CN111705154A - Kit and method for detecting transgenic soybean MON87712 - Google Patents
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Abstract
The invention discloses a kit and a method for transgenic soybean MON87712, which only generate fluorescent signals to a target soybean strain in a double real-time fluorescent PCR method through a primer probe combination, can carry out specific amplification and is used for screening and identifying soybean transgenic products. According to the MON87712 linear plasmid standard molecule established by the invention, the endogenous Lectin gene of the transgenic soybean and the specific detection gene of the strain of the transgenic soybean MON87712 are integrated on the same plasmid vector, so that a positive matrix standard substance can be replaced, strain specific qPCR detection is carried out on the transgenic soybean MON87712, the content of the transgenic ingredients in the soybean can be accurately detected, and the quantitative detection limit can reach 10 copy/mu L.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection kit for transgenic soybeans, and more particularly relates to a kit and a method for transgenic soybeans MON 87712.
Background
Soybean is a main oil crop and vegetable protein source of human beings, is also an important industrial raw material, and plays an important role in food safety and national economy in China. Soybean is a "land intensive" product, which is ripe once a year. China has more people and less land, and the absolute yield of the soybeans is very low, so the method has no advantage in the aspect of soybean planting. Chinese soybean is mainly imported, and the imported soybean accounts for 60% of the world soybean trade volume. According to data released by the customs administration in 2020 and 14 months, the imported quantity of the Chinese soybean in 12 months in 2019 is 954.3 ten thousand tons, which corresponds to 67% of the violent increase.
Since the first glyphosate-resistant transgenic soybean emerges in the world, the cultivation of new varieties of transgenic crops is continuously developed, the obvious effect is played in improving the crop production, and the huge potential is also shown. With the continuous popularization and application of transgenic crops and products thereof, the supervision of the transgenic crops is gradually strengthened in various countries, and the establishment of an accurate transgenic organism detection method is a precondition for realizing the supervision, so that the establishment of an accurate, reliable and wide-applicability quantitative detection method is urgently needed as a corresponding technical support.
According to the official statistics report of the international agricultural biological application technology consulting service center (ISAAA), 41 transgenic soybean lines approved for commercialization/planting and import (food and feed) are published in the world, and detection and identification methods for 26 of them are published by the joint research center of European Union reference laboratory for GM food and feed. Currently, there is no report on the research of the detection method of the transgenic soybean MON87712 in the world.
Transgenic soybean of the MON87712 line was developed by montmorindo usa and introduced for transgenic traits by agrobacterium tumefaciens mediated plant transformation. The transgenic trait is glyphosate herbicide tolerance and enhanced photosynthesis/yield. Was first approved for planting in the united states in 2013. Only the united states approved it for use as feed and processing feedstock by 2020. Only patent inventions by Cole, Robert h. et al were reported on the MON87712 study. There has been no study of the detection of MON87712 due to the lack of standards and corresponding detection methods. When a PCR method is used for detecting a transgenic product, a positive control is required to be arranged to monitor the detection activity, and the genomic DNA extracted from a transgenic matrix standard substance is usually used as a positive quality control. However, transgenic soybeans such as MON87712 currently have no positive matrix standard substance sold in the world, which also becomes a bottleneck for establishing a corresponding detection method. The plasmid standard substance is a recombinant plasmid molecule containing a target gene to be detected and an internal standard gene, can be used as a substitute of a standard positive substance, can calculate copy number according to molecular weight conversion, and is widely applied to quantitative PCR detection. At present, no report about plasmid standard molecules for detecting the transgenic soybean MON87712 exists.
In recent years, with the promotion and commercialization degree of transgenic technology, transgenic components contained in imported products or commercially available soybean products become more and more complex, and in the actual detection process, the situations of sample mixing, low purity or multi-copy integration and the like are often encountered. The research establishes a double real-time fluorescent quantitative PCR detection method for the transgenic soybean strain, and verifies parameters such as detection specificity, sensitivity, quantitative range and the like. The result shows that the primer probe combination of the transgenic soybean MON87712 strain designed by the experiment only generates a fluorescent signal to the target soybean strain in the double real-time fluorescent PCR method, can carry out specific amplification and is used for screening and identifying the transgenic soybean product, the method can also accurately detect the content of the transgenic component in the soybean, and the quantitative detection limit can reach 10 copy/mu L. The establishment of the method provides powerful technical support for the transgenic supervision work in China, and simultaneously adds new technical reserve for the identification threshold management of transgenic products in China in the future.
Disclosure of Invention
Based on the background art, the technical problem to be solved by the invention is to provide a kit and a method for detecting transgenic soybean MON87712, which can avoid the interference of other transgenic soybeans and has a high-sensitivity detection limit. In order to realize the purpose of the invention, the following technical scheme is adopted:
the invention relates to a kit for detecting transgenic soybean MON87712, which comprises soybean endogenous gene primer SEQ ID NO.02, Lectin upstream primer SEQ ID NO.03 and downstream primer SEQ ID NO.04, and MON87712 strain specificity detection genes SEQ ID NO.05, SEQ ID NO.06 and SEQ ID NO. 06.
In a preferred embodiment of the invention, the kit further comprises a linear plasmid standard molecule.
In a preferred embodiment of the invention, the linear plasmid standard molecule comprises a nucleotide sequence of SEQ id No. 01.
In a preferred embodiment of the invention, both ends of the Lectin downstream primer SEQ ID NO.04 are respectively connected with HEX and BHQ 1.
In a preferred embodiment of the invention, both ends of the MON87712 strain-specific detection gene SEQ id No.07 are linked with FAM and BHQ1, respectively.
The invention also relates to a method for detecting the transgenic soybean MON87712, which is characterized in that the soybean sample is detected by using the kit.
In a preferred embodiment of the present invention, the method is a dual real-time fluorescent quantitative PCR detection method.
In another preferred embodiment of the present invention, the method is a quantitative detection method comprising the step of obtaining a standard curve based on a linear plasmid standard molecule.
In a preferred embodiment of the present invention, the detection limit of the quantitative detection method is 10 copy/uL.
In a preferred embodiment of the present invention, the sample to be tested further contains other transgenic soybeans.
Advantageous effects
The primer probe combination of the transgenic soybean MON87712 strain only generates a fluorescent signal to a target soybean strain in a double real-time fluorescent PCR method, can carry out specific amplification, and is used for screening and identifying a soybean transgenic product. According to the MON87712 linear plasmid standard molecule established by the invention, the endogenous Lectin gene of the transgenic soybean and the specific detection gene of the strain of the transgenic soybean MON87712 are integrated on the same plasmid vector, so that a positive matrix standard substance can be replaced, strain specific qPCR detection is carried out on the transgenic soybean MON87712, the content of the transgenic ingredients in the soybean can be accurately detected, and the quantitative detection limit can reach 10 copy/mu L.
Drawings
FIG. 1A schematic representation of a linear plasmid standard for transgenic soybean MON 87712.
FIG. 2 Soybean endogenous gene and transgenic soybean MON87712 strain specific detection gene electropherograms.
Fig. 3 real-time fluorescent PCR amplification profile of transgenic soybean MON87712 strain specific genes: wherein A1 and A2 are soybean endogenous gene Lectin amplification curves; b1 and B2 are specific qPCR amplification curves for detecting the strain specificity of the transgenic soybean MON 87712.
FIG. 4 soybean endogenous gene Lectin qPCR amplification curve and standard curve: wherein the upper graph is a soybean endogenous gene Lectin qPCR amplification curve; the lower graph shows soybean endogenous gene Lectin qPCR standard curve and parameters.
Fig. 5 transgenic soybean MON87712 strain specific detection gene qPCR amplification curve (top panel) and standard curve (bottom panel).
Detailed Description
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. In the present invention, the percentages and ratios referred to herein refer to mass percentages or mass ratios, unless otherwise specified.
Example 1
Materials and methods
Materials and instruments
1.1.1 test materials
The 45 test materials used in this study are shown in Table 1 for specific information.
TABLE 1 transgenic Material sources
1.1.2 test reagents
DNA extraction kit of Genetic Modified Organization (GMO) detection Ver2.0(D9093, TaKaRa, Japan); Tris-EDTA (TE) solution (RYX262, Jieshikang technologies, Inc., Qingdao); premix Ex Taq (Probe qPCR) (2X Conc.) (RR390A, TaKaRa, Japan).
1.1.3 primers and probes
Transgenic soybean MON87712 specific primers and probes were designed using Oligo7 software; relevant DNA sequence data was obtained from public databases, patent and laboratory internal DNA sequence analysis. HEX (hexachloro-6-methyl fluorescein) is used for the probe 5 'fluorescent labeling of the soybean internal standard gene Lectin, and FAM (6-carboxyl fluorescein) is used for the probe 5' fluorescent labeling of the MON87712 flanking sequence. The primer and probe sequences and the fluorescent labels used are shown in table 2. Primer and probe synthesis was synthesized by Takara Bio Inc.
TABLE 2 primers and probes
1.1.4 test apparatus
ND-1000 ultramicro spectrophotometer (Nanodrop, USA); 5910R high speed refrigerated centrifuge (Eppendorf, germany); QS6 model and ABI7500 model real-time fluorescent PCR reactors (AB, USA).
1.2 test methods
1.2.1 extraction of genomic nucleic acid
The test material was ground in liquid nitrogen, and then genomic DNA was extracted using a DNA extraction kit of Genetic Modified Organization (GMO) detection Ver2.0 kit of TaKaRa. The extracted genomic DNA was dissolved in 100. mu.L of Tris-EDTA (TE) solution. The concentration of the purified DNA sample was measured by an ND-1000 ultramicro spectrophotometer.
1.2.2 bioinformatics analysis and detection of plasmid Standard molecular Synthesis
MON87712 transgenic soybean was prepared by transformation of a conventional soybean A3525 variety with Agrobacterium-mediated plasmid vector PV-GMAP5770 (see Monsanto (2011) fertility for the determination of transformed soybean for MON87712 soybean. USDA APHIS fertilization number 11-202-01 p.). The plasmid vector PV-GMAP5770 is approximately 11.4kb and contains two T-DNAs delineated by left and right border regions to facilitate this transformation. The first T-dna (i) comprises the BBX32 coding sequence under the control of the E35S promoter and the E63' untranslated region. The protein regulates the day and night physiological process of plant, so as to increase the utilization rate of assimilate, prolong the photosynthesis activity time and raise yield obviously. The second T-DNA (T-DNAII) contains a cp4 epsps coding sequence and related promoters, directors, introns, targeting sequences and 3' UTR as marker genes, can synthesize and generate 5-enolpyruvate-3-phosphate synthase (epsps), reduces the binding affinity to glyphosate and improves the tolerance to glyphosate herbicide.
Since there is no transgenic soybean MON87712 standard on the market, synthesis of the detection plasmid can only be performed by an artificial synthesis method. According to soybean endogenous gene lectin sequences (GenBank accession number: K00821) and 3 'end and 5' end flanking sequences of MON87712 soybean, molecular cloning technologies such as gene synthesis, connection, transformation, plasmid extraction, enzyme digestion and the like are utilized to sequentially clone a lectin gene fragment (150bp), an exogenous gene BBX32 gene inserted in the MON87712 transgenic soybean and a boundary sequence (610bp) thereof onto a pMD-19T vector (no-load length of 2692bp), so as to construct a plasmid detection standard molecule. Then, XbaI digestion is carried out, and linearization treatment is carried out. The sequence of the prepared linearized plasmid standard molecule suitable for detection is SEQ ID NO.01, and the concentration of the linearized plasmid standard molecule is 77.1 ng/mul.
1.2.3 detection method
1.2.3.1 reaction composition and reaction conditions
1.2.3.2 primer specificity analysis
The specificity of the MON87712 primer probe was verified using a dual real-time fluorescent quantitative qPCR method and 46 test materials, where the MON87712 transgenic soybean content was 100 copy/. mu.L. The reaction composition and reaction conditions were carried out according to the method 1.2.3.1.
1.2.3.3 stability, sensitivity and quantitation Limit tests
The concentration of the linear plasmid standard molecule was 77.1 ng/. mu.l.the copy number was calculated from its molecular weight (average molecular weight of DNA 660) and diluted to 5 × 10 with a TE buffer gradient6、5×105、5×104、5×103、5×10250, 25, 12.5, 5, 0.5, 0.05 copy/. mu.L, etc. And selecting different concentrations according to test requirements to carry out stability, sensitivity and quantitative limit tests respectively. The reaction composition and reaction conditions were carried out according to the method 1.2.3.1. Each concentration gradient was set 6 replicates. Real-time quantitative fluorescence qPCR data analysis A Relative Standard Deviation (RSD) analysis method of Ct value is adopted.
1.2.3.4 Standard Curve construction
At a concentration of 5 × 106、5×105、5×104、5×103、5×102The method comprises the steps of taking 50 and 5 copy/. mu.L MON87712 linear plasmid standard molecular gradient diluent as a template, carrying out double real-time fluorescence quantitative qPCR detection by adopting a soybean endogenous Lectin gene primer probe and a transgenic soybean MON87712 strain specific detection gene primer probe, and carrying out reaction composition and reaction conditions according to a method of 1.2.3.1, wherein each concentration gradient is set for 6 times in parallel, and a standard curve equation of MON87712 specific sequence and Lectin is respectively established by using a formula Ct ═ K × lg A + B, wherein Ct is the cycle number of a fluorescence signal exceeding a corresponding threshold value in the fluorescence qPCR reaction, and K is the cycle number of the fluorescence signal exceeding the corresponding threshold value in the fluorescence qPCR reactionThe slope of the curve, A is the copy number and B is the intercept.
2 results and analysis
2.1 plasmid Standard molecules
Since there is no transgenic soybean MON87712 standard on the market, synthesis of plasmid standard molecules can only be detected by artificial synthesis methods. The Lectin gene fragment (150bp), the foreign gene BBX32 gene inserted into the MON87712 transgenic soybean and the boundary sequence (610bp) thereof are sequentially cloned to a pMD-19T vector (no-load length 2692bp), and a detection plasmid standard molecule is constructed. Then, XbaI digestion is carried out, and linearization treatment is carried out. And preparing the linearized plasmid standard molecule suitable for detection. The sequence information of the full length 760bp of the linearized plasmid standard molecule is shown in SEQ ID NO. 01. A schematic diagram of the gene sites and primer probe sites of the linearized plasmid standard molecule is shown in FIG. 1.
2.2 primer specificity
2% agarose gel electrophoresis was performed on the amplified product of the soybean endogenous gene and the transgenic soybean MON87712 strain specific detection gene, and the results are shown in fig. 2. The length of the soybean endogenous gene primer amplification fragment is 74bp, and the length of the transgenic soybean MON87712 strain specific gene amplification fragment is 107 bp.
The results of the qPCR method specificity verification show that: except for amplifying MON87712 transgenic soybean plasmid standard molecules, 45 test materials such as soybean, corn, rape, alfalfa, red bean, mung bean, peanut, rice, transgenic cottonseed, barley, wheat and the like do not have amplification curves in the specific detection primer probe of the transgenic soybean MON87712 strain; except for the MON87712 plasmid standard molecule and other soybean strains, the soybean endogenous Lectin gene primer probe has no amplification curve in other 26 test materials such as corn, rape, alfalfa, red bean, mung bean, peanut, rice, transgenic cottonseed, barley, wheat and the like. The above results indicate that the primer probes tested have very good specificity (as shown in Table 3). The double real-time fluorescent PCR amplification map of the transgenic soybean MON87712 plasmid standard molecule is shown in FIG. 3.
TABLE 3 double real-time fluorescent quantitative PCR detection specificity results for transgenic soybean MON87712 strain
2.3 establishment of double real-time fluorescence quantitative qPCR detection method
2.3.1 Standard Curve
According to the detailed specification of EURL on various parameters of a qPCR standard regression curve, the linear correlation coefficient R2 of the standard curve is more than or equal to 0.98, the range of the amplification Efficiency E [ Efficiency is 10 (-1/slope) -1] is 90-110%, and the range of the slope (slope) is-3.1-3.6. The standard curve equation of soybean endogenous gene Lectin in the research is-3.093 lg X +39.77, the standard curve equation of the transgenic soybean MON87712 strain specificity detection gene is-3.196 lg X +39.81, and all parameters in the standard curve equation of qPCR are in a specified range, so that the method is suitable for quantitative analysis of the specificity of the transgenic soybean MON87712 strain. The soybean endogenous gene Lectin qPCR amplification curve and the standard curve are shown in figure 4, and the transgenic soybean MON87712 strain specificity detection gene qPCR amplification curve and the standard curve are shown in figure 5.
2.3.2 stability analysis
The stability of the qPCR method is an important indicator affecting the quantitative results of transgenes. To 105、104、103、102Carrying out real-time fluorescence qPCR (quantitative polymerase chain reaction) tests on 7 plasmid standard molecules with different transgene contents of 50, 25 and 10 copy/mu L, wherein the SD range of the detected Ct value of the soybean endogenous Lectin gene is 0.04-0.29, and the RSD range is 0.16-0.82%; the SD range of the Ct value detected by the specific gene of the transgenic soybean MON87712 strain is 0.13-0.90, the RSD range is 0.40-2.65%, the RSD range meeting the EURL regulation is not more than 25% of the threshold range, and the test stability is better. The results are shown in Table 4.
TABLE 4 transgenic Soybean MON87712 plasmid Standard molecule double qPCR detection Effect
2.3.3 sensitivity and quantitation Limit test
To 105、104、103、102And the double qPCR test is carried out on 9 MON87712 plasmid standard molecules with the concentrations of 50, 25, 10, 1, 0.1 copy/. mu.L and the like, and samples with the content of 105-0.1 copy/. mu.L can be detected, which shows that the method has better sensitivity and the detection limit can reach 0.1 copy/. mu.L. However, under the condition of 1 and 0.1 copy/. mu.L concentration of the template, the relative standard deviation RSD of 6 times of repetition is higher than the threshold range of 25 percent, which indicates that the precision of the quantitative result at the relative content is poor, and the precision of the quantitative result of the content above 10 copy/. mu.L concentration is less than 25 percent, so the lower limit of the quantitative determination of the method can reach 10 copy/. mu.L. The results are shown in Table 5.
TABLE 5 qPCR method sensitivity test Lectin and MON87712
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
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Claims (10)
1. A kit for detecting transgenic soybean MON87712 comprises soybean endogenous gene primer SEQ ID NO.02, Lectin upstream primer SEQ ID NO.03 and downstream primer SEQ ID NO.04, and MON87712 strain specificity detection genes SEQ ID NO.05, SEQ ID NO.05 and SEQ ID NO. 07.
2. The kit of claim 1, further comprising a linear plasmid standard molecule.
3. The kit of claim 1, wherein the linear plasmid standard molecule comprises a nucleotide sequence of SEQ ID No. 01.
4. The kit of claim 1, wherein HEX and BHQ1 are respectively connected to two ends of the Lectin downstream primer SEQ ID NO. 04.
5. The kit of claim 1, wherein both ends of the MON87712 strain-specific detection gene SEQ ID No.07 are linked to FAM and BHQ1, respectively.
6. A method for detecting transgenic soybean MON87712 is characterized in that the kit is used for detecting soybean samples.
7. The method of claim 6, wherein the method is a dual real-time fluorescent quantitative PCR detection method.
8. The method of claim 6, which is a quantitative detection method comprising the step of obtaining a standard curve based on a linear plasmid standard molecule.
9. The method of claim 6, wherein the limit of detection is 10 copy/uL.
10. The method of claim 6, wherein the test sample further comprises additional transgenic soybean.
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| WO2012051199A2 (en) * | 2010-10-12 | 2012-04-19 | Monsanto Technology Llc | Soybean plant and seed corresponding to transgenic event mon87712 and methods for detection thereof |
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