sequencing on the amplification product to obtain a high-flux library;
s4, analyzing the gene sequences in the high-throughput library to obtain a result of detecting the transgenic alfalfa.
In a fourth aspect, the use of a alfalfa primer pair combination for the preparation and detection of alfalfa transgenic ingredients and transgenic lines;
the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 24.
In particular, high throughput sequencing can be second generation sequencing or 3 generation sequencing, and the resulting high throughput library can analyze the composition of the transgene from multiple dimensions, including but not limited to transgenic elements in the embodiments. The method is applied to qualitative and quantitative detection of transgenic components and transgenic lines of alfalfa and products thereof.
By using the method, all transgenic components and transgenic strains of multiple samples can be detected at one time, and the method has the advantages of high flux, high sensitivity, accuracy, rapidness and the like, and can be applied to qualitative and quantitative detection of transgenic components of alfalfa and products thereof.
In the method of the embodiment of the present application, the amplification reaction comprises a general PCR amplification reaction or a real-time fluorescent PCR amplification reaction; the environment/procedure of the amplification reaction includes: the environment/procedure of the amplification reaction includes: pre-denaturation at 94 ℃ for 5 min; the first amplification reaction, denaturation at 94℃for 15s, annealing at 62℃to 56℃for 30s,12 Touch Down cycles, (0.5℃decrease in temperature for each cycle of annealing and extension); the second amplification step was 15s denatured at 94℃and 30S annealed at 57℃for 22 cycles.
In addition, the reaction system includes, but is not limited to: 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 remainder was replenished with water.
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 remainder was replenished with water.
In some embodiments, the high throughput library is at a concentration of ≡2ng/ul.
The concentration of the high-flux library is controlled, the systematic deviation caused by cloning is avoided, the experimental operation is simplified, and the sequencing efficiency is improved.
The PCR amplification primer pair group is used for preparing a detection kit, and the detection kit is used for detecting transgenic alfalfa and transgenic lines;
the primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 26.
The kit provided by the invention can sensitively detect the alfalfa 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=98.7%.
The kit provided by the invention detects various alfalfa transgenic strains in complex templates and has high specificity.
In conclusion, the defects of time and labor waste and high cost in the prior art are overcome, the alfalfa transgenic line detection kit is simple to operate, quick and sensitive, large in detection flux, good in repeatability of detection results, low in cost of multi-sample multi-target sequence detection, and important in application to detection of transgenic products on entry and exit ports of seed stations, academy of agricultural science and customs.
The method of the present invention will be described in detail with reference to examples, comparative examples and experimental data.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
screening common alfalfa transgenic product detection elements and internal reference genes as detection targets. Including 13 commonly used transgene elements for detection: p35S, t35S, pNOS, tNOS, tPIN II, pZmUBI, pRBCS4, tE9, bar, PAT, HPT, GUS, cry1Ab-Ac; sequence of alfalfa internal reference genes: SPS.
Next, the present invention developed multiplex PCR primer compositions for detecting the transgenic elements and alfalfa internal genes, wherein 13 pairs for 13 transgenic elements and 2 pairs for two internal genes. 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.
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=98.7%.
The kit has high specificity for detecting various transgenic components in complex templates.
A kit for the comprehensive detection of transgenic alfalfa components and its application of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 selection of target transgenic elements, lines and design of multiplex PCR amplification primers
In the embodiment of the application, the target transgenic component mainly refers to transgenic elements and strains, and alfalfa internal genes used in the transgenic elements and strains and the embodiments are mainly collected in a common transgenic database, a national standard, an industry standard or an existing literature, so that the specificity and the accuracy of detection are ensured. The names of the transgenic elements, lines and internal reference genes screened are shown in Table 1.
Table 1 the target molecules screened according to the invention and their primer sequences.
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. 24.
Example 2 detection of alfalfa samples containing transgenic Components
1. Experimental materials: transgenic material ms_j101. The experimental material was transformed into pFMV35S, cp4epsps, the transgene content was 10%, and the experimental material was used as a research material.
2. Preparation of DNA template: the extraction of plant genome adopts a high-efficiency plant genome DNA extraction kit (DP 350) of CTAB or Tiangen biochemical technology (Beijing) limited company. In this example, three biological replicates were performed for each sample of sample DNA extracted using the root DNA extraction kit.
PCR amplification, library construction and sequencing
Amplifying genomic DNA of the sample using 12 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 line in the test sample and the signal index P of the control transgenic line, 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, respectively, of the transgenic line in the control.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of the transgenic line 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 | testing the transgene content of the sample |
| tTi | Sequencing of each transgenic element and line in the test sampleNumber of sequences |
| tRi | Sequence order of sequencing of each internal reference gene fragment detected in the test sample |
| m | -total number of reference gene fragments detected in the test sample |
| n | Total number of transgenic elements and line fragments detected in the standard |
According to this example, 2 samples, 1 transgenic line ms_j101 and one negative sample were tested, three biological replicates were made for each sample, and the results are shown in table 2: several sequences were also detected in negative alfalfa by promoters and terminators commonly used in negative samples, requiring that fewer than 5 sequencing reads 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.
TABLE 2 transgene test results for the test sample of example 2
As can be seen from the table, each transgenic element corresponding to Ms_J101 was effectively detected in three repeated experiments, and the content was close to that of each transgenic element; from this table it is demonstrated that alfalfa transgenic kits can be used to detect transgenic products.
Example 3 accuracy, specificity and sensitivity assessment
Transgenic alfalfa varieties ms_j101 and ms_j163 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 alfalfa ms_j101 and ms_j163 were diluted with negative alfalfa 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 ms_j101 and diluted sample numbers (B1, B2, B3, B4, B5, B6) of transgenic line ms_j163, 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 replicates per sample, and the results are shown in table 3.
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%.
As can be seen from the table, 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, 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%.
Examples
In order to verify the accuracy of the invention and the role in transgene detection of batch samples, a laboratory selects 79 alfalfa 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 79 test samples, the results of only 1 sample are inconsistent, and the consistency of the detection result is as high as 98.7%, so that the accuracy of the method is better demonstrated.
Still another advantage of the present invention includes:
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 9 transgenic elements and 2 transgenic lines which are commonly used at present in alfalfa, can conveniently add new detection target sequences, 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 flux and 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 alfalfa and products thereof.
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.
Sequence listing
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Claims (8)
1. A primer pair composition for detecting transgenic alfalfa, comprising:
a primer pair for specifically amplifying p35S, the nucleotide sequence of which is shown in SEQ ID NO.1 to SEQ ID NO. 2;
a primer pair for specifically amplifying t35S, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.3 to SEQ ID NO. 4;
a primer pair for specifically amplifying pNOS, the nucleotide sequence of which is shown in SEQ ID NO.5 to SEQ ID NO. 6;
a primer pair for specifically amplifying pFMV35S, the nucleotide sequence of which is shown in SEQ ID NO.7 to SEQ ID NO. 8;
a primer pair for specifically amplifying tNOS, the nucleotide sequence of which is shown as SEQ ID NO.9 to SEQ ID NO. 10;
a primer pair for specifically amplifying PAT, the nucleotide sequence of which is shown in SEQ ID NO.11 to SEQ ID NO. 12;
a primer pair for specifically amplifying bar, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.13 to SEQ ID NO. 14;
a primer pair for specifically amplifying NPtII, the nucleotide sequence of which is shown in SEQ ID NO.15 to SEQ ID NO. 16;
and a primer pair for specifically amplifying cp4epsps, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.17 to SEQ ID NO. 18.
2. The primer pair composition of claim 1, wherein the primer pair composition further comprises:
a primer pair for specifically amplifying Ms_J163, the nucleotide sequence of which is shown in SEQ ID NO.19 to SEQ ID NO. 20;
and/or a primer pair for specifically amplifying Ms_J101, wherein the nucleotide sequence of the primer pair is shown in SEQ ID NO.21 to SEQ ID NO. 22.
3. The primer pair composition of claim 1, further comprising a primer pair that specifically amplifies ms_acc, wherein the primer pair has a nucleotide sequence set forth in SEQ ID No.23 to SEQ ID No. 24.
4. A kit for detecting a primer pair composition of transgenic alfalfa, comprising: a primer pair composition according to any one of claims 1 to 3.
5. The kit of claim 4, further comprising a multiplex PCR premix.
6. The kit of claim 4, further comprising a first container containing the primer pair composition therein.
7. A method for detecting transgenic alfalfa using the primer pair composition of any one of claims 1-3, comprising the steps of:
obtaining DNA of alfalfa to be detected and the primer pair composition;
adding the primer pair composition into a reaction system by taking the DNA as a template, and performing an amplification reaction to obtain an amplification product;
carrying out high-throughput sequencing on the amplification product to obtain a high-throughput library;
and analyzing the gene sequence in the high-flux library to obtain a result of detecting the transgenic alfalfa.
8. Use of a alfalfa primer pair composition, wherein the primer pair composition is used for detecting alfalfa transgenic components and transgenic lines;
the primer pair composition comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 24.
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| CN102690835A (en) * | 2012-05-30 | 2012-09-26 | 曹际娟 | Transgenic soybean molecular standard sample and establishing method and application thereof |
| CN104745686B (en) * | 2015-01-23 | 2017-02-22 | 中华人民共和国黄埔出入境检验检疫局 | Primers and method for specific loop-mediated isothermal amplification (LAMP) detection of genetically modified alfalfa strain J163 and application of method |
| CN106498049B (en) * | 2016-10-21 | 2019-10-01 | 上海泽衡生物科技有限公司 | A kind of probe combinations, liquid-phase chip, kit and its application for transgenic corns detection |
| 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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