CN114107552B - Primer pair combination, kit, detection method and application for detecting rape transgenic line - Google Patents
Primer pair combination, kit, detection method and application for detecting rape transgenic line Download PDFInfo
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Abstract
The application relates to the technical field of biology, in particular to a primer pair combination, a kit, a detection method and an application method for detecting a rape transgenic line. The primer pair combination for detecting the rape transgenic line comprises a primer group which is designed by amplifying the specific sequences of 12 common rape transgenic lines and 1 primer pair which is designed by the PE3-PEPCase sequence of the rape internal reference gene. The application also relates to a kit for detecting the rape transgenic line and a detection method. The technical scheme of the application 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 rape and products thereof, and has better application prospect.
Description
Technical Field
The application relates to the technical field of biology, in particular to a primer pair combination, a kit, a detection method and application for detecting a rape transgenic line.
Background
Transgenic rape is one of four transgenic crops worldwide, and the planting area of the transgenic rape accounts for 5.3% of the transgenic crops worldwide. Rape has a large planting area in China and is an important origin crop in China. Because transgenic rape is not commercially planted in China yet, a large amount of rapeseeds need to be imported every year in China to be used as processing raw materials in order to meet the consumption of edible oil and rapeseed meal. However, a large number of transgenic products are introduced into the market, and environmental and safety problems are beginning to attract attention, so that 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 sensitivity of detection 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 number of the genes is generally not more than 6, and if the number of the genes is not more than 6, the interference among primers is larger, so that 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 a rape transgenic line in order to solve the technical problems of low flux based on a quantitative PCR technology and high transgene detection cost based on a gene chip and a digital PCR technology.
According to the technical scheme for surface detection of the rape transgenic line, 12 specific nucleotide sequences of common rape transgenic lines OXY-235, T45, MS1 (B91-4), RT73 (GT 73) a, RT73 (GT 73) B, MON88302, rf1, rf2, rf3, topas19, MS8 and DP-073496-4, namely target molecules screened by the application, and internal reference genes PE3-PEPCase are used as detection targets, and nucleotide sequences of multiple PCR amplification primer pairs are designed; 13 pairs of primers were developed which do not affect each other and which allow efficient amplification by multiplex PCR. The multiplex PCR primer combination can be used for developing a rape transgenic line detection kit.
The specific technical scheme is that in the first aspect, the application provides a primer pair combination for detecting a transgenic rape line, namely 12 pairs of primers with the numbers of BnGMO1, bnGMO2, bnGMO3, bnGMO4, bnGMO5, bnGMO6, bnGMO7, bnGMO8, bnGMO9, bnGMO10, bnGMO11 and BnGMO12, wherein each pair of primers consists of a forward primer and a reverse primer, and the specific primer pair combination nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 24.
Also provided is a combination of two pairs of primers with the numbers of BnGMO13 and BnGMO14 for amplifying the rape internal reference gene PE3-PEPCase, 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.25-SEQ ID NO. 28.
These primers were used to amplify specific nucleotide sequences of the following transgenic lines of canola, OXY-235, T45, MS1 (B91-4), RT73 (GT 73) a, RT73 (GT 73) B, MON88302, rf1, rf2, rf3, topas19, MS8, DP-073496-4, respectively, i.e., the target molecules screened by the present application. The specific nucleotide sequences of the primers and the amplified rape transgenic line, 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 application 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 a transgenic line of rape, which is characterized by comprising the primer pair combination for detecting the transgenic line of rape according to claim 1 and the primer pair combination for amplifying a reference gene PE3-PEPCase of rape according to claim 2.
Preferably, the detection kit further comprises a multiplex PCR premix.
The application 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 rape and related products.
The application also provides a method for detecting the rape transgenic line, which is characterized by comprising the following steps:
1) The rape transgenic strain and the rape internal reference gene are used for reference to obtain a multiplex PCR primer;
2) Obtaining the DNA of rape to be detected; 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 amplified product to obtain a high-throughput library; and analyzing the gene sequence in the high-flux library to realize detection of the rape transgenic line.
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 rape transgenic line in the sample, when the rape transgenic line is selected, a detection primer for the rape internal reference gene is 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 groups for amplifying the transgenic rape line and the internal reference gene, each primer is premixed according to the proportion 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 1-14 is appropriately adjusted according to the specific sample to be tested. The later period can be increased periodically according to the newly collected transgenic line, 3000 pairs of primer combinations are tried, and the amplification effect is still good. In order to realize the detection of transgenic rape, I collect 12 common rape transgenic lines and cover almost all transgenic rape lines on the market, and the logarithmic range of the multiplex PCR primers is as follows: 1-14 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 transgenic lines common in canola from multiple dimensions, including but not limited to, the canola transgenic lines in our examples.
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 transgenic components of transgenic rape lines and products thereof.
The kit provided by the application can sensitively detect the rape transgenic product with the transgenic content of 0.05% in the sample.
In the reproducibility test of the application, the reproducibility rate r=100% and the accuracy rate a=100% of detection results among different libraries and different library-building batches of each sample are obtained.
The kit provided by the application detects various rape transgenic lines in a complex template and has high specificity.
The beneficial effects of the application 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 strain of rape, 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 with a second generation sequencing platform to sequence the amplified product, so that the detection throughput 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 rape and products thereof.
Therefore, the application overcomes the defects of time and labor waste and high cost in the prior art, and the provided rape 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 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.
Detailed Description
In order that the application may be readily understood, a more particular description of the application will be rendered by reference to specific embodiments that are illustrated below. This application 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 application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present application are commercially available or may be prepared by existing methods.
Example 1 screening of target transgenic lines and design of multiplex PCR amplification primers
S1, screening of target transgenic lines
In the embodiment of the application, the target transgenic component, namely the target transgenic strain, and the rape internal reference gene used in 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 lines and 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 non-specific amplification of non-target sequences, and 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. 1-28.
Example 2 detection of whether a canola sample contains transgenic lines
1. Experimental materials: transgenic materials OXY-235, T45, rf1, rf2, rf3, with transgene content of 10%,1%, 10%,1% and 1%, respectively, were used as our study 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 14 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 controlling the quality of the high-throughput sequencing data. According to the detection accuracy, sensitivity and other requirements, key parameters such as amplification cycle number, sequencing depth and the like are researched and adjusted; 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 of the test sample s=nt/Nt, where, 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 element strains and internal reference genes. Absolute quantification of the transgene component in the sample is achieved based on the number of sequenced sequences at each target location. When the sequencing sequences on the reference gene and the transgenic line are compared and exceed a specified threshold value, qualitatively judging that the sample contains transgenic components; 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 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 sequenced sequences for each transgenic line in 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 line fragments detected in Standard
According to this example, we examined 6 samples in total, 5 transgenic lines and one negative sample, three biological replicates per sample, and the results are shown in table 2: the promoters and terminators commonly used in negative samples also detect several sequences in negative canola species, which in this case we require that the number of sequencing reads be less than 5, be filtered out. The application 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 line in OXY-235, T45, rf1, rf2, rf3 samples was effectively detected in three repeated experimental china and the content was close to that thereof; from this table it was demonstrated that the canola transgene kit of our application can be used to detect transgenic products.
TABLE 2 transgene test results for the test sample of example 2
Note that: + represents detection
Example 3 accuracy, specificity and sensitivity assessment
Herbicide resistant rape variety RT-73 and Topas19 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 canola RT-73 and Topas19 were diluted with negative canola 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) for transgenic line RT-73 and diluted sample numbers (B1, B2, B3, B4, B5, B6) for transgenic line Topas 19. The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree to which the average value of multiple determinations accords with a true value, and is expressed by an error. 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 detect no transgenic strain 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 application
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 application and the role in transgene detection of batch samples, a laboratory selects 659 rape leaf samples of unknown genotypes of a company to detect, the detection method of the embodiment 2 is adopted, the detection result is compared with the preservation type of the company, and the consistency of the result is counted. The analysis result shows that in 659 test samples, the results of only 1 sample are inconsistent, and the consistency of the detection result is as high as 99.8%, so that the accuracy of the method disclosed by the application is 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.
The foregoing is only a specific embodiment of the application to enable those skilled in the art to understand or practice the application. 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 application. Thus, the present application 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 (8)
1. A primer pair composition for detecting a transgenic rape line is characterized by comprising 12 pairs of primers with the numbers of BnGMO1, bnGMO2, bnGMO3, bnGMO4, bnGMO5, bnGMO6, bnGMO7, bnGMO8, bnGMO9, bnGMO10, bnGMO11 and BnGMO12, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of the primer pair composition is shown as SEQ ID NO.1-SEQ ID NO. 24.
2. The primer pair composition according to claim 1, further comprising two primer pair compositions numbered BnGMO13 and BnGMO14 for amplifying the rape internal reference gene PE3-PEPCase, wherein each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequence of each primer pair is shown as SEQ ID NO.25-SEQ ID NO. 28.
3. A kit for detecting a transgenic line of canola, comprising the primer pair composition of claim 2.
4. The test kit of claim 3, further comprising a multiplex PCR premix.
5. Use of the primer pair composition for detecting transgenic lines of rape according to claim 1 for detecting transgenic rape and related products.
6. A method for detecting a transgenic line of canola, the method comprising the steps of:
1) The characteristic nucleotide sequence of the rape transgenic line and the rape internal reference gene are used for reference to obtain a multiplex PCR primer;
2) Obtaining the DNA of rape to be detected; adding a multiplex PCR primer of the primer pair composition for detecting the rape transgenic line in claim 1 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; and analyzing the gene sequence in the high-flux library to realize detection of the rape transgenic line.
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 high throughput library is qualified at a concentration greater than 2 ng/ul.
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| CN103757127A (en) * | 2014-02-05 | 2014-04-30 | 黄广平 | Rape transgenosis detecting kit |
| CN105586418A (en) * | 2016-01-29 | 2016-05-18 | 江汉大学 | Detection method of transgenic components in rapeseed oil |
| CN105624298A (en) * | 2016-01-29 | 2016-06-01 | 江汉大学 | Method for detecting genetically modified components of rape |
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| CN103757127A (en) * | 2014-02-05 | 2014-04-30 | 黄广平 | Rape transgenosis detecting kit |
| CN105586418A (en) * | 2016-01-29 | 2016-05-18 | 江汉大学 | Detection method of transgenic components in rapeseed oil |
| CN105624298A (en) * | 2016-01-29 | 2016-06-01 | 江汉大学 | Method for detecting genetically modified components of rape |
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