CN102586442A - Specific primer and probe for assaying genetically modified corn MIR162 and application thereof - Google Patents

Abstract

The invention discloses a specific primer for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:1 and SEQ ID NO:2. The invention also discloses a fluorescently labeled probe for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:3, the 5' end of the probe is connected with a fluorescent reporter FAM, and the 3' end of the probe is connected with a fluorescent quencher Eclipse. The invention also discloses an assay method which utilizes the primer and the probe to carry out gene-specific qualitative PCR (Polymerase Chain Reaction), SYBR Green I real-time fluorogenic quantitative PCR and TaqMan probe real-time fluorogenic quantitative PCR to assay whether corn and related products thereof contain MIR162 transformation events, and an experiment proves that the assay method is sensitive, accurate, simple and reliable, and has a high application value and a wide market prospect.

Description

Specific primers, probes and applications for detecting transgenic maize MIR162

technical field

The invention relates to specific primers and probes for detecting transgenic corn MIR162, and its use in gene-specific qualitative PCR, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR to detect whether corn and related products contain MIR162 Application in transformation events, belongs to the field of molecular biology. the

Background technique

In the early 1990s, the first genetically modified food on the market appeared in the United States, which was a kind of preserved tomato. Over the next ten years, genetically modified products were born one after another, especially in food and medicine. Since the ecological safety and food safety of genetically modified products have been controversial, more than 40 countries and regions have successively established and implemented genetically modified labeling systems. With the establishment and continuous improvement of these systems, people put forward strict requirements on the accuracy and sensitivity of genetically modified detection technology, and the detection technology of various genetically modified products has also become a research hotspot. the

According to the different detection purposes of different genetically modified products, the main ways to identify genetically modified products include screening methods, gene-specific detection methods, transformant-specific detection methods and construction-specific detection methods. Among them, transformant-specific detection is the most specific and most widely used detection method. From a technical point of view, PCR technology is the most widely used in the above detection, including qualitative PCR, nested PCR, multiplex PCR, competitive quantitative PCR and fluorescent quantitative PCR. the

Transgenic maize MIR162 is a new Lepidoptera-resistant insect variety developed by Syngenta Corporation of the United States. After searching the existing patents and other documents, the applicant has not found any reports on the specific qualitative PCR of transgenic maize MIR162 transformants, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR detection. the

Contents of the invention

For the above-mentioned prior art, the present invention provides specific primers and probes for detecting transgenic corn MIR162, and its use in gene-specific qualitative PCR, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR to detect corn Whether or not its related products contain MIR162 transformation events, belongs to the field of molecular biology. the

The present invention is achieved through the following technical solutions:

A kind of specific primer for detecting transgenic corn MIR162, said primer is made up of upstream primer and downstream primer, and its sequence is:

MIR162-F: 5′-CTGTCTAATAGTTTGAGTGA-3′;

MIR162-R: 5′-GTGACTCCCTTAATTCTC-3′;

As shown in SEQ ID NO.1 and SEQ ID NO.2. the

The specific primers for detecting transgenic corn MIR162 can be used to qualitatively detect whether corn and its related products contain MIR162 transformation events. In specific applications, the steps are:

(1) Extract the genomic DNA of the sample to be detected, and use this as a template to perform PCR amplification using the combination of MIR162-F and MIR162-R primers;

(2) PCR amplified products were separated by agarose gel electrophoresis, and identified whether there were amplified products after EB staining; if there were amplified products, the sample to be detected contained MIR162 transformation events; The detection sample does not contain MIR162 transformation events. the

In the step (1), the PCR program is: pre-denaturation at 95°C for 5 minutes; 35 cycles at 94°C for 30s, 50°C for 30s, and 72°C for 30s; and extension at 72°C for 7 minutes. the

The specific primers for detecting transgenic corn MIR162 can be used for SYBR Green I real-time fluorescent quantitative PCR to detect whether corn and related products contain MIR162 transformation events. In specific applications, the steps are:

(1) Establish the SYBR Green I real-time fluorescent quantitative PCR standard curve of MIR162 transformant-specific primers and the SYBR Green I real-time fluorescent quantitative PCR standard curve of the zSSIIb gene:

① Extract the genomic DNA of the transgenic maize MIR162 standard product for gradient dilution, and then add specific primers for the MIR162 transformation event and national standard primers for the zSSIIb gene to each dilution for amplification;

② After amplification, measure the Ct value of the relevant fluorescent marker in each dilution, the Ct value is linear with the natural logarithm of the copy number, and draw the quantitative PCR standard curve of the MIR162 transformation event and the quantitative PCR standard of the zSSIIb gene accordingly curve;

(2) Extract the genomic DNA of the sample to be detected to obtain the genomic DNA extract, then add the specific primers of the MIR162 gene and the national standard primers of the zSSIIb gene to the genomic DNA extract to amplify; after the amplification, measure the genomic DNA The Ct value of the relevant fluorescent marker in the extract, then according to the quantitative PCR standard curve of the above-mentioned drawn MIR162 gene and the quantitative PCR standard curve of the zSSIIb gene, calculate the corresponding copy number, then the copy number of the transgenic maize MIR162 gene is the same as that in the maize The ratio of the copy number of the standard gene zSSIIb gene is the percentage content of the transgenic maize MIR162 in the sample to be detected. the

In described step (1) 1. and (2), zSSIIb primer sequence (national standard) is as follows:

zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATTCTC-3'. the

In the steps (1)① and (2), the amplification parameters are as follows: the amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 10uL, the concentration is 0.4uL of Forward primer of 10umol/L, and the concentration is 10umol /L Reverse primer 0.4uL, ddH2O 8.2uL, DNA template 1uL;

Amplification reaction conditions: pre-denaturation at 95°C for 10 minutes; 15s at 95°C, 30s at 50°C, 30s at 72°C, and fluorescence signal collection at 72°C, a total of 40 cycles. the

A fluorescently labeled probe for detecting transgenic corn MIR162, the sequence is: MIR162-P: FAM-5'-CAGATTGTCGTTTCCCGCCTTC-3'-Eclipse, as shown in SEQ ID NO.3, the 5' end of the probe is connected to a The fluorescent reporting group FAM is connected to a fluorescent quenching group Eclipse at the 3′ end. the

The above-mentioned specific primers for detecting transgenic corn MIR162 and fluorescently labeled probes for detecting transgenic corn MIR162 can be used for real-time fluorescent quantitative PCR of TaqMan probes to detect whether corn and related products contain MIR162 transformation events. In specific applications, The steps are:

(1) Establish the TaqMan probe real-time fluorescent quantitative PCR standard curve of the MIR162 transformant specific primer and the TaqMan probe real-time fluorescent quantitative PCR standard curve of the zSSIIb gene:

① Extract the genomic DNA of the transgenic corn MIR162 standard product for gradient dilution, and then add specific primers and fluorescently labeled probes for the MIR162 transformation event, as well as national standard primers and fluorescently labeled probes for the zSSIIb gene, to each dilution for amplification;

② After amplification, measure the Ct value of the relevant fluorescent marker in each dilution, the Ct value is linear with the natural logarithm of the copy number, and draw the quantitative PCR standard curve of the MIR162 transformation event and the quantitative PCR standard of the zSSIIb gene accordingly curve;

(2) Extract the genomic DNA of the sample to be detected to obtain a genomic DNA extract, then add specific primers and fluorescently labeled probes of the MIR162 gene, and national standard primers and fluorescently labeled probes of the zSSIIb gene to the genomic DNA extract, and carry out Amplification; after amplification, measure the Ct value of the relevant fluorescent marker in the genomic DNA extract, then calculate the corresponding copy number according to the quantitative PCR standard curve of the MIR162 gene drawn above and the quantitative PCR standard curve of the zSSIIb gene, then The ratio of the copy number of the transgenic maize MIR162 gene to the copy number of the maize standard gene zSSIIb is the percentage of the transgenic maize MIR162 in the sample to be tested. the

In described step (1) 1. and (2), zSSIIb primer and probe sequence (national standard) are as follows:

zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATCCTC-3';

zSSIIb-P: FAM-5'-TAAGGAGCACTCGCCGCCGCATCTG-3'-TAMRA. the

In the steps (1)① and (2), the amplification parameters are as follows: the amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 12.5uL, the concentration is 10umol/L Forward primer 1uL, the concentration is 10umol /L Reverse primer 1uL, TaqMan probe 0.5uL with a concentration of 10umol/L, ddH2O 4uL, DNA template 1uL;

Amplification reaction conditions: pre-denaturation at 95°C for 10 minutes; 15s at 95°C, 30s at 50°C, 30s at 72°C, and fluorescence signal collection at 72°C for a total of 45 cycles. the

The present invention designs primers and probe sequences aimed at the side genes of the transgenic corn MIR162 transformation event, and establishes a specific qualitative PCR detection method for the transgenic corn MIR162 transformant, a SYBR Green I real-time fluorescent quantitative PCR detection method and a TaqMan detection method. A real-time fluorescent quantitative PCR detection method, which has been verified by experiments, is sensitive, accurate, simple and reliable, and has broad application value and market prospects. the

Description of drawings

1 is a schematic diagram of the electrophoresis of the specific qualitative PCR amplification results of the transgenic maize MIR162 transformant in Example 1. the

Fig. 2 is the SYBR Green I real-time fluorescent quantitative PCR standard curve of internal standard gene zSSIIb in embodiment 2. the

Fig. 3 is the real-time fluorescent quantitative PCR standard curve of transgenic maize MIR162 transformant SYBR Green I in embodiment 2. the

Fig. 4 is the TaqMan probe real-time fluorescence quantitative PCR standard curve of the internal standard gene zSSIIb in Example 3. the

Fig. 5 is the real-time fluorescent quantitative PCR standard curve of the TaqMan probe of the transgenic maize MIR162 transformant in Example 3. the

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings. the

Embodiment 1 transgenic maize MIR162 transformant-specific qualitative PCR detection method

Primers were synthesized by Dalian Takara Company and diluted to 10umol/L for use. The synthetic primer sequences are as follows:

MIR162-F: 5′-CTGTCTAATAGTTTGAGTGA-3′;

MIR162-R: 5'-GTGACTCCCTTAATTCTC-3'. the

Extract transgenic corn MIR162, MON810, NK603, Bt176, MON88017, GA21, TC1507, MIR604, Bt11, transgenic soybean GTS40-3-2, MON87701, non-transgenic soybean 1138-2, transgenic cotton MON88913, LLcotton25, MON531, MON1445, transgenic Rice TT51, Kefeng 6, and non-transgenic maize Zhengdan 958 gene DNA were used as templates, and PCR amplification was performed with MIR162-F and MIR162-R primer combinations, respectively. The PCR program was: 95°C 5min pre-denaturation; 94°C 30s, 50°C 30s, 72°C 30s, 35 cycles; 72°C extension 7min. PCR amplified products were separated by agarose gel electrophoresis, and the existence of amplified products was identified after EB staining. the

Results: PCR amplification was carried out using the designed primers and the extracted genome as a template. The results are shown in Figure 1. It can be seen from the figure that only the transgenic maize MIR162 genome successfully amplified a 94bp specific product, while other transgenic and non-transgenic samples None of the templates had observable amplification products. It is thus proved that the primer has good specificity and is suitable for the qualitative detection of the specificity of the transgenic maize MIR162 transformant. the

Example 2 Transgenic maize MIR162 transformant-specific SYBR Green I real-time fluorescent quantitative PCR detection method

Extract the genomic DNA of the transgenic corn MIR162 standard product, and dilute it to ⁵⁰ , ^5-1 , ^5-2 , ^5-3 , ^5-4 according to the 5-fold ratio; the corresponding copy numbers are set to 100000, 20000, 4000, 800, 160, 3 replicates were set for each concentration to check the repeatability of the amplification.

Primers were synthesized by Dalian Takara Company and diluted to 10umol/L for use. The synthetic MIR162 primer sequence is as follows:

MIR162-F: 5′-CTGTCTAATAGTTTGAGTGA-3′;

MIR162-R: 5'-GTGACTCCCTTAATTCTC-3'. the

Maize internal standard primer (zSSIIb) adopts national standard primer, its sequence is as follows:

zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATTCTC-3'. the

The amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 10uL, Forward primer 0.4uL with a concentration of 10umol/L, Reverse primer 0.4uL with a concentration of 10umol/L, ddH2O 8.2uL, DNA template 1uL. Amplification reaction conditions: pre-denaturation at 95°C for 10 minutes; 15s at 95°C, 30s at 50°C, 30s at 72°C, and fluorescence signal collection at 72°C for a total of 45 cycles. Each sample was repeated 3 times, and a sterile double distilled water (ddH ₂ O, blank) and a non-transgenic sample (Zhengdan 958, negative) controls were set up at the same time.

Results: Through SYBR Green I real-time fluorescent quantitative PCR amplification of DNA with copy numbers of 100,000, 20,000, 4,000, 800, and 160 copies of different MIR162 standard products, the computer software automatically generates a standard curve, with the ordinate being Ct and the abscissa being the start Natural logarithm of copy number. According to the linear calculation formula obtained by the standard curve, through the determination of the transgenic maize MIR162 standard substance (1%, 0.5%, 0.1%, 0.05%, 0.01%), positive samples, negative samples and blank control with different transgenic contents, the samples Substituting the Ct value of the formula into the formula, the percentage of genetically modified ingredients in the sample to be tested can be obtained. the

The transformant-specific primers of the standard gene zSSIIb in maize and the transgenic maize MIR162 were amplified by SYBR Green I real-time fluorescence quantitative PCR on the same set of standard DNA and different percentages of transgenic maize DNA, and PCR amplification curves were obtained respectively, and The standard curve is automatically generated by the system software. The standard curve of the standard gene zSSIIb in maize is shown in Figure 2, the slope of the curve is -3.25, and the correlation coefficient ^R2 is 0.999. The standard curve of the transformant-specific primers of transgenic maize MIR162 is shown in Figure 3. The slope of the curve is -3.332, and the correlation coefficient R2 is 0.999. Therefore, as long as the Ct value of the unknown sample is obtained, the Ct value can be calculated from the standard curve. The starting copy number of the sample. According to the formula:

Content of transgenic corn MIR162 in the sample = (copy number of exogenous gene MIR162/copy number of internal standard gene zSSIIb)×100%

The percentage content of the transgenic component MIR162 in the test sample can be calculated. the

Example 3 Transgenic maize MIR162 transformant-specific TaqMan probe real-time fluorescent quantitative PCR detection method

Extract the genomic DNA of the transgenic corn MIR162 standard product, and dilute it to ⁵⁰ , ^5-1 , ^5-2 , ^5-3 , ^5-4 according to the 5-fold ratio; the corresponding copy numbers are set to 100000, 20000, 4000, 800, 160, three replicates were set for each concentration to check the repeatability of the amplification.

Primers and probes were synthesized by Dalian Takara Company and diluted to 10umol/L for use. The synthetic MIR162 primer and probe sequences are as follows:

MIR162-F: 5′-CTGTCTAATAGTTTGAGTGA-3′;

MIR162-R: 5′-GTGACTCCCTTAATTCTC-3′;

MIR162-P: FAM-5'-CAGATTGTCGTTTCCCGCCTTC-3'-Eclipse. the

Maize internal standard primers and probes (zSSIIb) adopt national standard primers and probes, and their sequences are as follows:

zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3';

zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATCCTC-3';

zSSIIb-P: FAM-5'-TAAGGAGCACTCGCCGCCGCATCTG-3'-TAMRA. the

The amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 12.5uL, the concentration of 10umol/L Forwardprimer 1uL, the concentration of 10umol/L Reverse primer 1uL, the concentration of 10umol/L TaqMan probe 0.5uL, ddH2O 4uL , DNA template 1uL; amplification reaction conditions: 95°C 10min pre-denaturation; 95°C 15s, 50°C 30s, 72°C 30s, collect fluorescence signal at 72°C, a total of 45 cycles, repeat 3 times for each sample, set up at the same time Sterile double distilled water (ddH ₂ O, blank) and non-transgenic samples (Zhengdan 958, negative) were used as controls.

Results: Through TaqMan real-time fluorescent quantitative PCR amplification of DNA with copy numbers of 100000, 20000, 4000, 800, and 160 for different MIR162 standard products, the computer software automatically generates a standard curve, the vertical axis is Ct, and the horizontal axis is the initial copy number The natural logarithm of . According to the linear calculation formula obtained by the standard curve, through the determination of the transgenic maize MIR162 standard substance (1%, 0.5%, 0.1%, 0.05%, 0.01%), positive samples, negative samples and blank control with different transgenic contents, the samples Substituting the Ct value of the formula into the formula, the percentage of genetically modified ingredients in the sample to be tested can be obtained. the

The specific primers for transformants of the standard gene zSSIIb in maize and transgenic maize MIR162 were used to perform TaqMan real-time fluorescent quantitative PCR amplification on the same set of standard DNA and different percentages of transgenic maize DNA, and PCR amplification curves were obtained respectively, and analyzed by the system The software automatically generates a standard curve. The standard curve of the standard gene zSSIIb in maize is shown in Figure 4, the slope of the curve is -3.557, and the correlation coefficient R ² is 0.999. The standard curve of the transformant-specific primers of transgenic maize MIR162 is shown in Figure 5. The slope of the curve is -3.556, and the correlation coefficient R2 is 0.997. Therefore, as long as the Ct value of the unknown sample is obtained, the Ct value can be calculated from the standard curve. The starting copy number of the sample. According to the formula:

Content of transgenic corn MIR162 in the sample = (copy number of exogenous gene MIR162/copy number of internal standard gene zSSIIb)×100%

The percentage content of the transgenic component MIR162 in the test sample can be calculated. the

The above results can prove that the present invention provides a simple and reliable assay method for qualitative PCR, SYBR Green I real-time fluorescent quantitative PCR and TaqMan probe real-time fluorescent quantitative PCR detection of transgenic maize MIR162, and can be used for the detection of transgenic maize MIR162. The invention provides a strong technical support for transgenic labeling, and provides a necessary means for the control of genetically modified products. the

Claims (10)

1. A specific primer for detecting transgenic corn MIR162, characterized in that: said primer is made up of upstream primer and downstream primer, and its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2. 2. The application of the specific primers for detecting transgenic maize MIR162 described in claim 1 in the qualitative detection of whether maize and its related products contain MIR162 transformation events. 3. the application of the specific primer for detecting transgenic corn MIR162 described in claim 1 in whether SYBR Green I real-time fluorescent quantitative PCR detects corn and related products thereof contains MIR162 conversion event. 4. A fluorescently labeled probe for detecting transgenic corn MIR162, characterized in that: the sequence of the probe is as shown in SEQID NO.3, the 5' end of the probe is connected to a fluorescent reporter group FAM, and the 3' end Connect Eclipse with a fluorescence quencher group. 5. the specific primer for detecting transgenic corn MIR162 described in claim 1 and the fluorescently labeled probe for detecting transgenic corn MIR162 described in claim 4 detect corn and its related products in TaqMan probe real-time fluorescent quantitative PCR Does it contain the application of MIR162 transformation events. 6. A method for qualitatively detecting whether corn and its related products contain MIR162 transformation events, characterized in that: the steps are: (1) Extract the genomic DNA of the sample to be detected, and use this as a template to perform PCR amplification using the combination of MIR162-F and MIR162-R primers; (2) PCR amplified products were separated by agarose gel electrophoresis, and identified whether there were amplified products after EB staining; if there were amplified products, the sample to be detected contained MIR162 transformation events; The detection sample does not contain MIR162 transformation events. 7. A kind of SYBR Green I real-time fluorescent quantitative PCR detects the method whether corn and related products contain MIR162 conversion event, it is characterized in that: the step is: (1) Establish the SYBR Green I real-time fluorescent quantitative PCR standard curve of the MIR162 transformant-specific primers and the SYBR Green I real-time fluorescent quantitative PCR standard curve of the zSSIIb gene: ① Extract the genomic DNA of the transgenic maize MIR162 standard product for gradient dilution, and then add specific primers for the MIR162 transformation event and national standard primers for the zSSIIb gene to each dilution for amplification; ② After amplification, measure the Ct value of the relevant fluorescent marker in each dilution, the Ct value is linear with the natural logarithm of the copy number, and draw the quantitative PCR standard curve of the MIR162 transformation event and the quantitative PCR standard of the zSSIIb gene accordingly curve; (2) Extract the genomic DNA of the sample to be detected to obtain the genomic DNA extract, then add the specific primers of the MIR162 gene and the national standard primers of the zSSIIb gene to the genomic DNA extract to amplify; after the amplification, measure the genomic DNA The Ct value of the relevant fluorescent marker in the extract, then according to the quantitative PCR standard curve of the above-mentioned drawn MIR162 gene and the quantitative PCR standard curve of the zSSIIb gene, calculate the corresponding copy number, then the copy number of the transgenic maize MIR162 gene is the same as that in the maize The ratio of the copy number of the standard gene zSSIIb gene is the percentage content of the transgenic maize MIR162 in the sample to be detected. 8. The method according to claim 7, characterized in that: in the steps (1) ① and (2), the parameters of the amplification are as follows: the amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 10uL , 0.4uL of Forwardprimer with a concentration of 10umol/L, 0.4uL of Reverse primer with a concentration of 10umol/L, 8.2uL of ddH2O, and 1uL of DNA template; amplification reaction conditions: 95℃10min pre-denaturation; 95℃15s, 50℃30s, 72 °C for 30s, and collect fluorescence signals at 72 °C for a total of 40 cycles. 9. A TaqMan probe real-time fluorescent quantitative PCR detection method for whether corn and related products contain MIR162 transformation events, characterized in that: the steps are: (1) Establish the TaqMan probe real-time fluorescent quantitative PCR standard curve of the MIR162 transformant specific primer and the TaqMan probe real-time fluorescent quantitative PCR standard curve of the zSSIIb gene: ① Extract the genomic DNA of the transgenic corn MIR162 standard product for gradient dilution, and then add specific primers and fluorescently labeled probes for the MIR162 transformation event, as well as national standard primers and fluorescently labeled probes for the zSSIIb gene, to each dilution for amplification; ② After amplification, measure the Ct value of the relevant fluorescent marker in each dilution, the Ct value is linear with the natural logarithm of the copy number, and draw the quantitative PCR standard curve of the MIR162 transformation event and the quantitative PCR standard of the zSSIIb gene accordingly curve; (2) Extract the genomic DNA of the sample to be detected to obtain a genomic DNA extract, then add specific primers and fluorescently labeled probes of the MIR162 gene, and national standard primers and fluorescently labeled probes of the zSSIIb gene to the genomic DNA extract, and carry out Amplification; after amplification, measure the Ct value of the relevant fluorescent marker in the genomic DNA extract, then calculate the corresponding copy number according to the quantitative PCR standard curve of the MIR162 gene drawn above and the quantitative PCR standard curve of the zSSIIb gene, then The ratio of the copy number of the transgenic maize MIR162 gene to the copy number of the maize standard gene zSSIIb is the percentage of the transgenic maize MIR162 in the sample to be tested. 10. The method according to claim 9, characterized in that: in the steps (1) ① and (2), the parameters of amplification are as follows: the amplification reaction volume is 20uL, 2×Premix Ex Taq (Rox) 12.5 uL, Forwardprimer 1uL with a concentration of 10umol/L, Reverse primer with a concentration of 10umol/L 1uL, TaqManprobe with a concentration of 10umol/L0.5uL, ddH2O 4uL, DNA template 1uL; Amplification reaction conditions: pre-denaturation at 95°C for 10 minutes; 15s at 95°C, 30s at 50°C, 30s at 72°C, and fluorescence signal collection at 72°C for a total of 45 cycles.

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