A specific probe, primer, kit and method

Abstract

The present invention provides a probe, a probe and primer combination, a standard product, a detection kit, a Real-time PCR detection method and applications thereof in qualitative or quantitative detection of nucleic acid samples.

Description

Technical Field

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

[0002] Rice transformant H23 is a rice transgenic material that is insect-resistant and resistant to glufosinate-ammonium herbicides. It has excellent resistance to brown planthoppers and good tolerance to glufosinate-ammonium. This transformant can be used to cultivate insect-resistant and herbicide-resistant rice varieties. Establishing a transformant-specific detection method can provide an effective detection method 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 common 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 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 performed.

[0004] Real-time PCR methods can be divided into two types: dye method and probe method. The fluorescent dye in the dye method Real-time PCR can bind to double-stranded DNA to emit fluorescence, and its binding is non-specific. The primer dimers and DNA templates in the system will bind to it, so 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. It does not contain non-specific amplification components, and its 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 if different targets of the same sample are to be detected, the probe method is the most commonly used.

[0005] Rice transformant H23 and its detection method have been disclosed, and the patent application number is 202011498142.7. The disclosed detection method is ordinary PCR, in which the length of the PCR product between the designed detection primers is too large (908bp and 901bp), which is not suitable for direct use in Real-time PCR detection, especially Real-time PCR detection using probe method. Therefore, it is necessary to further establish a special Real-time PCR detection system. Summary of the invention

[0006] To solve the above problems, the present invention provides probes, probe and primer combinations, standard products, detection kits, and real-time PCR detection methods for detecting nucleic acid samples. The above nucleic acid samples can be genomic DNA of rice H23 transformants, genomic DNA of H23 transformant derivatives, genomic DNA of mixed rice materials containing H23 transformants, or nucleic acid samples containing H23 identity information separated from the above samples by PCR amplification and other methods. Since the sequence shown in SEQ ID NO.1 is a specific sequence for confirming the identity information of H23 transformants, any sample containing nucleic acid molecules of the sequence shown in SEQ ID NO.1 can be qualitatively and quantitatively detected by the method provided by the present invention.

[0007] The present invention can accurately identify rice H23 transformants by designing highly sensitive and specific probes, upstream primers and downstream primers, and can separate them from conventional rice and other transgenic rice materials that do not contain H23. The detection method has the advantages of high specificity, sensitivity and convenient operation, which makes up for the shortcomings of the conventional PCR method, such as complicated procedures and low detection sensitivity.

[0008] The present invention provides a probe, characterized in that the nucleotide sequence of the probe is 5'-CTTCTTGAAGCTGCCCTGCCCGA-3'.

[0009] In some embodiments, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quencher group. When the probe is in a free state, the fluorescence emitted by the fluorescent group will be absorbed by the quencher group; during the PCR amplification process, the fluorescent group at the 5' end of the probe tightly bound to the template will be cleaved by the Taq enzyme, thus away from the quencher group at the 3' end, 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 amplified product in the sample.

[0010] 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;

[0011] 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;

[0012] In the test of randomly selected fluorescent groups and quenching groups, the probes labeled with the above fluorescent groups and quenching groups can 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.

[0013] The present invention also provides a primer and probe combination, characterized in that: it comprises the probe according to claim 1 and two primers, the nucleotide sequences of the primers are 5'-CCTGGCTTCATTTCATTAACTTTTG-3' and 5'-CGTCCGCAATGTGTTATTAAGTTG-3';

[0014] The above-mentioned probes and probe and primer combinations were selected through software design, experimental screening, manual recombination and re-PCR experimental verification, and were located on the genome at the upstream boundary of the exogenous insertion sequence of the H23 transformant.

[0015] The present invention also provides a detection standard, characterized in that: the standard is one or more nucleic acid samples with a concentration of not less than 100 copies / μL; the nucleic acid sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1;

[0016] In some embodiments, the detection standard is 6 plasmid samples with concentrations of 1.0×107 copies / μL, 106 copies / μL, 105 copies / μL, 104 copies / μL, 103 copies / μL, and 102 copies / μL, respectively; the nucleic acid sample contains the nucleic acid molecule shown in SEQ ID NO.1;

[0017] In some embodiments, the preparation method of the standard is: take 21.46 μL of a plasmid sample with a full length of 6455 bp containing the nucleic acid molecule shown in SEQ ID NO.1 at a concentration of 3.3 ng / μL, add 78.54 μL ddH2O, and then dilute 10 times, 102 times, 103 times, 104 times, 105 times, and 106 times respectively.

[0018] 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 product;

[0019] In some embodiments, the detection kit comprises:

[0020] Primer 1, sequence: 5′-CCTGGCTTCATTTCATTAACTTTTG-3′;

[0021] Primer 2, sequence: 5′-CGTCCGCAATGTGTTATTAAGTTG-3′;

[0022] probe, sequence: 5′-CTTCTTGAAGCTGCCCTGCCCGA-3′;

[0023] Standards, the detection standards are 6 plasmid samples with concentrations of 1.0×107 copies / μL, 1.0×106 copies / μL, 1.0×105 copies / μL, 1.0×104 copies / μL, 1.0×103 copies / μL, and 1.0×102 copies / μL, respectively; the nucleic acid sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1;

[0024] The 5' end of the probe is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group BHQ1.

[0025] The present invention also provides a real-time PCR detection method, characterized in that: the real-time PCR detection is performed using the above detection kit, 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.

[0026] The present invention also provides the use of the above-mentioned probe, probe and primer combination, detection standard, detection kit, and detection method in qualitative or quantitative detection of nucleic acid samples; wherein the nucleic acid sample contains a nucleic acid molecule with a sequence shown in SEQ ID NO.1.

[0027] The beneficial effects of the present invention are as follows: through software design, manual recombination and multiple test screening, a combination of 1 probe and 2 primers is obtained from more than 300 sets of probe-primer combinations, on this basis, a Real-time PCR detection method is established and optimized using standard products with appropriate concentration gradients, and the use of the above-mentioned probes, probe and primer combinations, standard products, detection kits, and Real-time PCR detection methods can specifically detect nucleic acid samples containing SEQ ID NO.1 with a concentration of not less than 100 copies / μL, and can effectively distinguish H23 materials from other rice materials that do not contain H23, with extremely high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0029] Figure 1Electrophoresis results after testing 15 sets of primers using a common PCR amplification reaction. M represents DNA Ladder Marker, and A1-A15 represent primer pairs in combination A1-A15. It can be seen that the primer combinations A4, A5, A11, A12, A14, and A15 can obtain a clear single band that meets the expected size.

[0030] Figure 2 The amplification results of primer pairs A4, A5, A11, A12, A14 and A15 were detected by real-time PCR reaction using SYBR Green dye. The figure below is the amplification curve, and the figure above is the melting curve. Each curve represents a sample. It can be seen that the amplification of combination A15 failed, and the Ct value of combination A14 was greater than 35; the amplification curves of combinations A4, A5, A11, and A12 were normal, with Ct values ​​less than 35, among which the melting curves of combinations A4 and A5 were single peaks, while the melting curves of combinations A11 and A12 formed double peaks.

[0031] Figure 3 The amplification results of the primers and probes of the new combinations B1 and B2 were tested by real-time PCR reaction using the probe method. It can be seen that both combinations B1 and B2 were successfully amplified, forming a steadily rising amplification curve with Ct values ​​of 29.11 and 35.26, respectively.

[0032] Figure 4 Sensitivity test curve. Where A: 1.0×10 6 copies / μL; B: 1.0×10 5 copies / μL; C: 1.0×10 4 copies / μL; D: 1.0×10 3 copies / μL; E: 1.0×10 2 copies / μL; F: 1.0×10 copies / μL.

[0033] Figure 5 Amplification curve of standard product. Where A: 1.0×10 6 copies / μL; B: 1.0×10 5 copies / μL; C: 1.0×10 4 copies / μL; D: 1.0×10 3 copies / μL; E: 1.0×10 2 copies / μL.

[0034] Figure 6 Plot the standard curve and linear equation.

[0035] Figure 7 Specific sample detection test. Among them, A: transformant H23; B: receptor control Guangzhan 63-4S; C: transformant material H21; D: blank control.

[0036] Figure 8 Amplification curves of probes with different fluorescent groups. DETAILED DESCRIPTION

[0037] 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.

[0038] The term "rice" refers to Asian cultivated rice (Oryza sativa L.), and includes all plant species that can be crossed with rice, including wild rice species.

[0039] The term “including” means “including but not limited to”.

[0040] The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, 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, calli, embryos, and flowers, stems, fruits, leaves and roots, which are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of the present invention and are therefore at least partially composed of transgenic cells.

[0041] The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequences) and regulatory sequences after the coding sequence (3' non-coding sequences). "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a natural gene, which contains regulatory and coding sequences that are not found in nature. "Endogenous gene" refers to a natural gene that is located in its natural location in the genome of an organism. "Foreign gene" is a foreign gene that is now present in the genome of an organism and did not exist originally, and also refers to a gene introduced into a recipient cell through a transgenic step. Foreign genes can include natural genes inserted into non-natural organisms or chimeric genes. "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."

[0042] Transformation procedures that cause random integration of foreign DNA can result in transformants containing different flanking regions, which are specifically contained in each transformant. When recombinant DNA is introduced into plants by traditional hybridization, its flanking regions are usually not changed. Transformants will also contain a unique junction between a segment of heterologous insert DNA and genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA. "Joint" is the point where two specific DNA fragments connect. For example, the 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 connected together in a manner found in natural organisms. "Joint DNA" refers to the DNA that contains a junction point.

[0043] The H23 transformant is a plant and seed comprising transgenic rice H23 and plant cells or regenerable parts thereof, wherein the plant parts of H23 include but are not limited to cells, pollen, ovules, flowers, shoots, roots, stems, tassels, inflorescences, ears, leaves and products from the rice plant H23, such as rice, rice straw, rice husks, rice oil, cooked rice, rice flour, rice bran, rice husks and biomass remaining in the rice crop field.

[0044] The term "probe" is an isolated nucleic acid molecule to which a conventional detectable label or reporter molecule is bound, such as a radioisotope, a ligand, a chemiluminescent agent or an enzyme. The probe is complementary to a strand of a target nucleic acid. In the present invention, the probe is complementary to a DNA strand from the genome of transgenic rice H23, whether the genomic DNA is from transgenic rice H23 or seeds or from plants or seeds or extracts of transgenic rice H23 and other derivative strains, or a nucleic acid molecule containing H23 identity information isolated from H23. The probe of the present invention includes not only deoxyribonucleic acid or ribonucleic acid, but also polyamide 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.

[0045] The term "primer" is a separate nucleic acid molecule that anneals to a complementary target DNA strand through nucleic acid hybridization, forms a hybrid between the primer and the target DNA strand, and then extends along the target DNA strand under the action of a polymerase (e.g., DNA polymerase). The primer pair of the present invention relates to its use in amplification of a target nucleic acid sequence, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0046] Example 1 Design and screening of specific primers and probes for rice transformant H23

[0047] The specific detection method of transformants requires the design of primers and probes at the border 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, first of all, primers and probes should be designed according to the border sequences of the rice H23 insertion site.

[0048] 1. Use software to design primer and probe combinations

[0049] A portion of the upstream or downstream border sequence (100-600 bp) is input as a template into the software (such as ABI PrimerExpress 3.0). The template sequence must contain both the rice genome sequence (at least 50 bp in length) and the exogenous insertion sequence (at least 50 bp in length). The two templates SEQ ID NO. 2 and SEQ ID NO. 3 are shown below:

[0050] Rice transformant H23 upstream border template SEQ ID NO.2:

[0051]

[0052] Rice transformant H23 downstream border template SEQ ID NO.3:

[0053]

[0054] Lowercase letters represent exogenous sequences, and uppercase letters represent genomic sequences.

[0055] After setting the relevant parameters in the software according to the following requirements, more than 300 sets of primer and probe combinations were obtained, each of which contained 2 primers and 1 probe.

[0056] The probes and primers meet all of the following requirements:

[0057] ① The length of the primer is between 18 and 25 bp, and the length of the probe is between 18 and 30 bp;

[0058] ②The Tm value of the primer is between 58 and 60°C, and the Tm value of the probe is 8-10°C higher than that of the primer;

[0059] ③ Avoid generating complementary sequences of more than 3 bases inside the probe, inside the primer, or between the probe and the primer;

[0060] ④The first base at the 5' end of the probe is not G;

[0061] ⑤The forward primer and reverse primer are located in the rice genome and vector insertion sequence respectively.

[0062] ⑥The length of the PCR product between the forward primer and the reverse primer is between 80-300bp.

[0063] Finally, 15 sets of primers and probes with higher software scores were selected as candidate combinations for further screening. The candidate combinations are shown in Table 1.

[0064] Table 1 20 candidate primers and probes with higher software design scores

[0065]

[0066] 2. Primer synthesis and screening

[0067] The 15 sets of primers shown in Table 1 were synthesized and screened for specific primers. The screening process was as follows:

[0068] (1) Use ordinary PCR amplification reaction to test 15 candidate combinations of primers F and R. The electrophoresis results are as follows: Figure 1 As shown in Table 2. The results showed that combinations A2, A3, A6, A7 and A8 failed to amplify and had no bands, while the band sizes amplified by combinations A1, A9, A10 and A13 did not meet expectations, indicating nonspecific amplification; only the primers of combinations A4, A5, A11, A12, A14 and A15 amplified a single specific band that met the expected size (100-250 bp), so primers F and R of combinations A4, A5, A11, A12, A14 and A15 were class I specific primers. Combinations A1-A3, A6-A10 and A13 were eliminated.

[0069] Table 2. Common PCR screening of class I specific primers

[0070]

[0071] (2) Real-time PCR reaction was performed using SYBR Green dye to detect all class I specific primers, i.e. primer F and primer R of combinations A4, A5, A11, A12, A14 and A15. The real-time PCR reaction results are shown in Figure 2 As shown in Table 3. The results showed that combination A15 failed to amplify, with no steadily rising amplification curve and no Ct value; the amplification curve of combination A14 was normal, but the Ct value was >35; the amplification curves of combinations A4, A5, A11 and A12 were normal, and the Ct value was <35, among which the melting curves of combinations A4 and A5 were single-peaked, while the melting curves of combinations A11 and A12 formed two peaks. Therefore, primers F and R in combinations A4 and A5 were classified as class II specific primers. Combinations A11, A12, A14 and A15 were eliminated.

[0072] Table 3 Screening of class II specific primers by real-time PCR using SYBR Green dye

[0073]

[0074]

[0075] 3. Probe synthesis and screening

[0076] The probes in the synthetic combinations A4 and A5 were modified with the fluorescent labeling group FAM at the 5' end and the fluorescent quenching group BHQ1 at the 3' end.

[0077] The primers and probes in combinations A4 and A5 were tested by real-time PCR reaction using the probe method, and the reaction results are shown in Table 4. The results showed that combination A5 failed to amplify, with no steadily rising amplification curve and no Ct value; although combination A4 was successfully amplified, the Ct value was 35.78. Since combination A5 had no Ct value and combination A4 had a Ct>35, combinations A4 and A5 were eliminated.

[0078] Table 4 Probe method Real-time PCR screening specific primers and probe combinations

[0079]

[0080] After screening, no suitable primer and probe combination was found at both the upstream and downstream boundaries.

[0081] 4. Recombination of primers and probes

[0082] The primers and probes in combinations A4 and A5 were recombined, that is, the primers in combination A4 and the probes in combination A5 became a new combination B1, and the primers in combination A5 and the probes in combination A4 became a new combination B2 (Table 5).

[0083] Table 5 Recombined primers and probes

[0084]

[0085] 5. Screening of new combinations

[0086] The primers and probes in combinations B1 and B2 were tested by real-time PCR reaction using the probe method. The real-time PCR reaction results are shown in Figure 3 And as shown in Table 6.

[0087] The results showed that both combinations B1 and B2 were successfully amplified, forming a steadily rising amplification curve with Ct values ​​of 29.11 and 35.26 respectively. The Ct value of combination B1 was less than 35, so combination B1 was determined as the probe and primer combination for detecting rice transformant H23 material.

[0088] Table 6 Real-time PCR screening of new combinations using probe method

[0089]

[0090] The probe and primers of combination B1 are located at the upstream boundary of the exogenous insertion sequence. The specific sequences and positions are as follows:

[0091]

[0092] Lowercase letters represent vector sequences, uppercase letters represent genome sequences, the shaded part represents the location of the primers, and the boxed part represents the location of the probes.

[0093] Primer and probe sequences

[0094] Primer F: 5'-CCTGGCTTCATTTCATTAACTTTTG-3'

[0095] Primer R: 5′-CGTCCGCAATGTGTTATTAAGTTG-3′

[0096] Probe P: FAM-CTTCTTGAAGCTGCCCTGCCCGA-BHQ1

[0097] Example 2 Preparation of Standards

[0098] 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:

[0099] 1. The upstream border sequence fragment (SEQ ID NO. 2) in Example 1 was synthesized into a standard positive plasmid at BGI to prepare a standard product;

[0100] 2. Measure the absorbance of plasmid DNA using a UV spectrophotometer 260 and A 280 , calculate the concentration and purity of the plasmid respectively;

[0101] 3. When performing real-time PCR, the concentration of the standard template needs to be expressed in "copies / μL".

[0102] Calculation formula: Template copy number / μL = Avogadro constant × template molar number, where Avogadro constant = 6.02 × 10 23 copies / mol, template molecular weight = template DNA length (number of bases) × 660 (average molecular weight of bases).

[0103] 4. The measured plasmid concentration is 3.3 ng / μL. According to the above formula, the number of plasmid copies / μL = 6.02×1023 copies / mol×(3.3×10 -9 g / μL) / (6455×660g / mol), which is 4.66×10 8 copies / μL.

[0104] 5. Take 21.46 μL of the above plasmid solution and add 78.54 μL of ddH2O to obtain a concentration of 1.0×10 8 The standard was diluted 10-fold to obtain a concentration of 1.0×10 7 ~1.0×10 copies / μL standard. Store at -20℃ for future use.

[0105] Example 3 Establishment and Optimization of Probe Method Real-time PCR Reaction System

[0106] The present invention obtains usable probes and primers through the operation of Example 1, and obtains a series of standard products with concentration gradients through Example 2. However, whether the specific Real-time PCR reaction system can have a better effect is also affected by factors such as the concentration of primers and probes. Therefore, in order to obtain efficient and accurate quantitative results, the PCR reaction system needs to be further optimized.

[0107] 1. Establish a preliminary Real-time PCR reaction system

[0108] The reaction system was established by diluting the primer and probe combination B1 screened in Example 1, adding deionized water to dilute it to a working solution with a concentration of 10 μM, and performing probe method Real-time PCR amplification.

[0109] The PCR reaction system was as follows: 2×qPCR Mix 10 μL, 10 μM upstream primer 0.5 μL, 10 μM downstream primer 0.5 μL, 10 μM probe 0.25 μL, template DNA 1 μL, ddH2O was added to a total volume of 20 μL. Positive control (concentration of 1.0×10 5 100 copies / μL of plasmid DNA) was used as template and ddH2O was used as blank control.

[0110] The real-time PCR reaction program was: 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.

[0111] 2. Optimizing the Real-time PCR Reaction System

[0112] The final concentration of the primers was set to 0.1, 0.2, 0.3, 0.4 and 0.5 μM in 5 concentration gradients, and the corresponding probe concentration was 1 / 2 times the primer concentration. The real-time PCR test results of each treatment are shown in Table 7.

[0113] Table 7 Tests of different primer and probe concentrations

[0114]

[0115] The results showed that the PCR reaction system with a primer concentration of 0.3 μM and a probe concentration of 0.15 μM had the smallest Ct value and the highest fluorescence signal value. Therefore, the final concentration of the primers in the subsequent experiments was determined to be 0.3 μM and the probe concentration was 0.15 μM.

[0116] The optimized reaction system is:

[0117] 2×qPCR Mix 10μL, 10μM upstream primer 0.6μL, 10μM downstream primer 0.6μL, 10μM probe 0.3μL, template DNA 1μL, ddH2O to a total volume of 20μL. Positive control (concentration of 1.0×10 7 100 copies / μL of plasmid DNA) was used as template and ddH2O was used as blank control.

[0118] Example 4 Sensitivity test of real-time PCR system for detecting rice H23

[0119] Sensitivity refers to the minimum copy number of a sample that can be detected by the PCR amplification reaction, that is, the minimum detection limit. When Real-time PCR is used to detect standards of different concentrations, when a certain concentration of standard can form an amplification curve but the Ct value is >35, it is considered that the concentration of the standard exceeds the minimum detection limit of the PCR system.

[0120] The probe and primer combination B1 described in Example 1, the reaction system optimized in Example 3, and the standard sample (concentration 1.0×10 6 ~1.0×10 copies / μL) as template (three parallel tests for each concentration) and ddH2O as blank control, real-time PCR amplification was performed to determine the minimum detection limit of the detection method of the present invention. The amplification curve was obtained based on the fluorescence signal detected by the instrument. The results are shown in Figure 4 and Table 8. The results show that when the concentration of the standard is less than 100 copies / μL, the amplification curve Ct is greater than 35. Therefore, the detection limit of Real-time PCR is 100 copies / μL.

[0121] The above sensitivity test results show that when the sample does not show a typical amplification curve or the Ct value is greater than 35, that is, the transformant concentration in the sample is lower than 100 copies / μL, it is considered that no H23 transformant is detected in the sample and the test result is negative.

[0122] Table 8 Sensitivity test results

[0123]

[0124] Example 5 Drawing a standard curve

[0125] Real-time PCR is performed using multiple standards with gradient concentrations as templates and the Ct values ​​are recorded. A standard curve is drawn based on the initial template amount (logarithm of the copy number) and the Ct value to obtain a standard equation. When the initial template of the sample to be tested needs to be quantified, only the amplification curve needs to be obtained, the Ct value needs to be read, and the initial template amount of the sample to be tested can be calculated by inserting it into the standard equation.

[0126] The standard sample of Example 2 (concentration 1.0×10 6 ~1.0×10 2 100 copies / μL) as template (3 parallel tests for each concentration), ddH2O as blank control, using the probe and primer combination B1 of Example 1, and the reaction system of Example 2, Real-time PCR amplification was performed, and the amplification curve is shown in Figure 5 .

[0127] Draw a standard curve with the logarithm of the standard concentration as the horizontal axis and the Ct value as the vertical axis. Figure 6 The standard curve equation of the present invention is y=-2.267x+35.696 (y represents the Ct value, x is the logarithm of the copy number), and the standard curve has a good linear relationship, R 2 =0.9988, with a high correlation coefficient, which meets the requirements of Real-time PCR quantitative detection.

[0128] Example 6 Real-time PCR kit for detecting rice transformant H23

[0129] A kit for detecting rice H23 was prepared according to the following composition: 2×qPCR Mix, 10 μM upstream primer, 10 μM downstream primer, 10 μM probe, standard substance of Example 2 (concentration 1.0×10 6 ~1.0×10 2 copies / μL) and ddH2O.

[0130] The primers and probes were the combination B1 described in Example 1.

[0131] The reaction system of the kit can be: 2×qPCR Mix 10 μL, 10 μM upstream primer 0.6 μL, 10 μM downstream 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.

[0132] 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.

[0133] When the kit is used to detect samples, an amplification curve is obtained through the fluorescent 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.

[0134] Example 7 Specificity test and sample detection

[0135] The genomic DNA of rice transformant H23, receptor control Guangzhan 63-4S, and other transformant material H21 were extracted using the DNA extraction method (CTAB method) of Example 2.

[0136] The genomic DNA of the rice transformant H23, the receptor control Guangzhan 63-4S and H21 were used as templates, and ddH2O was used as a blank control. The kit described in Example 6 was used to perform real-time PCR amplification using the reaction system of Example 3 to perform specific test detection. The amplification curve was obtained based on the fluorescent signal detected by the instrument, as shown in FIG. Figure 7 shown.

[0137] The copy number of each sample was calculated according to the Ct value of the amplification curve, and the results are shown in Table 9: the Ct value of the amplification curve of the rice transformant H23 was 27.93, the copy number was 2665, and the detection result was positive; the Ct values ​​of the amplification curves of the receptor Guangzhan 63-4S and other transformant materials H21 were all greater than 35, and the copy numbers were all lower than the minimum detection limit of 100, so the detection results were all negative. It can be seen that the detection system established by the present invention has good specificity.

[0138] Table 9 Test results of specificity test of tested samples

[0139]

[0140] Example 8 Testing of different fluorescent groups

[0141] The primer and probe combination B1 screened in Example 1 were used, and the two ends of the probe were labeled with commonly used different types of fluorescent groups and quenching groups, as shown in Table 6. The kit described in Example 6 and the reaction system of Example 3 were used, and the standard sample of Example 2 (concentration of 1.0×10 5 Real-time PCR was performed using plasmid DNA from the human chromatin gene as a template to determine the most suitable fluorescent labeling group.

[0142] The amplification curve is obtained according to the fluorescence signal detected by the instrument, such as Figure 8 As shown in Table 10, the fluorescent labeling combinations numbered A, B, C, D, E and F can all be successfully amplified, and the average Ct value of the amplification curve is between 27.5 and 31.5. However, judging from the height of the y-axis of the amplification curve, the fluorescence collection efficiency of combinations A, B and C is relatively high. At the same time, among all the tested combinations, the probe labeling cost of combination A is the lowest, so combination A (5' end labeled with FAM, 3' end labeled with BHQ1) is determined as the preferred probe labeling scheme.

[0143] Table 10 Tests of probes labeled with different fluorescent groups at both ends

[0144]

[0145] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used 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 of equivalent changes 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. Sequence Listing <110> Wuhan Lekenbio Technology Co., Ltd. <120> A specific probe, primer, kit and method <130> 1 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 191 <212> DNA <213> Artificial synthesis (unknown) <400> 1 cctggcttca tttcattaac ttttgccttc ttgaagctgc cctgcccgac agaagatggc 60 aatgagccag cacacggtgt ctctttcaca gtctttctac tttggttttc cacaggcttt 120 cttgaacggt ggcggtttct gttgatgtaa acaaattgac gcttagacaa cttaataaca 180 cattgcggac g 191 <210> 2 <211> 558 <212> DNA <213> Synthetic (unknown) <400> 2 tactgtgatt cagtctgtag cactaaaata cttgcacgtt gagaatatta aaccaagttt 60 aaggatgtat taacctgttc aaactatctg tccaatagct gatactgcct ggcttcattt 120 cattaacttt tgccttcttg aagctgccct gcccgacaga agatggcaat gagccagcac 180 acggtgtctc tttcacagtc tttctacttt ggttttccac aggctttctt gaacggtggc 240 ggtttctgtt gatgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 300 tttaatgtta gactgaatta acgccgaatt aattcggggg atctggattt tagtactgga 360 ttttggtttt aggaattaga aattttattg atagaagtat tttacaaata caaatacata 420 ctaagggttt cttatatgct caacacatga gcgaaaccct ataggaaccc taattccctt 480 atctgggaac tactcacaca ttattatgga gaaactcgag tcaaatctcg gtgacgggca 540 ggaccggacg gggcggta 558 <210> 3 <211> 497 <212> DNA <213> Synthetic (unknown) <400> 3 tcgagtttct ccataataat gtgtgagtag ttcccagata agggaattag ggttcctata 60 gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa 120 tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtactaa aatccagatc 180 ccccgaatta attcggcgtt aattcagtct aacattaaaa acgtccgcaa tgtgttatta 240 agttgtctaa gcctcaagct gctctaacaa cttgtggtcg gccatggcct cctttgaaca 300 ccgccatttc ttgccatctg ttcgacggca tcttccaggt tctgggtcag agtctgcatt 360 tgggaagcct tgatagagag gcctccagct cactgcagta caataagtat gtcatgtttc 420 catttgattt acatatcacg ttgccttttg tcataataaa cctgaactgg tgagaactga 480 gaacgtaaca ttgatac 497

Claims

1. A probe and primer combination for detecting rice H23 transformants, characterized in that: The nucleotide sequence of the probe is 5'-CTTCTTGAAGCTGCCCTGCCCGA-3', The nucleotide sequences of the primers are 5'-CCTGGCTTCATTTCATTAACTTTTG-3' and 5'-CGTCCGCAATGTGTTATTAAGTTG-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 any one of FAM, TET, HEX, 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 kit for detecting rice H23 transformants, characterized in that: The kit comprises the probe and primer combination according to any one of claims 1 to 4, and a standard; wherein the standard is a plasmid sample with a concentration of not less than 100 copies / μL; and the plasmid sample contains the nucleic acid molecule shown in SEQ ID NO.

1.

6. The kit according to claim 5, characterized in that: The standard substances are 6 in concentration of 1.0×10 7 copies / μL, 1.0×10 6 copies / μL, 1.0×10 5 copies / μL, 1.0×10 4 copies / μL, 1.0×10 3 copies / μL, 1.0×10 2 Copies / μL of plasmid sample.

7. A method for detecting rice H23 transformants, characterized in that: Real-time PCR detection is performed using the 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. Use of the probe and primer combination according to any one of claims 1 to 4, the kit according to any one of claims 5 to 6, and the method according to claim 7 in qualitative or quantitative detection of rice H23 transformants; wherein, The H23 transformant contains the nucleic acid molecule shown in SEQ ID NO.1.

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