Specific probe, primer, kit and method for detecting nucleic acid sample

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 particularly relates to a probe for detecting a nucleic acid sample, a probe and primer combination, a standard product, a Real-time PCR detection kit, and a detection method. Background Art

[0002] The rice transformant RN85N-e10 is a transgenic rice material resistant to insects and glufosinate herbicide (Chinese Patent Application No. 2025100716880). It has excellent resistance to Lepidoptera pests such as Chilo suppressalis and good tolerance to glufosinate. Using this transformant can cultivate rice varieties resistant to insects and herbicides. Establishing a transformant-specific detection method can provide an effective detection means for the identification and supervision of genetically modified organisms and provide technical support for the safety management of agricultural genetically modified organisms.

[0003] The detection of transformant specificity mainly uses the ordinary PCR method, but its disadvantages are low sensitivity, inability to monitor the PCR process in real time, and inability to quantify. With the development of molecular biology technology, Real-time PCR (real-time fluorescence quantitative PCR) has been widely used in transgenic detection. Compared with the conventional PCR technology, it has the characteristics of short time consumption, simple operation, good specificity, and high sensitivity. The process can be monitored in real time, the results can be directly observed, and quantitative detection can be carried out.

[0004] The Real-time PCR method can be divided into two types: the dye method and the probe method. The fluorescent dye in the dye method Real-time PCR can bind to double-stranded DNA and emit fluorescence. Its binding is non-specific, and primer dimers, DNA templates, etc. in the system will all bind to it. Therefore, the specificity of the dye method is not high. The probe in the probe method can specifically bind to the template, and its amplification curve reflects the accumulation of specific products and does not contain non-specific amplification components. The sensitivity is 10 times higher than that of the dye method. In addition, the dye method only supports single-channel reactions. If multi-channel experiments are required, or different targets of the same sample need to be detected, the most commonly used method is still the probe method. Summary of the Invention

[0005] To solve the above problems, the present invention provides probes for detecting nucleic acid samples, probe and primer combinations, standards, detection kits, and Real-time PCR detection methods. The above nucleic acid samples can be genomic DNA of rice RN85N-e10 transformants, genomic DNA of RN85N-e10 transformant derivative lines, genomic DNA of hybrid rice materials containing RN85N-e10 transformants, or nucleic acid samples containing RN85N-e10 identity information isolated from the above samples by methods such as PCR amplification. Since the sequence shown in SEQ ID NO.1 is a specific sequence for confirming the identity information of RN85N-e10 transformants, any sample containing a nucleic acid molecule with the sequence shown in SEQ ID NO.1 can be detected by the method provided by the present invention.

[0006] By designing highly sensitive and specific probes and primer pairs, the present invention can accurately identify rice RN85N-e10 transformants and distinguish them from conventional rice without RN85N-e10 and other transgenic rice materials. This detection method has the advantages of high specificity, sensitivity, and operational convenience, making up for the disadvantages of the conventional PCR method, such as cumbersome procedures and low detection sensitivity.

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

[0008] In some embodiments, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. When the probe is in a free state, the fluorescence emitted by the fluorescent group will be absorbed by the quenching group; during the PCR amplification process, the 5' end fluorescent group of the probe tightly bound to the template will be cleaved by Taq enzyme, thus moving away from the 3' end quenching group, and the fluorescence emitted by the fluorescent group can be received by the instrument, and the generated fluorescence signal is proportional to the amount of amplification 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 quenching group includes any one of BHQ1, BHQ2, BHQ-X, TAMRA, DABCYL, or MGB;

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

[0011] In the test of randomly selected fluorescent groups and quenching groups, the probes labeled with the above fluorescent groups and quenching groups can all obtain specific detection results. At the same time, the probe labeled with FAM at the 5' end and BHQ1 at the 3' end has the lowest labeling cost and can be used as the most preferred probe labeling scheme.

[0012] The present invention also provides a primer and probe combination, characterized in that it includes the above probe and two primers, and the nucleotide sequences of the primers are 5'-TGTTGTTCCCTTCTTTTTCAGTTTC-3' and 5'-CCTGAATGGCGAATGCTAGAG-3';

[0013] The above probe and the probe and primer combination are selected through software design, experimental screening and verification, and are located at the downstream boundary of the exogenous insertion sequence in the RN85N-e10 transformant.

[0014] The present invention also provides a standard product, characterized in that: the standard product is one or more nucleic acid samples with a concentration of not less than 30 copies / μL; the nucleic acid sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.1;

[0015] In some embodiments, the standard product is 5 concentrations of 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL and 3×10 copies / μL of RN85N-e10 genomic DNA; the DNA sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.1;

[0016] In some embodiments, the preparation method of the standard product is: taking an RN85N-e10 genomic DNA sample with a concentration of 1.5 μg / μL and diluting it 10 times, 10 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 includes the above probe and primer combination and the above standard product;

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

[0019] Primer 1, with the sequence 5'-TGTTGTTCCCTTCTTTTTCAGTTTC-3';

[0020] Primer 2, with the sequence 5'-CCTGAATGGCGAATGCTAGAG-3';

[0021] Probe, with the sequence 5'-CGGGAAACGACAATCTGATCCAAGCTC-3';

[0022] Standard, consisting of 5 RN85N-e10 genomic DNA samples with concentrations of 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, and 3×10 copies / μL; the nucleic acid sample contains a nucleic acid molecule with the sequence shown in SEQ ID NO.1;

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

[0024] The present invention also provides a Real-time PCR detection method, which is characterized in that: using the above detection kit for Real-time PCR detection, wherein the final concentrations of primer 1 and primer 2 in the PCR reaction system are both 0.3 μM, and the final concentration of the probe is 0.15 μM.

[0025] The present invention also provides the application of the above probe, probe and primer combination, standard, detection kit, and detection method in qualitatively or quantitatively detecting 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: through software design and multiple experimental screenings, a combination of 1 probe and 2 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 products with appropriate concentration gradients. The above probe, probe and primer combination, standard, detection kit, and Real-time PCR detection method can specifically detect nucleic acid samples containing SEQ ID NO.1 with a concentration of not less than 30 copies / μL, and can effectively distinguish RN85N-e10 materials from other rice materials without RN85N-e10, with extremely high sensitivity and specificity. Description of the Drawings

[0027] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, objectives, and advantages of the present application will become more obvious:

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

[0029] Figure 2 Standard curve and linear equation.

[0030] Figure 3 Specific sample detection test. Among them, 1: Mixed sample of Nipponbare and RN85N-e10 (Sample 1); 2: Mixed sample of Nipponbare and RN85zN-e3 (Sample 2). Detailed implementation mode

[0031] The present invention will be further described below in conjunction with 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 the whole plant, 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 should be understood that the parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, and flowers, stems, fruits, leaves, and roots, and the above plant parts are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecule of the present invention and thus at least partially consist of transgenic cells.

[0033] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences (5' non-coding sequences) before the coding sequence and regulatory sequences (3' non-coding sequences) after the coding sequence. "Natural gene" refers to a gene that is naturally found with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a natural gene and contains regulatory and coding sequences that are not naturally found. "Endogenous gene" refers to a natural gene that is located at its natural position in the genome of an organism. "Foreign gene" refers to a foreign gene that currently exists in the genome of an organism and did not originally exist, and also refers to a gene introduced into a recipient cell through a transgenic step. A foreign gene can include a natural gene or a chimeric gene inserted into a non-natural organism. "Transgene" refers to a gene that has been introduced into the genome through a transformation procedure. The site where recombinant DNA has been inserted into the plant genome can be called an "insertion site" or a "target site".

[0034] Transformation procedures that cause random integration of foreign DNA result in transformants with different flanking regions, which are specific to each transformant. When recombinant DNA is introduced into a plant by traditional hybridization, its flanking regions generally do not change. Transformants also contain unique junctions between the heterologous insert DNA and segments of genomic DNA or between two segments of genomic DNA or between two heterologous DNAs. A "junction" is the point where two specific DNA fragments are joined. For example, a junction exists at the position where the insert DNA joins the flanking DNA. Junction points also exist in the transformed organism where two DNA fragments are joined together in a manner found in the natural organism. "Junction DNA" refers to the DNA that contains the junction point.

[0035] The RN85N-e10 transformant is a plant and seed including transgenic rice RN85N-e10 and its plant cells or its renewable parts. The plant parts of RN85N-e10 include, but are not limited to, cells, pollen, ovules, flowers, buds, roots, stems, inflorescences, leaves, and products from the rice plant RN85N-e10, such as rice, straw, rice husk, rice oil, cooked rice, rice flour, rice bran, rice bran layer, and biomass remaining in the rice crop field.

[0036] The term "probe" is a segment of isolated nucleic acid molecule to which a conventional detectable label or reporter molecule is attached, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to one strand of the target nucleic acid. In the present invention, the probe is complementary to one DNA strand from the genome of transgenic rice RN85N-e10, regardless of whether the genomic DNA is from transgenic rice RN85N-e10 or its seeds, or from plants or seeds or extracts of transgenic rice RN85N-e10 and other derivative lines, or a nucleic acid molecule containing the identity information of RN85N-e10 isolated from RN85N-e10. The probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence.

[0037] The term "primer" is a segment of isolated nucleic acid molecule that anneals and binds to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (such as DNA polymerase). The primer pairs of the present invention relate to their application in the amplification of target nucleic acid sequences, such as by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0038] Example 1 Design and Screening of Specific Primer / Probe Combinations

[0039] The specific detection method of the transformant requires designing primers and probes at the boundary sequences upstream and downstream of the insertion site, and the PCR amplification product needs to include the exogenous sequence and the rice genome sequence. Therefore, primers and probes should be designed first according to the boundary sequences of the rice RN85N-e10 insertion site.

[0040] 1. Design primer and probe combinations

[0041] Input a part (100 - 600 bp) of the upstream or downstream boundary sequence (or its reverse complementary sequence) as a template into software (such as ABIPrimer Express 3.0). This template sequence must contain both the rice genome sequence (with a length of at least 50 bp) and the exogenous insertion sequence (with a length of at least 50 bp). Two of the 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 in total, and each combination contains 2 primers and 1 probe.

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

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

[0045] ② The Tm value of the primers is between 58 - 60 °C, and the Tm value of the probe is 8 - 10 °C higher than that of the primers;

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

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

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

[0049] ⑥ The length of the PCR product is between 80 - 300 bp.

[0050] Finally, select 5 groups of primers and probes with higher software scores as candidate combinations for further screening. The candidate combinations are shown in Table 1.

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

[0052]

[0053] 2. Primer synthesis and screening

[0054] Synthesize 5 groups of primers shown in Table 1 and conduct specific primer screening. The screening process is as follows:

[0055] (1) Use ordinary PCR amplification reaction to detect Primer 1 and Primer 2 of 5 candidate combinations. The electrophoresis results are shown in Table 2. The results show that the amplified band sizes of combinations A4 and A5 do not meet the expectations; single specific bands of the expected size were obtained by amplification of combinations A1, A2, and A3. Therefore, combinations A4 and A5 are eliminated; Primer 1 and Primer 2 of combinations A1, A2, and A3 meet the requirements and are further tested.

[0056] Table 2 Screening of specific primers by ordinary PCR

[0057]

[0058] (2) Use SYBR Green dye method Real-time PCR reaction to detect Primer 1 and Primer 2 of combinations A1, A2, and A3. The Real-time PCR reaction results are shown in Table 3. The results show that: the amplification curves of combinations A1 and A2 are normal, and the Ct values < 35, and the melting curves are single peaks; no amplification curve was obtained for combination A3. Therefore, combination A3 is eliminated; Primer 1 and Primer 2 of combinations A1 and A2 meet the requirements and can be further tested.

[0059] Table 3 Screening of specific primers by SYBR Green dye method Real-time PCR

[0060]

[0061] 3. Probe synthesis and screening

[0062] Synthesize the probes of combinations A1 and A2, modify the 5' end with the fluorescent labeling group FAM and the 3' end with the fluorescent quenching group BHQ1.

[0063] Use probe method Real-time PCR reaction to detect the primers and probes of combinations A1 and A2. The reaction results are shown in Table 4. The results show that: both combinations A1 and A2 were amplified successfully, and the Ct values were 28.32 and 29.76 respectively, and the fluorescence signal value of combination A1 was higher.

[0064] Therefore, select combination A1 with the smallest Ct value and high fluorescence signal value as the primers and probes for quantitative detection of rice transformant RN85N-e10.

[0065] Table 4 Screening of specific primer and probe combinations by probe method Real-timePCR

[0066]

[0067] The probe and primer of combination A1 are located at the downstream boundary of the exogenous insertion sequence, and their reverse complementary sequences are as follows (lowercase letters are vector sequences, uppercase letters are genomic sequences), and the positions of the probe and primer of combination A1 are marked (underlined sequences represent primers, and bold sequences represent probes):

[0068]

[0069] The primer and probe sequences are as follows:

[0070] Primer 1: 5′-TGTTGTTCCCTTCTTTTTCAGTTTC-3′

[0071] Primer 2: 5′-CCTGAATGGCGAATGCTAGAG-3′

[0072] Probe P: FAM-CGGGAAACGACAATCTGATCCAAGCTC-BHQ1

[0073] Example 2 Preparation of Standards

[0074] To quantitatively analyze the initial template amount of a sample using real-time PCR, it is necessary to use a standard sample with a known copy number to make a standard curve, then obtain the Ct value of the sample to be tested through PCR, and finally calculate the copy number of the sample from the standard curve. Therefore, first of all, it is necessary to prepare a suitable standard sample, and the preparation method is as follows:

[0075] 1. Extraction of genomic DNA from rice RN85N-e10

[0076] The CTAB method was used for extraction. The specific steps are as follows:

[0077] 1) Take 0.2 g of rice RN85N-e10 leaves, grind them into powder, add 500-800 μL CTAB, and incubate at 65°C for 0.5 h;

[0078] 2) Add 700 μL of chloroform or chloroform:isoamyl alcohol (24:1), shake slowly, centrifuge at 12000 rpm for 15 min, and take 400-700 μL of the supernatant;

[0079] 3) Add 1 mL of pre-cooled anhydrous ethanol or isopropanol, mix thoroughly, centrifuge at 12000 rpm for 10 min, and discard the supernatant;

[0080] 4) Wash the precipitate with 75% alcohol, centrifuge at 12000 rpm for 5 min, discard the alcohol, and invert to absorb water and dry;

[0081] 5) Dissolve the DNA in 50 μL ddH2O, take 5 μL for electrophoresis, and use a UV spectrophotometer to determine the DNA concentration to be 1.5 μg / μL.

[0082] II. Preparation of Standard Products with a Series of Concentration Gradients

[0083] When performing Real-time PCR, the concentration of the standard product template needs to be in the unit of "copies / μL".

[0084] Calculation formula:

[0085] Template concentration (copies / μL) = Avogadro's constant × template mole number, where Avogadro's constant = 6.02×10 23 copies / mol, and template molecular weight = template DNA length (number of bases) × 660 (average molecular weight of bases).

[0086] According to the above formula, the RN85N-e10 genomic DNA solution with a concentration of 1.5 μg / μL is 6.02×10 23 copies / mol × (1.5×10 -6 g / μL) / (460×10 6 × 660 g / mol), which is 3×10 6 copies / μL

[0087] Take 1 μL of the above solution and perform 10-fold serial dilution to obtain standard products with concentrations of 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, 3×10 copies / μL, and 3 copies / μL. Store them at -20°C for standby.

[0088] Example 3 Establishment and Optimization of the Probe-based Real-time PCR Reaction System

[0089] In the present invention, an available probe and primer combination was obtained through the operation of Example 1, and a series of concentration gradients of standard products were obtained through Example 2. However, whether the specific Real-time PCR reaction system can have better effects is also affected by factors such as primer and probe concentrations. Therefore, to obtain efficient and accurate quantitative results, it is necessary to further optimize the PCR reaction system.

[0090] I. Establishment of a Preliminary Real-time PCR Reaction System

[0091] Dilute the primer and probe combination A1 screened in Example 1, and add deionized water to dilute its concentration to a working solution of 10 μM, and perform probe-based Real-time PCR amplification to establish a reaction system.

[0092] The PCR reaction system is as follows: 10 μL of 2×qPCR Mix, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 0.25 μL of 10 μM probe, 1 μL of template DNA, and ddH2O is added to make the total volume 20 μL. Using RN85N-e10 genomic DNA as the template and ddH2O as the blank control.

[0093] The Real-time PCR reaction program is as follows: 95°C for 10 min; 95°C for 10 s, 60°C for 20 s, 72°C for 40 s (collect fluorescence signal), and a total of 40 - 45 cycles are carried out.

[0094] II. Optimization of the Real-time PCR reaction system

[0095] The final concentrations of the primers are set at 5 concentration gradients, which are 0.2, 0.3, 0.4, 0.5, and 0.6 μM respectively, and the corresponding probe concentration is 1 / 2 times the primer concentration. The results of the Real-time PCR tests for each treatment are shown in Table 5.

[0096] Table 5 Tests of different primer and probe concentrations

[0097]

[0098] The results show that: in the PCR reaction system with a primer concentration of 0.3 μM and a probe concentration of 0.15 μM, the Ct value is the smallest and the fluorescence signal value is the highest. Therefore, the final concentration of the primer for the subsequent test is determined to be 0.3 μM, and the probe concentration is 0.15 μM.

[0099] The optimized reaction system is as follows:

[0100] 10 μL of 2×qPCR Mix, 0.6 μL of 10 μM forward primer, 0.6 μL of 10 μM reverse primer, 0.3 μL of 10 μM probe, 1 μL of template DNA, and ddH2O is added to make the total volume 20 μL. Using RN85N-e10 genomic DNA as the template and ddH2O as the blank control.

[0101] Example 4 Sensitivity test

[0102] Sensitivity refers to the lowest copy number of a sample that can be detected by the PCR amplification reaction, that is, the lowest detection limit. When detecting standard products with different concentrations by Real-time PCR, when a standard product at a certain concentration does not show a typical amplification curve, or can form an amplification curve but the Ct value > 35, it is considered that the standard product at this concentration exceeds the lowest detection limit of the PCR system.

[0103] Using the probe, primer combination A1 described in Example 1, the reaction system optimized in Example 3, and using the standard product in Example 2 (concentration of 3×105 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, 3×10 copies / μL and 3 copies / μL) were used as templates (3 parallel tests for each concentration), ddH2O was used as the blank control, and Real-time PCR amplification was carried out to determine the lowest detection limit of the detection method of the present invention. According to the fluorescence signals detected by the instrument, amplification curves were obtained, and the results are shown in Figure 1 and Table 6. The results showed that when the concentration of the standard product < 30 copies / μL, the Ct value of the amplification curve > 35. Therefore, the lower detection limit of Real-time PCR is 30 copies / μL.

[0104] The above sensitivity test results showed that when the concentration of the transformant in the sample was lower than 30 copies / μL, the Ct value of the amplification curve was greater than 35, that is, it was considered that the RN85N-e10 transformant was not detected in the sample, and the test result was negative.

[0105] Table 6 Results of the sensitivity test

[0106]

[0107] Example 5 Drawing the standard curve

[0108] Using multiple standard products with gradient concentrations as templates for Real-time PCR and recording the Ct values, a standard curve was drawn based on the initial template amount (logarithm of the copy number) and the Ct value to obtain the standard equation. When it is necessary to quantify the initial template of the sample to be tested, only the amplification curve needs to be obtained, the Ct value is read, and the initial template amount of the sample to be tested can be calculated by substituting it into the standard equation.

[0109] Using the standard products in Example 2 (concentration 3×10 5 copies / μL, 3×10 4 copies / μL, 3×10 3 copies / μL, 3×10 2 copies / μL, 3×10 copies / μL) were used as templates (3 parallel tests for each concentration), ddH2O was used as the blank control, and primer / probe combination A1 in Example 1 was used, and the reaction system in Example 2 was adopted for Real-time PCR amplification.

[0110] Taking the logarithm of the standard product concentration as the abscissa and the Ct value as the ordinate, the standard curve was drawn, as shown in Figure 2 . The standard curve equation of the present invention is y = -3.1117x + 36.386 (y represents the Ct value, x is the logarithm of the copy number), and the standard curve shows a good linear relationship, R 2= 0.9728, with a high correlation coefficient, meeting the requirements for Real-time PCR quantitative detection.

[0111] Detection Kit of Example 6

[0112] Prepare a kit for detecting rice RN85N-e10 according to the following composition: 2×qPCR Mix, 10 μM forward primer, 10 μM reverse primer, 10 μM probe, the standard product of Example 2, and ddH2O.

[0113] The primer and probe are the combination A1 described in Example 1.

[0114] The reaction system of this kit can be: 2×qPCR Mix 10 μL, 10 μM forward primer 0.6 μL, 10 μM reverse primer 0.6 μL, 10 μM probe 0.3 μL, template DNA 1 μL, ddH2O 7.5 μL, and the total reaction volume is 20 μL.

[0115] The reaction procedure of this 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 (collect fluorescence signal), for a total of 40 - 45 cycles.

[0116] When using this kit to detect a sample, an amplification curve is obtained from the fluorescence signal detected by the instrument, and the sample copy number is calculated according to the standard equation established with the standard product and the Ct value of the sample to be detected.

[0117] Specificity Test and Sample Detection of Example 7

[0118] Extract the genomic DNA of rice transformant RN85N-e10, receptor control Nipponbare, and other transformant material RN85zN-e3 using the DNA extraction method (CTAB method) of Example 2

[0119] Mix the genomic DNA of Nipponbare and RN85N-e10 as Sample 1, and mix the genomic DNA of Nipponbare and RN85zN-e3 as Sample 2. Use the kit described in Example 6 and the reaction system of Example 3 for Real-time PCR amplification to conduct a specificity test. An amplification curve is obtained from the fluorescence signal detected by the instrument, as Figure 3 shown.

[0120] The copy number of each sample was calculated based on its Ct value in the amplification curve, and the results are shown in Table 7: The Ct value of the amplification curve of Sample 1 was 23.51, the copy number was 13738, and the test result was positive; the Ct value of the amplification curve of Sample 2 was greater than 35, and the copy number was lower than the lowest detection limit of 30 copies / μL. Therefore, the test result was negative. Thus, it can be seen that the detection system established by the present invention has good specificity.

[0121] Table 7 Detection Results of Specificity Test of Test Samples

[0122]

[0123] The above description is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed above in a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make changes or modifications to the equivalent embodiments by using the technical content disclosed above without departing from the technical solution of the present invention. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall 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'-CGGGAAACGACAATCTGATCCAAGCTC-3', and the nucleotide sequences of the primers are 5'-TGTTGTTCCCTTCTTTTTCAGTTTC-3' and 5'-CCTGAATGGCGAATGCTAGAG-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 RN85N-e10 sample with a concentration of not less than 30 copies / μL; Among them, the RN85N-e10 sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.

1.

6. The detection kit according to claim 5, characterized in that: The standard substances are RN85N-e10 genomic DNA with five concentrations of 3×105 copies / μL, 3×104 copies / μL, 3×103 copies / μL, 3×102 copies / μL and 3×10 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 0.3 μM, and the final concentration of the probe is 0.15 μM.

8. The probe and primer combination according to any one of claims 1 to 4, the detection Use of the detection kit and the detection method according to claim 7 in the quantitative detection of RN85N-e10 samples; in, The RN85N-e10 sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1.

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