Quantitative detection method of nucleic acid sample and application

Abstract

The invention provides a probe and primer combination, a standard substance, a detection kit, a Real-time PCR detection method and application of the Real-time PCR detection method in detection of a nucleic acid sample.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and in particular relates to a probe, a probe and primer combination, a standard, a Real-time PCR detection kit and a detection method for detecting nucleic acid samples. Background Art

[0002] The rice transformant RN85zN-eH is a transgenic rice material that is insect-resistant and tolerant to the herbicide glufosinate (Chinese patent application number 202510099723X). It exhibits excellent resistance to lepidopteran pests such as the Chilo suppressalis (Stichopus suppressalis) and good tolerance to glufosinate. This transformant can be used to breed insect-resistant and herbicide-resistant rice varieties. Establishing a transformant-specific detection method can provide an effective means for the identification and regulation of genetically modified organisms, offering technical support for the safety management of agricultural genetically modified organisms.

[0003] Transformant-specific detection primarily relies on conventional PCR methods, but these methods suffer from low sensitivity and the inability to monitor the PCR process in real time, resulting in quantification. With the advancement of molecular biology techniques, real-time PCR (real-time fluorescence quantitative PCR) has become widely used in transgenic detection. Compared to conventional PCR, it boasts shorter processing times, simpler operation, better specificity, and higher sensitivity. The process can be monitored in real time, and the results can be directly observed, allowing for quantitative detection.

[0004] Real-time PCR methods can be divided into two types: dye-based and probe-based. The fluorescent dyes used in dye-based real-time PCR can bind to double-stranded DNA and emit fluorescence. However, this binding is non-specific, as primer dimers and DNA templates in the system will also bind to them, resulting in a low specificity for the dye-based method. In contrast, the probes in the probe-based method can specifically bind to the template, and their amplification curves reflect the accumulation of specific products without any non-specific amplification components. Their sensitivity is 10 times higher than that of the dye-based method. Furthermore, the dye-based method only supports single-channel reactions. Therefore, if multi-channel experiments are required, or if different targets in the same sample are to be detected, the probe-based method is the most commonly used method. Summary of the Invention

[0005] To solve the above problems, the present invention provides probes, probe and primer combinations, standards, detection kits, and Real-time PCR detection methods for detecting nucleic acid samples. The above-mentioned nucleic acid sample can be the genomic DNA of the rice RN85zN-eH transformant, or the genomic DNA of a derivative line of the RN85zN-eH transformant, or the genomic DNA of a mixed rice material containing the RN85zN-eH transformant, or a nucleic acid sample containing the RN85zN-eH identity information obtained by separating the above-mentioned samples through PCR amplification or other methods. Since the sequence shown in SEQ ID NO.1 is a specific sequence for confirming the identity information of the RN85zN-eH transformant, any sample containing the nucleic acid molecule shown in SEQ ID NO.1 can be detected using the method provided by the present invention.

[0006] By designing highly sensitive and specific probe and primer pairs, the present invention accurately identifies rice RN85zN-eH transformants and separates them from conventional rice that does not contain RN85zN-eH and other transgenic rice materials. This detection method offers advantages such as high specificity, sensitivity, and ease of use, overcoming the drawbacks of conventional PCR methods, which often require complex procedures and low sensitivity.

[0007] The present invention provides a probe, characterized in that the nucleotide sequence of the probe is 5'-AGTCGCACCCATGGCAATTAAGGACC-3'.

[0008] In some embodiments, the probe is labeled with a fluorescent group at its 5' end and a quencher at its 3' end. When the probe is in a free state, the fluorescence emitted by the fluorescent group is absorbed by the quencher. During PCR amplification, the fluorescent group at the 5' end of the probe, which is tightly bound to the template, is cleaved by the Taq enzyme, separating it from the quencher at the 3' end. The fluorescence emitted by the fluorescent group can be detected by the instrument, and the generated fluorescence signal is proportional to the amount of amplified product in the sample.

[0009] In some embodiments, the fluorescent group includes any one of FAM, TET, HEX, CY3, JOE, VIC, ROX, CY5, TAMRA, or Texas; the quencher group includes any one of BHQ1, BHQ2, BHQ-X, TAMRA, DABCYL, or MGB;

[0010] In some embodiments, the fluorescent group / quencher group combination is any one of FAM / BHQ1, FAM / BHQ2, CY3 / BHQ-X, HEX / DABCYL, JOE / TAMRA, or VIC / BHQ2;

[0011] In tests using randomly selected fluorophores and quenchers, the aforementioned probes all produced specific detection results. Furthermore, probes labeled with FAM at the 5' end and BHQ1 at the 3' end offered the lowest cost and were considered the most preferred probe labeling scheme.

[0012] The present invention also provides a primer and probe combination, characterized in that: it comprises the above-mentioned probe and two primers, the nucleotide sequences of the primers are 5'-GGCTGTGGGTGCATGGTT-3' and 5'-TTGTTTACACCACCCAGGGTTT-3';

[0013] The above-mentioned probes and probe and primer combinations were selected through software design and experimental screening and verification, and are located at the upstream boundary of the exogenous insertion sequence of the RN85zN-eH transformant.

[0014] The present invention also provides a standard, characterized in that: the standard is one or more nucleic acid samples with a concentration of not less than 20 copies / μL; the nucleic acid sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1;

[0015] In some embodiments, the standard comprises five standards with concentrations of 2×10 5 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 copies / μL and 2×10 copies / μL of RN85zN-eH genomic DNA; the DNA sample contains the nucleic acid molecule shown in SEQ ID NO.1;

[0016] In some embodiments, the preparation method of the standard is as follows: take a genomic DNA sample of RN85zN-eH with a concentration of 1.0 μg / μL, dilute it 10 times, 10 times, 2 times, 10 3 times, 10 4 times, 10 5 times.

[0017] The present invention also provides a detection kit, characterized in that: the detection kit comprises the above-mentioned probe and primer combination and the above-mentioned standard;

[0018] In some embodiments, the detection kit comprises:

[0019] Primer 1, sequence: 5′-GGCTGTGGGTGCATGGTT-3′;

[0020] Primer 2, sequence: 5′-TTGTTTACACCACCCAGGGTTT-3′;

[0021] probe, sequence: 5′-AGTCGCACCCATGGCAATTAAGGACC-3′;

[0022] Standards, 5 concentrations of 2 × 10 5 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 RN85zN-eH genomic DNA samples containing 10 copies / μL and 2×10 copies / μL; the nucleic acid sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.1;

[0023] The 5' end of the probe is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

[0024] The present invention also provides a real-time PCR detection method, characterized in that: the real-time PCR detection is performed using the above-mentioned detection kit, wherein the final concentrations of primer 1 and primer 2 in the PCR reaction system are both 0.4 μM, and the final concentration of the probe is 0.2 μM.

[0025] The present invention also provides the use of the above-mentioned probe, probe and primer combination, standard, detection kit, and detection method in the qualitative or quantitative detection of nucleic acid samples; wherein the nucleic acid sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.1.

[0026] The beneficial effects of the present invention are as follows: through software design and multiple experimental screening, a combination of one probe and two primers was obtained from a large number of probe-primer combinations. On this basis, a real-time PCR detection method was established and optimized using standard substances with an appropriate concentration gradient. The application of the above-mentioned probe, probe and primer combination, standard substance, detection kit, and real-time PCR detection method can specifically detect nucleic acid samples containing SEQ ID NO. 1 at a concentration of not less than 20 copies / μL, and can effectively distinguish RN85zN-eH materials from other rice materials that do not contain RN85zN-eH, with extremely high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Other features, objects and advantages of the present application will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0028] Figure 1 Sensitivity test curve. Among them, 1:2×10 5copies / μL; 2: 2×10 4 copies / μL; 3: 2×10 3 copies / μL; 4: 2×10 2 copies / μL; 5: 2×10 copies / μL; 6: 2 copies / μL.

[0029] Figure 2 Standard curve and linear equation.

[0030] Figure 3 Specific sample detection test: 1: a mixed sample of Nipponbare and RN85zN-eH (sample 1); 2: a mixed sample of Nipponbare and RN85zN-eJ (sample 2). DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings. The following examples are only used to illustrate the present invention but do not limit the scope of the present invention.

[0032] The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. It is understood that parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, which are derived from transgenic plants previously transformed with a DNA molecule of the present invention and thus consist at least in part of transgenic cells, or their progeny.

[0033] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). A "native gene" refers to a gene found in nature that has its own regulatory sequences. A "chimeric gene" refers to any gene that is not a natural gene and contains regulatory and coding sequences that are not found in nature. An "endogenous gene" refers to a natural gene that is located in its natural location in the genome of an organism. An "exogenous gene" is a foreign gene that is now present in the genome of an organism and did not originally exist, and also refers to a gene that is introduced into a recipient cell through a transgenic step. Exogenous genes can include natural genes inserted into non-natural organisms or chimeric genes. A "transgene" is a gene that has been introduced into the genome through a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted can be called an "insertion site" or "target site."

[0034] Transformation procedures that cause random integration of exogenous DNA will result in transformants containing different flanking regions, which are specifically contained in each transformant. When recombinant DNA is introduced into plants through traditional hybridization, its flanking regions usually do not change. Transformants will also contain unique junctions between segments of heterologous insert DNA and genomic DNA, or between two segments of genomic DNA, or between two segments of heterologous DNA. "Junction" is the point where two specific DNA fragments connect. For example, a junction is present at the position where the insert DNA connects to the flanking DNA. Junction points are also present in transformed organisms where two DNA fragments are linked together in the manner found in natural organisms. "Junction DNA" refers to the DNA that comprises a junction point.

[0035] RN85zN-eH transformants include plants and seeds of transgenic rice RN85zN-eH, and plant cells or regenerable parts thereof, wherein the plant parts of RN85zN-eH include but are not limited to cells, pollen, ovules, flowers, shoots, roots, stems, inflorescences, leaves and products from the rice plant RN85zN-eH, such as rice, rice straw, rice husks, rice oil, cooked rice, rice flour, rice bran, rice husks and biomass remaining in rice crop fields.

[0036] The term "probe" refers to an isolated nucleic acid molecule conjugated to a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid. In the present invention, the probe is complementary to a strand of genomic DNA from the transgenic rice RN85zN-eH, whether the genomic DNA is from the transgenic rice RN85zN-eH or its seeds, or from plants, seeds, or extracts of the transgenic rice RN85zN-eH or its derivatives, or a nucleic acid molecule isolated from RN85zN-eH containing information about the identity of RN85zN-eH. The probes of the present invention include not only deoxyribonucleic acids or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0037] The term "primer" refers to an isolated nucleic acid molecule that anneals to a complementary target DNA strand through nucleic acid hybridization, forming a hybrid between the primer and the target DNA strand, which is then extended along the target DNA strand by a polymerase (e.g., DNA polymerase). The primer pairs of the present invention relate to their use in amplifying a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0038] Example 1 Design and screening of specific primer / probe combinations

[0039] Specific detection of transformants requires designing primers and probes based on the border sequences upstream and downstream of the insertion site. The PCR amplification product must include both the exogenous sequence and the rice genomic sequence. Therefore, the primers and probes must first be designed based on the border sequences of the rice RN85zN-eH insertion site.

[0040] 1. Design primer and probe combinations

[0041] A portion (100-600 bp) of the upstream or downstream border sequence (or its reverse complementary sequence) is input as a template into software (e.g., ABIPrimer Express 3.0). The template sequence must contain both the rice genomic sequence (at least 50 bp in length) and the exogenous insert sequence (at least 50 bp in length). The two template sequences are shown as SEQ ID NO. 2 and SEQ ID NO. 3.

[0042] After setting the relevant parameters in the software according to the following requirements, dozens of primer and probe combinations were obtained, each combination containing 2 primers and 1 probe.

[0043] The probes and primers meet all of the following requirements:

[0044] ① The length of the primer is between 18 and 25 bp, and the length of the probe is between 18 and 30 bp;

[0045] ②The Tm value of the primer is 58-60℃, and the Tm value of the probe is 8-10℃ higher than that of the primer;

[0046] ③ Avoid generating complementary sequences of more than 3 bases within the probe, within the primer, or between the probe and the primer;

[0047] ④The first base at the 5' end of the probe is not G;

[0048] ⑤The amplification products of the two primers must contain no less than 11bp of rice genome sequence and no less than 11bp of exogenous insertion sequence.

[0049] ⑥The length of PCR products ranges from 80 to 300 bp.

[0050] Finally, five primer and probe combinations with higher software scores were selected as candidate combinations for further screening. The candidate combinations are shown in Table 1.

[0051] Table 1 Five candidate primers and probes with higher software design scores

[0052]

[0053] 2. Primer synthesis and screening

[0054] The five primer sets shown in Table 1 were synthesized and screened for specific primers. The screening process was as follows:

[0055] (1) Primer 1 and Primer 2 of the five candidate combinations were tested using a conventional PCR amplification reaction. The electrophoresis results are shown in Table 2. The results showed that combinations A2 and A4 amplified two bands, indicating nonspecific amplification. Combinations A1, A3, and A5 amplified a single specific band of the expected size. Therefore, combinations A2 and A4 were eliminated. Primer 1 and Primer 2 of combinations A1, A3, and A5 met the requirements and were further tested.

[0056] Table 2 Specific primers for conventional PCR screening

[0057]

[0058]

[0059] (2) Real-time PCR was performed using SYBR Green dye to test primers 1 and 2 for combinations A1, A3, and A5. The results of the real-time PCR reactions are shown in Table 3. The results showed that the amplification curves of all three combinations were normal, with Ct values ​​< 35, and the melting curves were all single peaks. Therefore, primers 1 and 2 for combinations A1, A3, and A5 met the requirements and were suitable for further testing.

[0060] Table 3 SYBR Green dye method Real-time PCR screening of specific primers

[0061]

[0062] 3. Probe synthesis and screening

[0063] Probes of combinations A1, A3, and A5 were synthesized, with the 5' end modified with a fluorescent labeling group FAM and the 3' end modified with a fluorescent quenching group BHQ1.

[0064] Real-time PCR was performed using probes to detect the primers and probes for combinations A1, A3, and A5. The results are shown in Table 4. The results showed that combinations A1, A3, and A5 were successfully amplified, with Ct values ​​of 27.29, 30.83, and 30.51, respectively. Combination A1 had the highest fluorescence signal value.

[0065] Therefore, combination A1 with the smallest Ct value and the highest fluorescence signal value was selected as the primer and probe for quantitative detection of rice transformant RN85zN-eH.

[0066] Table 4 Probe method Real-time PCR screening specific primer and probe combinations

[0067]

[0068] The probe and primers of combination A1 are located at the upstream boundary of the exogenous insertion sequence. The specific sequences and positions are as follows (lowercase letters are vector sequences, uppercase letters are genomic sequences, underlined sequences represent primers, and bold sequences represent probes):

[0069]

[0070] The primer and probe sequences are as follows:

[0071] Primer 1: 5′-GGCTGTGGGTGCATGGTT-3′

[0072] Primer 2: 5′-TTGTTTACACCACCCAGGGTTT-3′

[0073] Probe P: FAM-AGTCGCACCCATGGCAATTAAGGACC-BHQ1

[0074] Example 2 Preparation of Standards

[0075] To quantify the initial template amount in a sample using real-time PCR, a standard with a known copy number must be used to create a standard curve. The Ct value of the sample to be tested is then obtained through PCR, and the copy number of the sample is calculated from the standard curve. Therefore, appropriate standards must first be prepared, as follows:

[0076] 1. Extraction of Rice RN85zN-eH Genomic DNA

[0077] The CTAB method was used for extraction. The specific steps are as follows:

[0078] 1) Grind 0.2 g of rice RN85zN-eH leaves into powder, add 500-800 μL of CTAB, and incubate at 65°C for 0.5 h.

[0079] 2) Add 700 μL of chloroform or chloroform:isoamyl alcohol (24:1), shake slowly, centrifuge at 12,000 rpm for 15 minutes, and collect 400-700 μL of the supernatant;

[0080] 3) Add 1 mL of pre-cooled anhydrous ethanol or isopropanol, mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant;

[0081] 4) Wash the precipitate with 75% alcohol, centrifuge at 12,000 rpm for 5 minutes, discard the alcohol, and invert to absorb water and dry;

[0082] 5) Dissolve the DNA in 50 μL of ddH2O, take 5 μL for electrophoresis, and determine the DNA concentration using a UV spectrophotometer to be 1.0 μg / μL.

[0083] 2. Preparation of a series of concentration gradient standards

[0084] When performing real-time PCR, the concentration of the standard template needs to be expressed in "copies / μL".

[0085] Calculation formula:

[0086] Template concentration (copies / μL) = Avogadro constant × number of template moles, where Avogadro constant = 6.02 × 10 23 Copies / mol, template molecular weight = template DNA length (number of bases) × 660 (average molecular weight of bases).

[0087] According to the above formula, 1.0 μg / μL of RN85zN-eH genomic DNA solution is 6.02×10 23 copies / mol×(1.0×10 -6 g / μL) / (460×10 6 ×660g / mol), which is 2×10 6 copies / μL

[0088] Take 1 μL of the above solution and dilute it 10-fold to obtain a concentration of 2×10 5 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 The standard samples were 100 copies / μL, 2×10 copies / μL, and 2 copies / μL. Store at -20°C until use.

[0089] Example 3 Establishment and Optimization of Probe-Based Real-time PCR Reaction System

[0090] The present invention obtains a usable probe and primer combination through the operation of Example 1, and obtains a series of standard products with a concentration gradient through Example 2. However, whether a specific real-time PCR reaction system can achieve better results is also affected by factors such as primer and probe concentrations. Therefore, in order to obtain efficient and accurate quantitative results, further optimization of the PCR reaction system is required.

[0091] 1. Establish a preliminary real-time PCR reaction system

[0092] The primer and probe combination A1 screened in Example 1 was diluted, deionized water was added to dilute it to a working solution with a concentration of 10 μM, and probe method real-time PCR amplification was performed to establish a reaction system.

[0093] The PCR reaction system was as follows: 2× qPCR Mix 10 μL, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, 10 μM probe 0.25 μL, template DNA 1 μL, and ddH2O to a total volume of 20 μL. RN85zN-eH genomic DNA was used as the template, and ddH2O served as a blank control.

[0094] The real-time PCR reaction program was as follows: 95°C for 10 min; 95°C for 10 s, 60°C for 20 s, and 72°C for 40 s (collecting fluorescence signals), for a total of 40-45 cycles.

[0095] 2. Optimizing the Real-time PCR Reaction System

[0096] The final primer concentration was set at five concentration gradients: 0.2, 0.3, 0.4, 0.5, and 0.6 μM, respectively. The corresponding probe concentration was 1 / 2 times the primer concentration. The real-time PCR test results for each treatment are shown in Table 5.

[0097] Table 5 Tests of different primer and probe concentrations

[0098]

[0099] The results showed that the PCR reaction system with a primer concentration of 0.4 μM and a probe concentration of 0.2 μM had the lowest Ct value and the highest fluorescence signal value. Therefore, the final primer concentration in subsequent experiments was determined to be 0.4 μM and the probe concentration was 0.2 μM.

[0100] The optimized reaction system is:

[0101] 2× qPCR Mix (10 μL), 10 μM forward primer (0.8 μL), 10 μM reverse primer (0.8 μL), 10 μM probe (0.4 μL), template DNA (1 μL), and ddH₂O to a total volume of 20 μL. RN85zN-eH genomic DNA was used as the template, and ddH₂O was used as a blank control.

[0102] Example 4 Sensitivity test

[0103] Sensitivity refers to the minimum copy number of a sample that can be detected by a PCR amplification reaction, also known as the minimum detection limit. When using real-time PCR to detect standards of varying concentrations, if a standard concentration does not produce a typical amplification curve, or if an amplification curve is formed but the Ct value is >35, the standard at that concentration is considered to have exceeded the minimum detection limit of the PCR system.

[0104] The probe and primer combination A1 described in Example 1, the reaction system optimized in Example 3, and the standard sample of Example 2 (concentration of 2×105 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 Real-time PCR amplification was performed using 100 copies / μL, 2×10 copies / μL, and 2 copies / μL as templates (three parallel tests for each concentration) and ddH2O as a blank control to determine the minimum detection limit of the detection method of the present invention. Amplification curves were obtained based on the fluorescence signals detected by the instrument. The results are shown in Figure 1 and Table 6. The results show that when the standard concentration is less than 20 copies / μL, the amplification curve Ct is greater than 35. Therefore, the detection limit of real-time PCR is 20 copies / μL.

[0105] The above sensitivity test results show that when the transformant concentration in the sample is lower than 20 copies / μL, the Ct value of the amplification curve is greater than 35, which means that the RN85zN-eH transformant is not detected in the sample and the test result is negative.

[0106] Table 6 Sensitivity test results

[0107]

[0108] Example 5 Drawing a standard curve

[0109] Real-time PCR is performed using multiple standards at varying concentrations as templates, and the Ct values ​​are recorded. A standard curve is plotted based on the initial template amount (logarithm of the copy number) and the Ct values, generating a standard equation. To quantify the initial template amount in the test sample, simply generate the amplification curve, read the Ct values, and plug them into the standard equation to calculate the initial template amount in the test sample.

[0110] The standard sample of Example 2 (concentration 2×10 5 copies / μL, 2×10 4 copies / μL, 2×10 3 copies / μL, 2×10 2 Real-time PCR amplification was performed using the primer / probe combination A1 of Example 1 and the reaction system of Example 2, with 100 copies / μL, 2×10 copies / μL) as templates (three parallel tests for each concentration) and ddH2O as a blank control.

[0111] Use the logarithm of the standard concentration as the horizontal axis and the Ct value as the vertical axis to draw a standard curve. Figure 2 The standard curve equation of the present invention is y=-3.145x+36.826 (y represents the Ct value, x is the logarithm of the copy number), and the standard curve shows a good linear relationship. 2=0.9797, with a high correlation coefficient, which meets the requirements of Real-time PCR quantitative detection.

[0112] Example 6 Detection Kit

[0113] A kit for detecting rice RN85zN-eH was prepared according to the following composition: 2×qPCR Mix, 10 μM forward primer, 10 μM reverse primer, 10 μM probe, the standard of Example 2, and ddH 2 O.

[0114] The primers and probes were combination A1 described in Example 1.

[0115] The reaction system of the kit can be: 2×qPCR Mix 10 μL, 10 μM forward primer 0.8 μL, 10 μM reverse primer 0.8 μL, 10 μM probe 0.4 μL, template DNA 1 μL, ddH2O 7 μL, and the total reaction volume is 20 μL.

[0116] The reaction program of the kit for real-time PCR is: 95°C for 10 min; 95°C for 10 s, 60°C for 20 s, 72°C for 40 s (collecting fluorescence signals), for a total of 40-45 cycles.

[0117] When the kit is used to detect a sample, an amplification curve is obtained through the fluorescence signal detected by the instrument, and the sample copy number is calculated based on the standard equation established by the standard and the Ct value of the sample to be tested.

[0118] Example 7 Specificity test and sample detection

[0119] The genomic DNA of rice transformant RN85zN-eH, the control Nipponbare, and other transformant materials RN85zN-eJ were extracted using the DNA extraction method (CTAB method) of Example 2.

[0120] The genomic DNA of Nipponbare and RN85zN-eH was mixed as sample 1, and the genomic DNA of Nipponbare and RN85zN-eJ was mixed as sample 2. Real-time PCR amplification was performed using the kit described in Example 6 and the reaction system of Example 3 to perform specific test detection. Amplification curves were obtained based on the fluorescence signals detected by the instrument, as shown in FIG. Figure 3 shown.

[0121] The copy number of each sample was calculated based on the Ct value of the amplification curve. The results are shown in Table 7: Sample 1 had a Ct value of 28.50 and a copy number of 444, indicating a positive test result. Sample 2 had a Ct value greater than 35 and a copy number below the minimum detection limit of 20 copies / μL, indicating a negative test result. This demonstrates that the detection system established by the present invention has excellent specificity.

[0122] Table 7 Specificity test results of the tested samples

[0123]

[0124] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can use the technical content disclosed above to make changes or modifications to equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A probe and primer combination, characterized in that The nucleotide sequence of the probe is 5'-AGTCGCACCCATGGCAATTAAGGACC-3', and the nucleotide sequences of the primers are 5'-GGCTGTGGGTGCATGGTT-3' and 5'-TTGTTTACACCACCCAGGGTTT-3'.

2. The probe and primer combination according to claim 1, characterized in that: The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; The fluorescent group is FAM, TET, Any one of CY3, JOE, VIC, ROX, CY5, TAMRA or Texas; the quenching group is any one of BHQ1, BHQ2, BHQ-X, TAMRA, DABCYL or MGB.

3. The probe and primer combination according to claim 2, characterized in that: The combination of the fluorescent group and the quenching group is any one of FAM / BHQ1, FAM / BHQ2, CY3 / BHQ-X, HEX / DABCYL, JOE / TAMRA or VIC / BHQ2.

4. The probe and primer combination according to claim 3, characterized in that: The fluorescent group is FAM; the quenching group is BHQ1.

5. A detection kit, characterized in that: The detection kit comprises the probe and primer combination according to any one of claims 1 to 4 and a standard; Wherein, the standard is a RN85zN-eH sample with a concentration of not less than 20 copies / μL; Among them, the RN85zN-eH sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.

1.

6. The detection kit according to claim 5, characterized in that: The standard substances are RN85zN-eH genomic DNA with five concentrations of 2×105 copies / μL, 2×104 copies / μL, 2×103 copies / μL, 2×102 copies / μL and 2×10 copies / μL, respectively.

7. Real-time PCR detection method, characterized in that: Real-time PCR detection is performed using the detection kit according to any one of claims 5 to 6, wherein the final concentration of primers in the PCR reaction system is 0.4 μM, and the final concentration of the probe is 0.2 μM.

8. Use of the probe and primer combination according to any one of claims 1 to 4, the detection kit according to any one of claims 5 to 6, and the detection method according to claim 7 in detecting RN85zN-eH samples; in, The RN85zN-eH sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1.

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