Primer probe combination, kit and detection method for herbicide-resistant corn fluorescent quantitative PCR (Polymerase Chain Reaction) detection

Abstract

The invention provides a primer probe combination, a kit and a detection method for fluorescent quantitative PCR (Polymerase Chain Reaction) detection of herbicide-resistant corn, and belongs to the technical field of biological detection. The primer probe combination comprises an upstream primer GG2-QF, a downstream primer GG2-QR and a probe GG2-QP which are used for detecting genomic DNA of a transformant of the herbicide-resistant transgenic maize GG2, and an upstream primer zSSIIb-3F, a downstream primer zSSIIb-4R and a probe zSSIIb-P which are used for detecting a standard gene zSSIIb in the maize, wherein the nucleotide sequences of the upstream primer GG2-QF, the downstream primer GG2-QR and the probe GG2-QP are respectively shown as SEQ ID NO: 1-6. The primer probe combination disclosed by the invention can be used for qualitatively and quantitatively detecting the herbicide-resistant transgenic maize GG2 transformant, has high specificity, high stability and high sensitivity, and provides powerful technical support for supervision of safety of transgenic maize in China.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a primer-probe combination, a kit and a detection method thereof for fluorescent quantitative PCR detection of herbicide-resistant corn. Background Art

[0002] In recent years, the research on plant transgenics has progressed rapidly. Certain achievements have been made in plant disease resistance, insect resistance, herbicide resistance and changing certain traits of plants. A considerable number of transgenic plants have been obtained, some of which have become strains, providing ways to increase crop yields, stress resistance, improve quality, accelerate breeding processes, and reduce production costs. While the cultivation of genetically modified crops brings huge economic and ecological benefits, its potential risks have also been controversial. Therefore, many countries and regions have strictly regulated genetically modified crops. The 2020 "Food Labeling Supervision and Management Measures (Draft for Comments)" stipulates that my country implements a mandatory labeling system for genetically modified foods, implements a catalog of agricultural genetically modified organisms that are subject to labeling management, and all agricultural genetically modified organisms included in the labeling management catalog and used for sale must be labeled. Therefore, it is very necessary to establish a highly specific, sensitive and stable detection method for genetically modified corn transformants, which provides technical support for product traceability, labeling management and intellectual property protection after corn industrialization.

[0003] GG2, a new herbicide-tolerant corn strain developed by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, has a high potential for industrial application. So far, there is no quantitative PCR detection method for GG2 in China or abroad. Therefore, the development of a quantitative method for detecting GG2 is of great significance for the safety supervision of genetically modified organisms in my country and the promotion of the healthy development of my country's corn industry. Summary of the invention

[0004] The object of the present invention is to provide a primer-probe combination, a kit and a detection method for fluorescent quantitative PCR detection of herbicide-resistant corn, which improves the sensitivity of herbicide-resistant corn GG2 and can achieve quantitative detection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a primer-probe combination for herbicide-resistant corn fluorescence quantitative PCR detection, comprising the following components:

[0007] (1) Upstream primer GG2-QF, downstream primer GG2-QR and probe GG2-QP for detecting genomic DNA of herbicide-tolerant transgenic maize GG2 transformants;

[0008] The nucleotide sequence of the upstream primer GG2-QF is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer GG2-QR is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe GG2-QP is shown in SEQ ID NO: 3;

[0009] (2) upstream primer zSSIIb-3F, downstream primer zSSIIb-4R and probe zSSIIb-P for detecting the standard gene zSSIIb in maize;

[0010] The nucleotide sequence of the upstream primer zSSIIb-3F is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer zSSIIb-4R is shown in SEQ ID NO:5, and the nucleotide sequence of the probe zSSIIb-P is shown in SEQ ID NO:6.

[0011] Preferably, the 5' end of the probe GG2-QP and the probe GG2-QR is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; the fluorescent reporter group is selected from FAM or HEX, and the fluorescent quencher group is selected from TAMRA or BHQ1.

[0012] The present invention also provides a kit for PCR detection of herbicide-resistant corn GG2 transformants, comprising the primer-probe combination.

[0013] Preferably, each primer and probe is packaged separately, and the concentration of each primer is independently 9 to 11 μmol / L, and the concentration of each probe is independently 9 to 11 μmol / L.

[0014] Preferably, the kit further comprises a negative control and a positive control, wherein the negative control is genomic DNA of non-transgenic corn, and the positive control is genomic DNA of a transgenic corn GG2 transformant.

[0015] The present invention also provides a fluorescent quantitative PCR detection method for herbicide-resistant transgenic corn GG2 transformants, comprising the following steps:

[0016] S1. Extract genomic DNA of the sample to be tested;

[0017] S2. Using the extracted genomic DNA of the sample to be tested as a template, the primer probe combination or the kit is used to perform fluorescent quantitative PCR amplification, and based on the amplification results, a qualitative judgment and / or quantitative detection is performed on the presence of GG2 transformant DNA in the sample to be tested.

[0018] Preferably, the method of qualitative judgment is as follows:

[0019] (1) If the zSSIIb gene shows positive amplification, it means that the sample to be tested is corn, and the next step of judgment is required: if the GG2 transformant DNA shows positive amplification, it means that the sample is a sample containing the herbicide-resistant transgenic corn GG2 transformant; if the GG2 transformant DNA does not show positive amplification, it means that the sample does not contain the herbicide-resistant transgenic corn GG2 transformant;

[0020] (2) If the zSSIIb gene is not amplified, it means that the sample to be tested is not corn, but a sample that does not contain herbicide-tolerant transgenic corn GG2 transformants.

[0021] The principles for qualitative judgment of samples to be tested are:

[0022] If the real-time fluorescence quantitative PCR detection of the internal standard gene zSSIIb and the GG2 transformant DNA in the tested corn sample shows a typical amplification curve and the Ct value is ≤36, the tested sample is judged to be positive for the GG2 transformant;

[0023] If a typical amplification curve appears in the real-time fluorescence quantitative PCR test of the internal standard gene zSSIIb in the tested corn sample and the Ct value is ≤36, and there is no typical amplification curve or the Ct value is greater than 36 in the real-time fluorescence quantitative PCR test of the GG2 transformant DNA, the tested sample is judged to be negative for the GG2 transformant;

[0024] If no typical amplification curve or Ct value > 36 appears in the real-time fluorescence quantitative PCR detection of the internal standard gene zSSIIb in the tested corn sample, it is judged that the tested sample is negative for the GG2 transformant.

[0025] Preferably, the reaction system of fluorescent quantitative PCR is 25 μL: 2×TaqMan Fast Advanced Master Mix 12.5 μL, 10 μmol / L forward primer 1.0 μL, 10 μmol / L reverse primer 1.0 μL, 10 μmol / L probe 0.5 μL, 25 ng / μL DNA template 2.0 μL, and ddH2O 8.0 μL.

[0026] Preferably, the reaction procedure of fluorescent quantitative PCR is: 95°C, 5 min; 95°C, 15 s; 60°C, 60 s; 45 cycles.

[0027] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention obtains a primer and probe combination with the best sensitivity and specificity by specifically screening the primer probe for real-time fluorescence quantitative PCR detection of herbicide-resistant transgenic corn GG2 transformants, and on this basis, a real-time fluorescence quantitative PCR detection method for herbicide-resistant transgenic corn GG2 transformants is established and optimized using a standard product with a suitable concentration gradient. The present invention also provides a kit containing a primer pair and a probe combination obtained through screening, which has the advantages of simple operation, high sensitivity, and good reliability, and is used in conjunction with the standard gene zSSIIb in corn, which can not only qualitatively detect whether the corn sample to be tested contains GG2 transformants, but also quantitatively detect its content, and the minimum detection limit can reach 10 copies / reaction. This has important practical significance for promoting the sustainable and healthy development of my country's corn industry, and can also provide a reference for improving my country's transgenic product detection system and provide strong technical support for the supervision of my country's transgenic biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 1 is the fluorescence curve of real-time fluorescence quantitative PCR amplification of genomic DNA of positive standard of herbicide-resistant transgenic corn GG2 transformant by primer-probe combination No. 1 to No. 11 designed in Example 1; 12 is blank control and negative control.

[0029] Figure 2 The fluorescence curve of real-time fluorescence quantitative PCR amplification of the genomic DNA of the positive standard of herbicide-tolerant transgenic corn GG2 transformant under different concentration conditions is the optimal primer-probe combination.

[0030] Figure 3 The fluorescence amplification curve shows the specific results of the optimal primer-probe combination for detecting the genomic DNA of the positive standard of herbicide-resistant transgenic corn GG2 transformant.

[0031] Figure 4 The fluorescence curve and standard curve of real-time fluorescence quantitative PCR amplification of genomic DNA of positive standard of herbicide-tolerant transgenic corn GG2 transformant at different concentrations are the best primer-probe combination.

[0032] Figure 5 The fluorescence amplification curve shows the sensitivity of the optimal primer-probe combination for detecting the genomic DNA of the positive standard of herbicide-resistant transgenic corn GG2 transformants.

[0033] Figure 6 The fluorescence curve and standard curve are obtained by real-time fluorescence quantitative PCR amplification of the genomic DNA of the internal standard gene zSSIIb of maize using the internal standard primer and probe combination.

[0034] Figure 7Real-time fluorescence quantitative PCR amplification fluorescence curve and standard curve for the optimal primer-probe combination to detect genomic DNA of herbicide-tolerant transgenic corn GG2 transformants. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] 1. The instruments, reagents and biological materials used in the present invention are as follows:

[0037] 1. Experimental instruments

[0038] Spectrophotometer: ThermoND-1000;

[0039] Centrifuge: Thermo Biofuge Primo R;

[0040] Real-time fluorescence quantitative PCR instrument: BioRad CFX96;

[0041] Other instruments include water bath, precision balance, fume hood, biological safety cabinet, etc.

[0042] 2. Enzymes and Reagents

[0043] FastAdvancedMasterMix (Thermo Fisher scientific, cat. no. 4444557), which contains dNTPS and taq polymerase;

[0044] Plant genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.;

[0045] The primer pairs and probes used in the examples were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0046] 3. Reference substances and / or standards

[0047] Homozygous herbicide-tolerant transgenic maize GG2 genomic DNA was used as a positive control and / or standard;

[0048] Non-transgenic corn genomic DNA was used as a negative control; double distilled water was used as a blank control.

[0049] 4. Other plant materials

[0050] 1) Other genetically modified corn mixtures (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON87460, DAS40278-9, 4114, MON87427, 5307 mixed in equal mass ratio to form one sample);

[0051] 2) genetically modified soybean mixture (GTS40-3-2, MON89788, A5547-127, A2704-12, 356043, 305423, CV127, MON87701, MON87708, MON87769, MON87705, FG72, and DAS-81419-2 were mixed in equal mass ratios to form one sample);

[0052] 3) genetically modified cotton mixture (MON1445, MON531, MON15985, LLCOTTON25, MON88913, GHB614, and COT102 in equal mass ratios were mixed to form one sample);

[0053] 4) genetically modified rapeseed mixture (MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19 / 2, MON88302, and 73496 were mixed in equal mass ratios to form one sample);

[0054] 5) genetically modified rice mixture (TT51-1, KF-6, KMD-1, M12, KF-8, KF-2, G6H1, and T1C-19 in equal mass ratios were mixed to form one sample);

[0055] 6) Non-GMO corn mixture (Zheng 58, Zhengdan 958 and Jingdan 28 in equal mass ratios are mixed to form one sample).

[0056] 2. The common methods and criteria in the following embodiments are as follows:

[0057] 1. PCR amplification qualification criteria:

[0058] a. In the lowest concentration calibrator, zSSIIb gene and GG2 transformant DNA both had typical amplification curves, and Ct values ​​were ≤36;

[0059] b. In the negative control, the zSSIIb gene has a typical amplification curve and the Ct value is ≤36; the GG2 transformant DNA has no typical amplification curve, or the Ct value is ≥40;

[0060] c. In the blank control, there was no typical amplification curve for zSSIIb gene and GG2 transformant DNA, or the Ct value was ≥40;

[0061] d. The determination coefficient of the standard curve is R2 ≥ 0.98, -3.1 ≥ the slope of the standard curve (b) ≥ -3.6.

[0062] 2. Principles for qualitative judgment of samples to be tested:

[0063] If the real-time fluorescence quantitative PCR detection of the internal standard gene zSSIIb and the GG2 transformant DNA in the tested corn sample shows a typical amplification curve and the Ct value is ≤36, the tested sample is judged to be positive for the GG2 transformant;

[0064] If a typical amplification curve appears in the real-time fluorescence quantitative PCR test of the internal standard gene zSSIIb in the tested corn sample and the Ct value is ≤36, and there is no typical amplification curve or the Ct value is greater than 36 in the real-time fluorescence quantitative PCR test of the GG2 transformant DNA, the tested sample is judged to be negative for the GG2 transformant;

[0065] If no typical amplification curve or Ct value > 36 appears in the real-time fluorescence quantitative PCR detection of the internal standard gene zSSIIb in the tested corn sample, it is judged that the tested sample is negative for the GG2 transformant.

[0066] 3. Principles of quantitative detection of samples to be tested:

[0067] If typical amplification curves appear in the detection of the internal standard zSSIIb gene and the GG2 transformant DNA in the tested corn sample, and the GG2 transformant DNA content is higher than the quantitative limit, the GG2 transformant content is calculated;

[0068] If typical amplification curves appear in the detection of both the internal standard zSSIIb gene and the GG2 transformant DNA in the tested corn sample, but the GG2 transformant DNA content is lower than the quantitative limit, the GG2 transformant content will not be calculated.

[0069] 4. The sequence information of the primer pair and probe for detecting herbicide-tolerant maize GG2 transformants and the primer pair and probe for detecting the standard gene zSSIIb gene in maize are shown in Table 1 below:

[0070] Table 1 Primer pairs and probe sequences for detecting maize GG2 transformants and internal standard gene zSSIIb

[0071]

[0072] The amplified fragment of primer pair GG2-QF / QR is 103 bp, as shown in SEQ ID NO: 7:

[0073] SEQ ID NO: 7:

[0074]

[0075] The direction of the sequence shown in SEQ ID NO: 7 is 5′-3′. The 5′ underlined part is the GG2-QF primer sequence, the 3′ underlined part is the reverse complementary sequence of the GG2-QR primer, and the inside of the box is the GG2-QP probe sequence. 1 to 75 are partial sequences of the exogenous insert fragment, and 76 to 103 are partial sequences of the maize genome.

[0076] The amplified fragment of primer pair zSSIIb-3F / 4R is 88 bp, as shown in SEQ ID NO: 8:

[0077] SEQ ID NO: 8:

[0078]

[0079] The direction of the sequence shown in SEQ ID NO: 8 is 5′-3′. The 5′ underlined part is the zSSIIb-3F primer sequence, the 3′ underlined part is the reverse complementary sequence of the zSSIIb-4R primer, and the inside of the box is the reverse complementary sequence of the zSSIIb-P probe. This sequence is a partial sequence of the maize zSSIIb gene.

[0080] Example 1

[0081] According to the sequence information of herbicide-resistant transgenic corn GG2 material (see SEQID NO.3 in patent CN 113186337 B), the primer design software Primer Premier 5 was used to comprehensively consider the GC content of the nucleotide sequence and the primer Tm value, and multiple primers and probes for herbicide-resistant transgenic corn GG2 transformants were designed for the 5' and 3' end sequences of the insertion position. By arranging and combining the obtained primer and probe sequences, a total of 11 sets of primer-probe combinations were obtained. The specific primer-probe combination information is shown in Table 2:

[0082] Table 2 Primer probe combination information

[0083]

[0084]

[0085]

[0086] Wherein: the probe is a TaqMan probe, the 5′ end of which is labeled with a fluorescent reporter group FAM, and the 3′ end is labeled with a corresponding fluorescent quencher group BHQ1.

[0087] Using the genomic DNA of the positive standard of the herbicide-tolerant transgenic corn GG2 transformant as a template, the primer probe combinations in Table 2 were used to perform real-time fluorescence quantitative PCR amplification to select and determine the best real-time fluorescence quantitative PCR primer probe combination. 25 μL of a reaction system for real-time fluorescence quantitative PCR amplification detection can be added to a 0.2 mL EP reaction tube, which includes the reagents shown in Table 3 below. After the reaction system is prepared, it is placed in a real-time fluorescence quantitative PCR instrument for reaction. The reaction procedure is shown in Table 4.

[0088] Table 3 25 μL reaction system for real-time fluorescence quantitative PCR amplification detection

[0089]

[0090] Table 4 Real-time fluorescence quantitative PCR reaction program

[0091]

[0092] After the second stage of annealing and extension (60°C), the fluorescence signal was collected and the amplification curve was read. Each reaction was set up in parallel for 3 times. The amplification curve is shown in Figure 1 The Ct value of each reaction and the average Ct value are shown in Table 5.

[0093] Table 5 Real-time fluorescence quantitative PCR amplification Ct values ​​under different primer-probe combinations

[0094]

[0095]

[0096] The basic principle for screening primer probes is to compare the shapes of the obtained amplification curves and the levels of the fluorescence signals, and select primer probe combinations with "S"-shaped amplification curves, higher fluorescence signals, and smaller Ct values ​​as candidate primer probe combinations for subsequent experimental verification.

[0097] Figure 1 The fluorescence curves of the real-time fluorescence quantitative PCR amplification of the genomic DNA of the positive standard of the herbicide-resistant transgenic corn GG2 transformant by the primer-probe combinations No. 1 to No. 11 in the figure correspond to the amplification curves obtained by the real-time fluorescence quantitative PCR amplification using the primer-probe combinations No. 1 to No. 11 in Table 2. Figure 1 The amplification curves and Ct values ​​in Table 5 were analyzed, among which the amplified fragment size of primer-probe combination 3F2 / 3R1 / 3P1 was appropriate, the amplification signal was strong, and the Ct value was the smallest. Therefore, the primer-probe combination 6 shown in Table 2 was selected as the primer-probe combination for real-time fluorescence quantitative PCR of herbicide-tolerant transgenic corn GG2.

[0098] Example 2: Selection of primer-probe combination concentrations for real-time fluorescence quantitative PCR of herbicide-tolerant transgenic corn GG2

[0099] The primer-probe combination No. 6 determined in Example 1 was used to perform real-time fluorescence quantitative PCR amplification on the genomic DNA of the positive standard of the herbicide-resistant transgenic corn GG2 transformant under different concentration conditions to screen and determine the optimal reaction concentration of the real-time fluorescence quantitative PCR primer probe for detecting the herbicide-resistant transgenic corn GG2 transformant provided by the present invention. In this embodiment, 1 to 5 groups of primer-probe combinations with different concentrations were designed, and the corresponding final primer concentrations and probe concentrations in each reaction system are shown in Table 6. The final concentrations of the primers and probes in the 25 μL reaction system are different from those in Example 1, and the other reagents are as shown in Table 3 in Example 1. The reaction procedure is as shown in Table 4 in Example 1. During the reaction, the fluorescence signal was collected and the amplification curve was read. Three parallels were set for each reaction, and the amplification curve was shown in Table 6. Figure 2 The Ct value of each reaction and the average Ct value are shown in Table 6.

[0100] Table 6 Different primer-probe concentration combinations and corresponding real-time fluorescence quantitative PCR amplification Ct values Figure 2 The fluorescence curves of real-time fluorescence quantitative PCR amplification of the genomic DNA of the positive standard of the herbicide-tolerant transgenic corn GG2 transformant using the primer probe combination No. 6 determined in Example 1 under different concentration conditions, wherein the amplification curves No. 1 to No. 5 in the figure correspond to the amplification curves obtained under the primer probe concentrations of combinations No. 1 to No. 5 in Table 6, respectively. Figure 2 The amplification curve and Ct value analysis in Table 6 show that the greater the primer probe concentration, the higher the fluorescence signal value. Combined with the Ct value data in Table 6, and referring to the "Announcement No. 2031 of the Ministry of Agriculture of the People's Republic of China-8-2013 Qualitative PCR Method for Detecting Standard Genes in Corn for Components of Transgenic Plants and Their Products", the primer probe concentration combination of combination 3 in Table 6 is determined as the optimal primer probe concentration in the real-time fluorescence quantitative PCR method for herbicide-resistant transgenic corn GG2. It can be seen that the optimal primer probe concentration combination obtained after screening is: the final concentration of the primer is 0.4 μmol / L, and the final concentration of the probe is 0.2 μmol / L. Therefore, the optimal reaction system and reaction procedure for real-time fluorescence quantitative PCR of herbicide-resistant transgenic corn GG2 can be determined as shown in Tables 7 and 8.

[0103] Table 7 25 μL reaction system for real-time fluorescence quantitative PCR amplification detection

[0104]

[0105] Table 8 Real-time fluorescence quantitative PCR reaction program

[0106]

[0107] Example 3. Specific detection of real-time fluorescence quantitative PCR primer-probe combination for herbicide-tolerant transgenic corn GG2

[0108] The primer probe combination No. 6 determined in Example 1 was used to perform real-time fluorescence quantitative PCR detection on the genomic DNA of the positive standard of herbicide-resistant transgenic corn GG2 transformant, genomic DNA of 5 other transgenic samples (other transgenic corn mixed samples, transgenic soybean mixed samples, transgenic cotton mixed samples, transgenic rapeseed mixed samples, transgenic rice mixed samples) and non-transgenic corn mixed samples as templates, and blank control ddH2O to evaluate the specificity of the real-time fluorescence quantitative PCR primer probe combination and detection method for detecting herbicide-resistant transgenic corn GG2 transformants provided by the present invention. The composition of the 5 other transgenic samples and non-transgenic corn mixed samples is as follows:

[0109] 1. Other genetically modified corn mixtures (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON87460, DAS40278-9, 4114, MON87427, 5307 in equal mass ratios are mixed to form one sample);

[0110] 2) genetically modified soybean mixture (GTS40-3-2, MON89788, A5547-127, A2704-12, 356043, 305423, CV127, MON87701, MON87708, MON87769, MON87705, FG72, and DAS-81419-2 were mixed in equal mass ratios to form one sample);

[0111] 3) genetically modified cotton mixture (MON1445, MON531, MON15985, LLCOTTON25, MON88913, GHB614, and COT102 in equal mass ratios were mixed to form one sample);

[0112] 4) genetically modified rapeseed mixture (MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19 / 2, MON88302, and 73496 were mixed in equal mass ratios to form one sample);

[0113] 5) genetically modified rice mixture (TT51-1, KF-6, KMD-1, M12, KF-8, KF-2, G6H1, and T1C-19 in equal mass ratios were mixed to form one sample);

[0114] 6) Non-GMO corn mixture (Zheng 58, Zhengdan 958 and Jingdan 28 in equal mass ratios are mixed to form one sample).

[0115] The 25 μL reaction system is as shown in Table 7 of Example 2, the reaction procedure is as shown in Table 8 of Example 2, and the fluorescence signal is collected during the reaction to obtain the amplification curve. Figure 3 .

[0116] Figure 3 This is the specific result fluorescence amplification curve of the primer-probe combination No. 6 determined in Example 1 for detecting the genomic DNA of the positive standard of the herbicide-resistant transgenic corn GG2 transformant. Figure 3 It can be seen that an amplification curve appeared in the detection of the positive standard sample of the herbicide-resistant transgenic corn GG2 transformant, while no amplification curve appeared in the detection results of the other five transgenic and non-transgenic mixture samples and the blank control sample. This result shows that the real-time fluorescent quantitative PCR primer probe combination and detection method for herbicide-resistant transgenic corn GG2 provided by the present invention have good specificity.

[0117] Example 4. Linear interval and sensitivity detection of primer-probe combination for real-time fluorescence quantitative PCR of herbicide-tolerant transgenic corn

[0118] 1. Detection of linear interval

[0119] The primer-probe combination No. 6 determined in Example 1 above was used to detect different concentrations of the positive standard genomic DNA of the herbicide-resistant transgenic corn GG2 transformant, so as to determine the linear range of the primer-probe combination for the real-time fluorescence quantitative PCR of the herbicide-resistant transgenic corn GG2 genome provided by the present invention. The preparation method of the positive standard genomic DNA of the herbicide-resistant transgenic corn GG2 transformant of different concentrations is as follows: dilute the positive standard genomic DNA of the herbicide-resistant transgenic corn GG2 transformant to 25 ng / μL, and then perform gradient dilution with ddH2O to obtain a series of concentrations of the positive standard genomic DNA of the corn GG2 transformant (see Table 9) as a template for real-time fluorescence quantitative PCR reaction. The 25μL reaction system is shown in Table 7 in Example 3, and the reaction procedure is shown in Table 8 in Example 2. Each test under the same conditions was repeated twice in parallel. During the reaction, the fluorescence signal was collected, and the amplification result was detected. The obtained fluorescence amplification curve and Ct value results are shown as follows. Figure 4 and as shown in Table 10.

[0120] Table 10 Standard curve linearity repeated measurement data statistics

[0121]

[0122] Figure 4 The fluorescence curve and standard curve of the real-time fluorescence quantitative PCR amplification of the genomic DNA of the positive standard of herbicide-resistant transgenic corn GG2 transformant at different concentrations by the primer-probe combination No. 6 determined in Example 1, wherein 1, standard S1 (20,000 copies / reaction); 2, standard S2 (4,000 copies / reaction); 3, standard S3 (800 copies / reaction); 4, standard S4 (160 copies / reaction); 5, standard S5 (32 copies / reaction); 6, standard S6 (16 copies / reaction). Figure 4 It can be seen from the results in Table 10 that in the two repeated experiments, the standard curves met the amplification efficiency of 90% to 110%, the slope was -3.6 to -3.1, and R2 ≥ 0.98, which is in line with the relevant provisions of "Ministry of Agriculture Announcement No. 2259-5-2015 Guidelines for the Development of Real-time Fluorescence Quantitative PCR Methods for Detection of Components of Genetically Modified Plants and Their Products" (hereinafter referred to as "Quantitative Standards"), indicating that this method has good linearity in the copy number range of 40,000 to 20.

[0123] 2. Sensitivity detection

[0124] According to the above linear interval test results, the positive standard genomic DNA standard of herbicide-resistant transgenic corn GG2 transformant was diluted to 10 copies (equivalent to 0.05% GG2 by mass fraction) for sensitivity test, and the sample was subjected to real-time fluorescence quantitative PCR amplification test. The 25 μL reaction system is shown in Table 7 of Example 3, and the reaction procedure is shown in Table 8 of Example 3. The parallel test was repeated 60 times. The fluorescence signal was collected during the reaction, and the amplification result was detected. The fluorescence amplification curve obtained is as shown in FIG. Figure 5 shown.

[0125] Figure 5 The fluorescence amplification curve is the sensitivity result of the primer-probe combination No. 6 determined in Example 1 for detecting the genomic DNA of the positive standard of the herbicide-resistant transgenic corn GG2 transformant. Figure 5 It can be seen that typical amplification curves can be obtained in all 60 parallel experiments, from which the minimum detection limit can be determined to be 10 copies or 0.05% (calculated by adding 50 ng of DNA template into the PCR detection reaction system).

[0126] Example 5: Limit of Quantitation Test of Real-time Fluorescence Quantitative PCR Primer Probe Combination for Herbicide-Tolerant Transgenic Corn

[0127] In this example, the genomic DNA of the positive standard of the herbicide-resistant transgenic corn GG2 transformant was diluted to 100 copies as the sample to be tested, and the primer probe combination No. 6 determined in Example 1 was used to perform real-time fluorescence quantitative PCR amplification on the sample to be tested. The 25 μL reaction system is shown in Table 7 of Example 2, and the reaction procedure is shown in Table 8 of Example 2. Each test under the same conditions was repeated three times in parallel. During the reaction, the fluorescence signal was collected to obtain the amplification Ct value, and the results are shown in Table 11.

[0128] Table 11 Quantitative detection limit test results

[0129]

[0130] It can be seen from the results in Table 11 that the deviation of the measurement results of 100 copies of the sample is 1.00%, which does not exceed 25% of the standard value, and the RSDr (relative standard deviation) is 4.15%, which is less than 25%, meeting the requirements of the quantitative standard. Therefore, the quantitative limit of the detection method is determined to be 100 copies.

[0131] Example 6: Quantitative detection of herbicide-tolerant transgenic corn GG2 transformant genes in actual samples

[0132] 1. Establishment of standard curve

[0133] The internal standard gene zSSIIb and GG2 transformant gene standards with 20000, 4000, 800, 160, and 32 copies were used as positive templates, and ddH2O was used as a negative control template. The reaction system shown in Table 7 and the reaction program shown in Table 8 in Example 2 were used to perform real-time fluorescence quantitative PCR amplification of the internal standard gene zSSIIb and GG2 transformant genes, respectively, to establish a standard curve for real-time fluorescence quantitative PCR for detecting herbicide-resistant transgenic corn GG2 transformants. The standard curve was drawn with the Log value of the copy number of each standard as the horizontal axis and the corresponding Ct value as the vertical axis, and the correlation coefficient and amplification efficiency of the standard curve were analyzed. Each standard was tested twice at the same time. The fluorescence amplification curve and standard curve results of each standard were shown as follows. Figures 6-7 As shown in Table 12.

[0134] Table 12 Real-time fluorescence quantitative PCR results of transgenic corn GG2 transformants

[0135]

[0136] Note: The standard curve formula is y=ax+b, where the Ct value is used as the y-axis and the logarithm of the copy number is used as the x-axis.

[0137] Figure 6The fluorescence curve and standard curve are obtained by real-time fluorescence quantitative PCR amplification of the zSSIIb genomic DNA of the internal standard gene of maize using an internal standard primer probe combination (sequences shown in SEQ ID NOs: 4-6). Figure 7 The real-time fluorescence quantitative PCR amplification fluorescence curve and standard curve of the primer-probe combination No. 6 determined in Example 1 for detecting genomic DNA of herbicide-resistant transgenic corn GG2 transformants, wherein: 1. Standard S1 (20,000 copies); 2. Standard S2 (4,000 copies); 3. Standard S3 (800 copies); 4. Standard S4 (160 copies); 5. Standard S5 (32 copies).

[0138] 2. Quantitative detection and determination methods of samples to be tested

[0139] According to the standard curve of the GG2 transformant DNA standard and the internal standard zSSIIb gene drawn above, the Ct value of the sample to be tested is substituted into the standard curve, the copy number and ratio of the GG2 transformant DNA and zSSIIb gene in the sample to be tested are calculated, and the content of the GG2 transformant in the sample to be tested is obtained.

[0140] 3. Testing of actual samples

[0141] The herbicide-resistant transgenic corn GG2 powder was mixed with the non-transgenic corn powder to prepare samples with mass fractions of 5%, 2%, and 0.5%, respectively, as test samples, and the accuracy and precision of the primer probe combination No. 6 and the detection system and detection method determined in Example 1 of the present invention were tested. The internal standard gene used the primer probe in the standard of "Announcement No. 2031 of the Ministry of Agriculture-8-2013 Qualitative PCR Method for Detecting Internal Standard Genes in Corn for Transgenic Plants and Their Product Components" (sequences are shown in SEQ ID NOs: 4 to 6).

[0142] According to the standard curve drawn above, the copy number of the sample to be tested was calculated, and the transgenic content was calculated: transgenic content (%) = GG2 copy number / zSSIIb copy number × 100%. The statistical results are shown in Table 13.

[0143] Table 13 Calculation results of genetically modified ingredient content

[0144]

[0145] Table 13 shows that the deviations of the sample determination results of 5%, 2% and 2% content are -0.80%, -1.50% and 8.00% respectively, and the RSDr is 2.28%, 1.08% and 2.62% respectively, which meets the requirements of the quantitative standard. It can be seen that the herbicide-resistant transgenic GG2 transformant-specific real-time fluorescence quantitative PCR method established by the present invention can be used for the determination of the content of herbicide-resistant transgenic corn GG2 components in transgenic samples.

[0146] It can be seen from the above examples that the present invention provides a primer-probe combination, a kit and a detection method for fluorescent quantitative PCR detection of herbicide-resistant corn, which can realize qualitative and quantitative detection of herbicide-resistant transgenic corn GG2 transformants with a detection limit of 10 copies / reaction.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe combination for fluorescent quantitative PCR detection of herbicide-resistant corn, characterized in that: Includes the following components: (1) Upstream primer GG2-QF, downstream primer GG2-QR and probe GG2-QP for detecting genomic DNA of herbicide-tolerant transgenic maize GG2 transformants; The nucleotide sequence of the upstream primer GG2-QF is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer GG2-QR is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe GG2-QP is shown in SEQ ID NO: 3; (2) upstream primer zSSIIb-3F, downstream primer zSSIIb-4R and probe zSSIIb-P for detecting the standard gene zSSIIb in maize; The nucleotide sequence of the upstream primer zSSIIb-3F is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer zSSIIb-4R is shown in SEQ ID NO:5, and the nucleotide sequence of the probe zSSIIb-P is shown in SEQ ID NO:

6.

2. The primer-probe combination according to claim 1, characterized in that: The 5' end of the probe GG2-QP and the probe GG2-QR is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group; the fluorescent reporter group is selected from FAM or HEX, and the fluorescent quencher group is selected from TAMRA or BHQ1.

3. A kit for PCR detection of herbicide-resistant corn GG2 transformants, characterized in that: The method comprises the primer-probe combination according to claim 1 or claim 2.

4. The kit according to claim 3, characterized in that Each primer and probe is packaged separately, and the concentration of each primer is independently 9 to 11 μmol / L, and the concentration of each probe is independently 9 to 11 μmol / L.

5. The kit according to claim 4, characterized in that The kit also includes a negative control substance and a positive control substance. The negative control substance is non-transgenic corn genomic DNA, and the positive control substance is genomic DNA of a transgenic corn GG2 transformant.

6. A fluorescent quantitative PCR detection method for herbicide-resistant transgenic corn GG2 transformants, characterized in that: The following steps are involved: S1. Extract genomic DNA of the sample to be tested; S2. Using the extracted genomic DNA of the sample to be tested as a template, the primer-probe combination described in claim 1 or 2 or the kit described in any one of claims 3 to 5 is used to perform fluorescent quantitative PCR amplification, and based on the amplification results, a qualitative judgment and / or quantitative detection is performed on the presence of GG2 transformant DNA in the sample to be tested.

7. The fluorescent quantitative PCR detection method according to claim 6, characterized in that: The method of qualitative judgment is as follows: (1) If the zSSIIb gene shows positive amplification, it means that the sample to be tested is corn, and the next step of judgment is required: if the GG2 transformant DNA shows positive amplification, it means that the sample is a sample containing the herbicide-resistant transgenic corn GG2 transformant; if the GG2 transformant DNA does not show positive amplification, it means that the sample does not contain the herbicide-resistant transgenic corn GG2 transformant; (2) If the zSSIIb gene is not amplified, it means that the sample to be tested is not corn, but a sample that does not contain herbicide-tolerant transgenic corn GG2 transformants.

8. The fluorescent quantitative PCR detection method according to claim 6, characterized in that: The quantitative detection method is as follows: PCR amplification is performed on GG2 transformant DNA standard solutions of different concentrations, thereby establishing a standard curve between the Ct values ​​obtained after amplification and the logarithmic value of the corresponding GG2 transformant DNA concentration, substituting the Ct value of the DNA extracted from S1 after amplification into the standard curve, and quantitatively obtaining the GG2 transformant DNA concentration in the corn sample to be tested.

9. The fluorescent quantitative PCR detection method according to any one of claims 6 to 8, characterized in that: The reaction system of fluorescence quantitative PCR was 25 μL: 2×TaqMan FastAdvanced Master Mix 12.5 μL, 10 μmol / L forward primer 1.0 μL, 10 μmol / L reverse primer 1.0 μL, 10 μmol / L probe 0.5 μL, 25 ng / μL DNA template 2.0 μL, and ddH2O 8.0 μL.

10. The fluorescent quantitative PCR detection method according to claim 9, characterized in that: The reaction program of fluorescence quantitative PCR was as follows: 95°C, 5 min; 95°C, 15 s; 60°C, 60 s; 45 cycles.

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