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

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 cotton, and belongs to the technical field of biological detection. The primer probe combination for the fluorescent quantitative PCR detection of the herbicide-resistant cotton GGK2 comprises a primer pair and a probe for detecting the genome DNA of a transformant of the herbicide-resistant transgenic cotton GGK2 and a primer pair and a probe for detecting a standard gene ACP in the cotton. The method not only can qualitatively detect whether the to-be-detected cotton sample contains the GGK2 transformant, but also can quantitatively detect the content of the GGK2 transformant, and the lowest detection limit can reach 10 copies / reaction. The method has important practical significance for promoting the sustainable and healthy development of the cotton industry, can provide reference for perfecting a transgenic product detection system, and provides powerful technical support for monitoring the safety of transgenic organisms.

Description

Technical Field

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

[0002] Since the advent of the first genetically modified crop, kanamycin-resistant tobacco, in 1983, genetically modified technology has attracted widespread attention from many countries and organizations in the world. Subsequently, genetically modified tomatoes with delayed maturity began to be commercialized in 1994, and genetically modified crops began to be planted on a large scale in 1996. In 2023, the global planting area of ​​genetically modified crops reached 206.3 million hectares. With the widespread planting of genetically modified crops around the world, it has not only brought huge social, economic and ecological benefits, but also brought huge safety issues. Therefore, many countries and regions in the world have begun to implement the genetically modified product labeling management system and set labeling thresholds. Among them, the establishment of a specific, sensitive and accurate fluorescence quantitative detection method for genetically modified crop transformants provides effective technical guarantees for the effective supervision of genetically modified products and the smooth implementation of the labeling system.

[0003] Cotton is a high-value-added cash crop rich in protein, oil and fiber. It is an important strategic material related to the national economy and people's livelihood and an industrial raw material for the cotton textile industry. The industrial chain involves cotton processing, circulation, textiles, clothing, exports and other industries. By the end of 2019, the global genetically modified cotton planting area had reached 25 million hectares, accounting for 76% of the world's total cotton planting area. Since the 1990s, cotton bollworm disasters have broken out in the northern cotton-growing areas of my country for many years, and the lint production has been greatly reduced, causing serious economic losses. And with the increasing demand for cottonseed (meal) in my country, my country's imports of cotton are also increasing year by year. Therefore, my country pays great attention to the agricultural production of domestic cotton and actively develops new varieties of genetically modified cotton in order to promote the further development of the cotton industry.

[0004] Herbicide-resistant cotton GGK2 is a new type of transgenic cotton. By co-expressing two glyphosate-resistant genes GR79EPSPS and GAT with different mechanisms of action, it has obtained the characteristics of high glyphosate resistance and low residue. Among them, GR79 EPSPS protein has low affinity for glyphosate molecules, which can overcome the competitive inhibition of herbicides, thereby avoiding the lethal effect of glyphosate herbicides on plants. The invention patent with application number "201610329846.9" and invention name "Identification of molecular markers of bivalent herbicide-resistant cotton GGK2 and application" discloses a method for identifying bivalent herbicide-resistant cotton GGK2, but it has the defects of high detection limit and can only perform qualitative detection, not quantitative detection. In actual work, in order to improve the detection efficiency, several cotton samples are usually mixed for detection, which requires high detection sensitivity. Therefore, it is particularly important to develop a kit and detection method for quantitative detection of herbicide-resistant cotton GGK2. Summary of the invention

[0005] 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 cotton, which improves the sensitivity of herbicide-resistant cotton GGK2 and can achieve quantitative detection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a primer-probe combination for fluorescent quantitative PCR detection of herbicide-resistant cotton, comprising the following components:

[0008] (1) Upstream primer GGK2-QF, downstream primer GGK2-QR and probe GGK2-QP for detecting genomic DNA of herbicide-tolerant transgenic cotton GGK2 transformants;

[0009] The nucleotide sequence of the upstream primer GGK2-QF is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer GGK2-QR is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe GGK2-QP is shown in SEQ ID NO: 3;

[0010] (2) upstream primer ACP-QF, downstream primer ACP-QR and probe ACP-QP for detecting the standard gene ACP in cotton;

[0011] The nucleotide sequence of the upstream primer ACP-QF is shown in SEQ ID NO: 4, the nucleotide sequence of the downstream primer ACP-QR is shown in SEQ ID NO: 5, and the nucleotide sequence of the probe ACP-QP is shown in SEQ ID NO: 6.

[0012] Preferably, the 5' end of the probe GGK2-QP and the probe ACP-QP 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 ROX, FAM, HEX or CY5, and the fluorescent quencher group is selected from TAMRA, BHQ1 or BHQ2.

[0013] The present invention also provides a kit for PCR detection of herbicide-resistant transgenic cotton GGK2 transformants, comprising the primer-probe combination.

[0014] Preferably, each primer and probe is packaged separately, the concentration of each primer is independently 10 μmol / L, and the concentration of each probe is independently 10 μmol / L.

[0015] Preferably, the kit further comprises a negative control and a positive control, wherein the negative control is non-transgenic cotton genomic DNA, and the positive control is genomic DNA of a transgenic cotton GGK2 transformant target sequence; the transgenic cotton GGK2 transformant target sequence is the amplified fragment of the upstream primer GGK2-QF and the downstream primer GGK2-QR.

[0016] The present invention also provides a fluorescent quantitative PCR detection method for herbicide-resistant transgenic cotton GGK2 transformants, the detection method comprising the following steps:

[0017] S1. Extracting genomic DNA of cotton samples to be tested;

[0018] S2. Using the extracted genomic DNA of the cotton sample to be tested as a template, the primer probe combination or the kit is used to perform fluorescent quantitative PCR amplification, and qualitatively judge and / or quantitatively detect the presence of GGK2 transformant DNA in the sample to be tested based on the amplification result.

[0019] Preferably, the method of qualitative judgment is as follows:

[0020] (1) If the ACP gene is amplified, it indicates that the sample to be tested is cotton, and the next step of judgment is required: if the GGK2 transformant DNA in the sample is amplified, it indicates that the sample is a sample containing the herbicide-resistant transgenic cotton GGK2 transformant; if the GGK2 transformant DNA in the sample is not amplified, it indicates that the sample does not contain the herbicide-resistant transgenic cotton GGK2 transformant;

[0021] (2) If the ACP gene is not amplified, it means that the sample to be tested is not cotton and does not contain herbicide-tolerant transgenic cotton GGK2 transformants.

[0022] Preferably, the quantitative detection is as follows: PCR amplification is performed on GGK2 transformant DNA standard solutions of different concentrations, thereby establishing a standard curve between the Ct values ​​obtained after amplification and the logarithmic values ​​of the corresponding GGK2 transformant DNA concentrations, substituting the Ct value of the DNA extracted from S1 after amplification into the standard curve, and quantitatively obtaining the GGK2 transformant DNA concentration in the cotton sample to be tested.

[0023] Preferably, the reaction system of fluorescent quantitative PCR is 25 μL: FastAdvanced MasterMix 12.5μL, 10μmol / L forward primer 1.0μL, 10μmol / L reverse primer 1.0μL, 10μmol / L probe 0.5μL, 25ng / μL genomic DNA of the sample to be tested 2μL, ddH2O 8.0μL.

[0024] Preferably, the reaction procedure of fluorescent quantitative PCR is: 95°C, 5 min; 95°C, 15 s; 60°C, 60 s; 45 cycles.

[0025] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention specifically screens the primer probe for real-time fluorescence quantitative PCR detection of herbicide-resistant cotton GGK2 transformants, obtains a pair of primers and probe combinations with the best sensitivity and specificity from a large number of primer probe groups, and on this basis, uses a standard product with a suitable concentration gradient to establish and optimize the real-time fluorescence quantitative PCR detection method of herbicide-resistant cotton GGK2 transformants. The present invention also provides a kit containing the primer pair and 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 ACP gene in cotton, which can not only qualitatively detect whether the cotton sample to be tested contains GGK2 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 cotton industry, and can also provide a reference for improving my country's genetically modified product detection system and provide strong technical support for the supervision of genetically modified biosafety in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 cotton GGK2 transformant by the primer-probe combinations No. 1 to No. 19 designed in Example 1 were respectively obtained.

[0027] Figure 2 This is the fluorescence curve of real-time fluorescence quantitative PCR amplification of genomic DNA of positive standard of herbicide-tolerant cotton GGK2 transformant using primer-probe combination No. 8 under different concentration conditions.

[0028] Figure 3 This is the fluorescence amplification curve of the specific results of the No. 8 primer-probe combination for the detection of the positive standard genomic DNA of the herbicide-resistant cotton GGK2 transformant and the genomic DNA of other transgenic samples, the negative control and the blank control.

[0029] Figure 4 The fluorescence curve and standard curve are obtained by real-time fluorescence quantitative PCR amplification of genomic DNA of positive standard of herbicide-tolerant cotton GGK2 transformant at different concentrations using primer-probe combination No. 8.

[0030] Figure 5 The fluorescence amplification curve is the sensitivity result of primer-probe combination No. 8 for detecting 100% and 10 copies of herbicide-tolerant cotton GGK2 transformant positive standard genomic DNA, negative control and blank control.

[0031] Figure 6 The fluorescence curve and standard curve are obtained by real-time fluorescence quantitative PCR amplification of cotton internal standard gene ACP genomic DNA using internal standard primer and probe combination.

[0032] Figure 7 This is the fluorescence curve and standard curve of real-time fluorescence quantitative PCR amplification for detecting genomic DNA of herbicide-tolerant cotton GGK2 transformants using primer-probe combination No. 8. DETAILED DESCRIPTION

[0033] 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.

[0034] 1. The instruments, reagents and biological materials used in the present invention are as follows:

[0035] 1. Experimental instruments

[0036] Spectrophotometer: Thermo ND-1000;

[0037] Centrifuge: Thermo Biofuge Primo R;

[0038] Real-time fluorescence quantitative PCR instrument: BioRad CFX96, QuantStudio 3;

[0039] Other instruments include water bath, precision balance, fume hood, biological safety cabinet, etc.

[0040] 2. Enzymes and Reagents

[0041] 2×TaqMan Fast Advanced Master Mix (purchased from Applied Biosystems official website);

[0042] Plant genomic DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd.).

[0043] 3. Positive control and / or standard

[0044] Homozygous herbicide-tolerant cotton GGK2 genomic DNA was used as a positive control and / or standard.

[0045] Non-transgenic cotton genomic DNA was used as a negative control.

[0046] 4. Other plant materials

[0047] The samples for specific experimental verification are as follows:

[0048] Other genetically modified cotton mixtures (MON1445, MON531, MON15985, LLCOTTON25, MON88913, GHB614, and COT102 in equal mass ratios were mixed into one sample);

[0049] 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);

[0050] A mixture of genetically modified corn (Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON87460, DAS40278-9, 4114, MON87427, and 5307 were mixed in equal proportions to form one sample);

[0051] 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);

[0052] 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).

[0053] 2. The common methods and criteria in the following embodiments are as follows:

[0054] 1. PCR amplification qualification criteria:

[0055] a. In the positive control, the ACP gene and GGK2 transformant DNA both had typical amplification curves, and the Ct value was ≤36;

[0056] b. In the negative control, the ACP gene has a typical amplification curve and the Ct value is ≤36; the GGK2 transformant DNA has no typical amplification curve, or the Ct value is ≥40;

[0057] c. In the blank control, there is no typical amplification curve for ACP gene and GGK2 transformant DNA, or the Ct value is ≥40;

[0058] d. Determination coefficient R of the standard curve 2 ≥0.98, -3.1≥Standard curve slope (b) ≥-3.6.

[0059] 2. Qualitative judgment principles for samples to be tested:

[0060] If the real-time fluorescence quantitative PCR detection of the internal standard gene ACP gene and the GGK2 transformant DNA in the cotton sample to be tested shows a typical amplification curve and the Ct value is ≤36, the sample to be tested is judged to be positive for the GGK2 transformant;

[0061] If a typical amplification curve appears in the real-time fluorescence quantitative PCR test of the internal standard gene ACP gene in the cotton sample to be tested 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 GGK2 transformant DNA, the sample to be tested is judged to be negative for the GGK2 transformant;

[0062] If no typical amplification curve or Ct value > 36 appears in the real-time fluorescence quantitative PCR detection of the internal standard gene ACP gene in the cotton sample to be tested, it is judged that the sample to be tested is negative for GGK2 transformant.

[0063] 3. Principles of quantitative detection of samples to be tested:

[0064] If typical amplification curves appear in the detection of the internal standard gene ACP gene and the GGK2 transformant DNA in the cotton sample to be tested, and the GGK2 transformant DNA content is higher than the quantitative limit, the GGK2 transformant content is calculated;

[0065] If typical amplification curves appear in the detection of the internal standard gene ACP gene and the GGK2 transformant DNA in the cotton sample to be tested, but the GGK2 transformant DNA content is lower than the quantitative limit, the GGK2 transformant content will not be calculated.

[0066] 4. The sequence information of the primer pair and probe for detecting herbicide-tolerant cotton GGK2 transformants and the primer pair and probe for detecting the standard gene ACP gene in cotton are shown in Table 1 below:

[0067] Table 1 Primer pairs and probe sequences for detecting cotton GGK2 transformants and internal standard gene ACP gene

[0068]

[0069] Example 1. Design and optimization of primer-probe combination for real-time fluorescence quantitative PCR of herbicide-tolerant cotton

[0070] According to the exogenous insertion sequence of transgenic cotton GGK2 and its 5' and 3' flanking sequence information, the primer design software Primer Premier 5 was used to comprehensively consider the GC content of the nucleotide sequence and the primer Tm value. A number of primers and probes for herbicide-resistant cotton GGK2 transformants were designed for the 5' and 3' sequences of the insertion position. By arranging and combining the obtained primer and probe sequences, a total of 19 primer-probe combinations were obtained. The specific primer-probe combination information is shown in Table 2:

[0071] Table 2 Primer probe combination information

[0072]

[0073]

[0074]

[0075] 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.

[0076] Using the genomic DNA of the positive standard of the herbicide-tolerant cotton GGK2 transformant as a template, the primer-probe combinations in Table 2 were used to perform real-time fluorescence quantitative PCR amplifications to select and determine the best real-time fluorescence quantitative PCR primer-probe combination.

[0077] The 25 μL reaction system for real-time fluorescence quantitative PCR amplification detection includes Fast Advanced Master Mix (purchased from Thermo Fisher Scientific, catalog number 4444557), 10 μmol / L forward primer, 10 μmol / L reverse primer, 10 μmol / L probe, 25 ng / μL DNA template, add ddH2O to 25 μL, the specific reaction system is shown in Table 3, placed in a real-time fluorescence quantitative PCR instrument for reaction, and the reaction procedure is shown in Table 4.

[0078] Table 3 25 μL reaction system for real-time fluorescence quantitative PCR amplification detection

[0079]

[0080]

[0081] Table 4 Real-time fluorescence quantitative PCR reaction program

[0082]

[0083] 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 3 times, and the amplification curve obtained for each reaction is shown in Figure 1 The Ct value of each reaction and the average Ct value are shown in Table 5.

[0084] Table 5 Real-time fluorescence quantitative PCR amplification Ct values ​​under different primer-probe combinations

[0085]

[0086]

[0087] 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.

[0088] 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 cotton GGK2 transformant by the primer-probe combinations No. 1 to No. 19 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. 19 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 8 5QF3 / 5QR2 / 5P2 was appropriate, the amplification signal was strong, and the Ct value was the smallest. Therefore, the primer-probe combination 8 shown in Table 2 was selected as the primer-probe combination for real-time fluorescence quantitative PCR of herbicide-tolerant cotton.

[0089] Example 2. Selection of primer and probe combination concentrations for real-time fluorescence quantitative PCR of herbicide-tolerant cotton

[0090] In this embodiment, the primer-probe combination No. 8 determined in Example 1 is used to perform real-time fluorescence quantitative PCR amplification on the genomic DNA of the positive standard of the herbicide-resistant cotton GGK2 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 cotton GGK2 transformant provided by the present invention, wherein the 25 μL reaction system is basically as shown in Table 3 in Example 1, except that 1 to 5 groups of primer-probe combinations with different concentrations are designed in this embodiment, wherein the corresponding final primer concentration and final probe concentration in each group of reaction systems are shown in Table 6, and the reaction procedure is the same as Table 4 in Example 1. During the reaction, the fluorescence signal is collected and the amplification curve is read. Each reaction is set up in 3 parallels, and the amplification curve obtained for each reaction is shown in Table 6. Figure 2 The Ct value of each reaction and the average Ct value are shown in Table 6.

[0091] Table 6 Different primer-probe concentration combinations and corresponding real-time fluorescence quantitative PCR amplification Ct values

[0092]

[0093]

[0094] Figure 2 The fluorescence curves of real-time fluorescence quantitative PCR amplification of the genomic DNA of the positive standard of the herbicide-tolerant cotton GGK2 transformant using the primer-probe combination No. 8 determined in Example 1 under different concentration conditions. 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 national standard "Qualitative PCR Method for Detecting Standard Genes in Cotton for Genetically Modified Plants and Their Product Components" (Ministry of Agriculture Announcement No. 1943-1-2013), the primer probe concentration combination of combination 3 in Table 6 is determined to be the optimal primer probe concentration in the real-time fluorescence quantitative PCR method for herbicide-resistant cotton GGK2. It can be seen that the optimal primer probe concentration combination obtained after screening is: the final concentration of the primer is 0.4μmol / L, and the final concentration of the probe is 0.2μmol / L.

[0095] Example 3: Specific detection of real-time fluorescence quantitative PCR primer-probe combination for herbicide-tolerant cotton

[0096] The primer probe combination No. 8 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 cotton GGK2 transformants and the genomic DNA of five other transgenic samples (transgenic cotton mixed sample, transgenic soybean mixed sample, transgenic corn mixed sample, transgenic rapeseed mixed sample, transgenic rice mixed sample) as templates, negative controls 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 cotton GGK2 transformants provided by the present invention. The 25 μL reaction system is shown in Table 3 of Example 1, the reaction procedure is shown in Table 4 of Example 1, the fluorescence signal is collected during the reaction, and the amplification curve is obtained. The results are shown in Table 4 of Example 1. Figure 3 .

[0097] Figure 3 The specific fluorescence amplification curve of the primer-probe combination No. 8 determined in Example 1 for the detection of the positive standard genomic DNA of the herbicide-tolerant cotton GGK2 transformant and other transgenic sample genomic DNA, negative control and blank control. Figure 3 It can be seen that an amplification curve appeared in the detection of the positive standard sample of the herbicide-resistant cotton GGK2 transformant, while no amplification curve appeared in the detection results of the samples of the other five transgenic mixtures, the negative control and the blank control samples. This result shows that the primer probe combination and detection method for real-time fluorescence quantitative PCR for herbicide-resistant cotton provided by the present invention have good specificity.

[0098] Example 4: Linear interval and sensitivity detection of primer-probe combination for real-time fluorescence quantitative PCR of herbicide-tolerant cotton

[0099] 1. Detection of linear interval

[0100] The primer probe combination No. 8 determined in Example 1 was used to detect different concentrations of the positive standard genomic DNA of the herbicide-resistant cotton GGK2 transformant to determine the linear range of the primer probe combination for the herbicide-resistant cotton GGK2 genome real-time fluorescence quantitative PCR provided by the present invention. The preparation method of the positive standard genomic DNA of the herbicide-resistant cotton GGK2 transformant at different concentrations is as follows: the positive standard genomic DNA of the herbicide-resistant cotton GGK2 transformant is diluted to 25 ng / μL, and then gradiently diluted with ddH2O to obtain a series of concentrations of the positive standard genomic DNA of the cotton GGK2 transformant (see Table 7) as a template for real-time fluorescence quantitative PCR reaction.

[0101] Table 7 DNA quantity and copy number in standard products

[0102] Standard product number DNA(ng) / reaction Copy number / reaction S1 50 21900 S2 10 4380 S3 2 876 S4 0.4 175 S5 0.08 35 S6 0.04 18

[0103] The 25 μL reaction system is shown in Table 3 of Example 1, and the reaction procedure is shown in Table 4 of Example 1. Each test under the same conditions was repeated twice in parallel. During the reaction, the fluorescence signal was collected, and the amplification results were detected. The obtained fluorescence amplification curves and Ct value results are shown in Table 4. Figure 4 and as shown in Table 8.

[0104] Table 8 Standard curve linear repeated measurement data statistics

[0105]

[0106] 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 the herbicide-tolerant cotton GGK2 transformant at different concentrations by the primer-probe combination No. 8 determined in Example 1, wherein:

[0107] 1. Standard S1 (21900 copies / reaction);

[0108] 2. Standard S2 (4380 copies / reaction);

[0109] 3. Standard S3 (876 copies / reaction);

[0110] 4. Standard S4 (175 copies / reaction);

[0111] 5. Standard S5 (35 copies / reaction);

[0112] 6. Standard S6 (18 copies / reaction).

[0113] Depend on Figure 4 As can be seen from the results in Table 8, in the two repeated experiments, the standard curves all met the amplification efficiency of 90% to 110%, the slope was -3.6 to -3.1, and R 2 ≥0.98, which complies with the relevant provisions of the national standard “Guidelines for the Development of Real-time Fluorescence Quantitative PCR Methods for Detection of Components of Genetically Modified Plants and Their Products” (Ministry of Agriculture Announcement No. 2259-5-2015) (hereinafter referred to as “quantitative standard”), indicating that this method has good linearity in the copy number range of 21900-18.

[0114] 2. Sensitivity detection

[0115] According to the above linear interval test results, the genomic DNA standard of the positive standard of the herbicide-resistant cotton GGK2 transformant was diluted to 10 copies (equivalent to a mass fraction of 0.05% GGK2, and 50 ng of cotton genomic DNA template containing the specific sequence of the herbicide-resistant cotton GGK2 transformant was added to the PCR detection amplification system for measurement) for sensitivity testing, and the samples were subjected to real-time fluorescence quantitative PCR amplification test. The 25 μL reaction system is shown in Table 3 of Example 1, and the reaction procedure is shown in Table 4 of Example 1. Repeat 60 parallel tests. During the reaction, the fluorescence signal was collected, and the amplification results were detected. The obtained fluorescence amplification curve is as shown in FIG. Figure 5 shown.

[0116] Figure 5 The fluorescence amplification curve is the sensitivity result of the primer-probe combination No. 8 determined in Example 1 for detecting 100% and 10 copies of the herbicide-tolerant cotton GGK2 transformant positive standard genomic DNA, negative control and blank control. Figure 5 It can be seen that typical amplification curves were obtained in all 60 parallel tests, thereby determining the minimum detection limit to be 10 copies / reaction or 0.05% (calculated by adding 50 ng of cotton genomic DNA template containing the herbicide-resistant cotton GGK2 transformant-specific sequence into the PCR detection amplification system).

[0117] Example 5: Limit of Quantitation Test of Real-time Fluorescence Quantitative PCR Primer Probe Combination for Herbicide Tolerant Cotton

[0118] In this example, the genomic DNA of the positive standard of the herbicide-resistant cotton GGK2 transformant was diluted to 100 copies as the sample to be tested, and the primer probe combination No. 8 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 3 of Example 1, and the reaction procedure is shown in Table 4 of Example 1. 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 9.

[0119] Table 9 Quantitative detection limit test results

[0120]

[0121] It can be seen from the results in Table 9 that the deviation of the measurement results of 100 copies of the sample is -2.70%, which does not exceed 25% of the standard value, and the RSDr (relative standard deviation) is 3.66%, 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.

[0122] Example 6: Quantitative detection of herbicide-tolerant cotton GGK2 transformant genes in actual samples

[0123] 1. Establishment of standard curve

[0124] The internal standard gene ACP gene and GGK2 transformant gene standards with a copy number of 21900, 4380, 876, 175, and 35 were used as positive templates, and ddH2O was used as a negative control template. The reaction system shown in Table 3 in Example 1 and the reaction program shown in Table 4 were used to perform real-time fluorescence quantitative PCR amplification of the internal standard gene ACP gene and the GGK2 transformant gene, respectively, to establish a standard curve for real-time fluorescence quantitative PCR for detecting herbicide-resistant cotton GGK2 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. Figure 6 , Figure 7 As shown in Table 10.

[0125] Table 10 Real-time fluorescence quantitative PCR results of transgenic cotton GGK2 transformants

[0126]

[0127]

[0128] 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.

[0129] Figure 6 The fluorescence curve and standard curve are obtained by real-time fluorescence quantitative PCR amplification of the cotton internal standard gene ACP genomic DNA 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. 8 determined in Example 1 for detecting genomic DNA of herbicide-resistant cotton GGK2 transformants, wherein:

[0130] 1. Standard S1 (21900 copies); 2. Standard S2 (4380 copies); 3. Standard S3 (876 copies); 4. Standard S4 (175 copies); 5. Standard S5 (35 copies); 6. GGK2 (5%); 7. GGK2 (2%); 8. GGK2 (0.5%); 9. Limit of quantification (100 copies). 5%, 2% and 0.5% represent samples of herbicide-resistant cotton GGK2 powder mixed with non-transgenic cotton powder, with the mass fractions of herbicide-resistant cotton GGK2 being 5%, 2% and 0.5% respectively.

[0131] like Figures 6-7As shown in Table 10, the slope of the standard curve of the herbicide-tolerant cotton GGK2 transformant-specific real-time fluorescence quantitative PCR method is -3.6 to -3.1, the amplification efficiency is 90% to 110%, and the correlation coefficient R 2 ≥0.98, all met the requirements of the quantitative standard.

[0132] 2. Quantitative detection and determination methods of samples to be tested

[0133] According to the standard curve of the GGK2 transformant DNA standard and the internal standard ACP 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 GGK2 transformant DNA and ACP gene in the sample to be tested are calculated, and the content of the GGK2 transformant in the sample to be tested is obtained.

[0134] 3. Testing of actual samples

[0135] The herbicide-resistant cotton GGK2 powder was mixed with the non-transgenic cotton powder to prepare samples with a mass fraction of 5%, 2% and 0.5% of the herbicide-resistant cotton GGK2, respectively, as test samples, and the correctness and precision of the primer probe combination No. 8 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 national standard "Qualitative PCR Method for Detecting Internal Standard Genes in Transgenic Plants and Their Product Components in Cotton" (Ministry of Agriculture Announcement No. 1943-1-2013) (sequences are shown in SEQ ID NO: 4-6).

[0136] 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 (%) = GGK2 copy number / ACP copy number × 100%. The statistical results are shown in Table 11.

[0137] Table 11 Calculation results of genetically modified ingredient content

[0138]

[0139] Table 11 shows that the deviations of the determination results of the herbicide-resistant cotton GGK2 samples with mass fractions of 5%, 2% and 0.5% were 0.00%, 0.50% and -2.00% respectively, and the RSDr were 0.28%, 0.35% and 2.89% respectively, all of which met the requirements of the quantitative standard. It can be seen that the herbicide-resistant cotton GGK2 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 cotton GGK2 components in transgenic samples.

[0140] 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 cotton GGK2, which can achieve qualitative detection of herbicide-resistant cotton GGK2.

[0141] 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 cotton, characterized in that: Includes the following components: (1) Upstream primer GGK2-QF, downstream primer GGK2-QR and probe GGK2-QP for detecting genomic DNA of herbicide-tolerant transgenic cotton GGK2 transformants; The nucleotide sequence of the upstream primer GGK2-QF is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer GGK2-QR is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe GGK2-QP is shown in SEQ ID NO: 3; (2) upstream primer ACP-QF, downstream primer ACP-QR and probe ACP-QP for detecting the standard gene ACP in cotton; The nucleotide sequence of the upstream primer ACP-QF is shown in SEQ ID NO: 4, the nucleotide sequence of the downstream primer ACP-QR is shown in SEQ ID NO: 5, and the nucleotide sequence of the probe ACP-QP 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 GGK2-QP and the probe ACP-QP 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, HEX, ROX or CY5, and the fluorescent quencher group is selected from BHQ1, BHQ2 or TAMRA.

3. A kit for PCR detection of herbicide-resistant transgenic cotton GGK2 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 10 μmol / L, and the concentration of each probe is independently 10 μmol / L.

5. The kit according to claim 4, characterized in that The kit also includes a negative control and a positive control. The negative control is non-transgenic cotton genomic DNA, and the positive control is genomic DNA of a transgenic cotton GGK2 transformant target sequence. The transgenic cotton GGK2 transformant target sequence is an amplified fragment of an upstream primer GGK2-QF and a downstream primer GGK2-QR.

6. A fluorescent quantitative PCR detection method for herbicide-resistant transgenic cotton GGK2 transformants, characterized in that: The following steps are involved: S1. Extracting genomic DNA of cotton samples to be tested; S2. Using the extracted genomic DNA of the cotton 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 GGK2 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 ACP gene is amplified, it indicates that the sample to be tested is cotton, and the next step of judgment is required: if the GGK2 transformant DNA in the sample is amplified, it indicates that the sample is a sample containing the herbicide-resistant transgenic cotton GGK2 transformant; if the GGK2 transformant DNA in the sample is not amplified, it indicates that the sample does not contain the herbicide-resistant transgenic cotton GGK2 transformant; (2) If the ACP gene is not amplified, it means that the sample to be tested is not cotton and does not contain herbicide-tolerant transgenic cotton GGK2 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 GGK2 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 GGK2 transformant DNA concentration, substituting the Ct value of the DNA extracted from S1 after amplification into the standard curve, and quantitatively obtaining the GGK2 transformant DNA concentration in the cotton 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 genomic DNA of the sample to be tested 2 μL, and 8.0 μL of ddH2O.

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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