CN117187396A - Primer probe group, kit and system for quantitative detection of HER2 gene amplification - Google Patents
Primer probe group, kit and system for quantitative detection of HER2 gene amplification Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a primer probe group, a kit and a system for quantitative detection of HER2 gene amplification; the system comprises an acquisition module, a sampling module and a sampling module, wherein the acquisition module is used for acquiring a plasma sample; an extraction module for extracting a DNA sample from a plasma sample; the amplification module is used for carrying out PCR amplification on the DNA sample by adopting a primer probe group or a kit; the detection module is used for carrying out fluorescence detection on the amplified product; the analysis module is used for analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, the detection result is positive; the invention adopts fluorescent quantitative PCR based on circulating tumor DNA, designs specific primers and probes aiming at HER2 gene amplification, realizes quantitative and copy number detection, and has the advantages of rapid and practical detection and more suitable clinical detection.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a primer probe group, a kit and a system for quantitative detection of HER2 gene amplification.
Background
HER2 (Human Epidermal Growth Factor Receptor 2), chinese HER2/neu or C-erbB-2, is a transmembrane protein with tyrosine protein kinase (protein tyrosinekinase, PTK) activity. The HER2 gene is a protooncogene located in the long arm 2 zone 1 (17 q 21) of human chromosome 17, and is expressed under normal conditions or not, and can be converted into an oncogene and induce abnormal proliferation of cells when the external environment is changed to some extent. The HER2 gene coding product is HER2 protein with the molecular weight of 185kD, and consists of a ligand binding region, a single-chain transmembrane region and a tyrosine protein kinase region, and belongs to one of four receptors (EGFR/HER 1, HER2, HER3 and HER 4) in EGFR family, wherein HER2 monomers generally have no known ligand interaction, need to form dimers with other members in the family and generate interrelated signals, and then transduce and activate PI3K/Akt and Ras/Raf/MEK/MAPK channels, so that proliferation, differentiation and apoptosis of cells are regulated.
Research shows that HER2 is over-expressed in various cancer cells such as breast cancer, gastric cancer, lung cancer and the like, wherein the incidence rate of breast cancer patients is 20-30%, gastric cancer is 10-20%, and lung cancer is 1.9-14.3%; and relatively less in colorectal cancer, accounting for 2% -5%. anti-HER 2 targeting drugs that have been marketed at present include trastuzumab (HER 2 targeting mab marketed as the first 1998), pertuzumab, antibody-drug conjugate (T-DM 1), lapatinib, afatinib, pyrroltinib, and the like. However, in the case of variations in the HER2 gene, drugs of different mechanisms should be used for treatment. Because patients of different cancer species who are also amplified or overexpressed the HER2 gene do not respond well to anti-HER 2 drugs.
Because of the wide space-time heterogeneity of tumors, sampling of tumor tissue may result in tissue detection results that do not reflect the tumor's general appearance, and IHC detection of only major lesions may affect the treatment decisions of some patients. Advanced cancer patients with unavailable partial tissue samples may also miss the opportunity for targeted therapy due to the inability to take tissue biopsies. In addition, under-regulation of tissue sample processing, IHC and FISH detection techniques' own challenges may also lead to false negative results in tissue detection, thereby rendering such patients unacceptable and benefiting from targeted therapies.
Current methods for detection of HER2 amplification mainly include second generation sequencing NGS, fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), and digital PCR methods. The second generation sequencing method can simultaneously detect a plurality of samples, but has higher cost, longer experimental period and complex data analysis; fluorescence In Situ Hybridization (FISH) technology has high detection specificity, but the sample treatment period is long, the cost is high (the probe is expensive), and the result interpretation subjectivity and the speciality are strong; the digital PCR technology can realize absolute quantification, has higher sensitivity and high detection speed, and can realize rapid detection for 1-2 working days, but the platform is few in hospitals or units and has higher cost.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to solve the problems that: how to provide a primer probe group, a kit and a system for quantitative detection of HER2 gene amplification, so as to solve the problems of long period, high cost or complex analysis existing in the existing detection method.
In order to solve the problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer probe set for quantitative detection of HER2 gene amplification, which comprises primers and probes with nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 3.
As one embodiment, the kit further comprises primers and probes with nucleotide sequences shown as SEQ ID NO.4-SEQ ID NO. 6.
In a second aspect, the invention provides a kit for quantitative detection of HER2 gene amplification, which comprises a primer probe group for quantitative detection of HER2 gene amplification.
As one embodiment, the primer comprises 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.1, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.2, 0.5 part by volume of the probe of the nucleotide sequence shown in SEQ ID NO.3, 5.5 parts by volume of the non-nucleic acid water and 10 parts by volume of the PCR premix;
or, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.4, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.5, 0.5 part by volume of the probe of the nucleotide sequence shown in SEQ ID NO.6, 5.5 parts by volume of the non-nucleic acid water and 10 parts by volume of the PCR premix solution are also included.
In a third aspect, the invention provides a system for quantitative detection of HER2 gene amplification, comprising an acquisition module, an extraction module, an amplification module, a detection module and an analysis module;
the collection module is used for collecting a plasma sample;
the extraction module is used for extracting a DNA sample from the plasma sample;
the amplification module is used for carrying out PCR amplification on the DNA sample by adopting a primer probe group for quantitative detection of the HER2 gene amplification or a kit for quantitative detection of the HER2 gene amplification;
the detection module is used for carrying out fluorescence detection on the amplified product;
the analysis module is used for analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is S, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, the detection result is positive; if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is less than 2, and the detection result is negative.
As an embodiment, the PCR amplification procedure in the amplification module includes: activating at 95 ℃ for 300s; cycling for 45 times at 95 ℃, 15s,56 ℃ and 35 s; the temperature was kept at 25℃for 10s.
As an embodiment, in the analysis module, the detection is not effective if the Ct value of the reference gene is > 35 or there is no amplification.
In a fourth aspect, the invention provides a method for non-disease diagnostic purposes for quantitative detection of HER2 gene amplification, comprising:
collecting a plasma sample;
extracting a DNA sample from the plasma sample;
carrying out PCR amplification on a DNA sample by adopting a primer probe group for quantitative detection of the HER2 gene amplification or a kit for quantitative detection of the HER2 gene amplification;
carrying out fluorescence detection on the amplified product;
analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, and the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, determining that the detection result is positive; if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is less than 2, and the detection result is negative.
In a fifth aspect, the invention provides a primer probe set for quantitative detection of amplification of the HER2 gene or application of a kit for quantitative detection of amplification of the HER2 gene in preparation of a reagent for quantitative detection of amplification of the HER2 gene.
The invention has the beneficial effects that: the primer probe group, the kit and the system for quantitative detection of HER2 gene amplification provided by the invention are based on ctDNA detection, and circulating tumor DNA (ctDNA) can reflect in-vivo tumor load in a short time, monitor drug curative effect in real time and dynamically, can be used as effective supplement for tissue sample detection, are beneficial to avoiding false negative results of tissue sample detection caused by tumor heterogeneity, are easy to obtain liquid biopsy samples, can dynamically monitor and manage curative effect through ctDNA for late cancer patients incapable of obtaining tissue samples, and are significant in relieving clinical symptoms of late tumor patients, prolonging survival time and improving life quality by optimizing a treatment scheme.
The fluorescent quantitative PCR can also be used for detecting HER2 amplification, the detection speed is higher, the detection result can be obtained within 2 working days, the detection sensitivity is better, the kit adopts the Taqman-MGB probe design, the signal to noise ratio is improved (because the quenching group at the 3' end of the probe is a fluorescent group which does not emit light), the kit is closer to the position of the reporter gene in space, the experimental flux is high, a plurality of samples are detected at one time, the detection is quick, the price is more suitable for clinic, the detection steps can be standardized, the flow is standardized, and the sample selectivity is high: tissue and blood can be detected.
Drawings
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings, in which:
FIG. 1 is a graph showing amplification of HER2 plasmid standard in accordance with the examples of the present invention;
FIG. 2 is an amplification plot of ACTB plasmid standard in accordance with the examples of the present invention;
FIG. 3 is a graph showing the positive amplification of HER2 gene in a sample according to the embodiment of the invention;
fig. 4 is a graph showing HER2 gene negative amplification of a sample according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be noted that these examples are only for illustrating the present invention, and not for limiting the present invention, and simple modifications of the method under the premise of the inventive concept are all within the scope of the claimed invention.
Example 1
Quantitative detection of HER2 gene amplification
1. The method comprises the steps of designing an upstream primer and a downstream primer aiming at the HER2 genome DNA, specifically amplifying a sequence of the HER2 genome by the primer, and specifically combining with the HER2 by the probe.
2. In order to quantitatively detect the amplification proportion of HER2, the invention designs an internal reference, and designs an upstream primer, a downstream primer and a specific probe aiming at the DNA of the ACTB genome of the internal reference gene, wherein the primer can specifically amplify a section of sequence of the ACTB genome, and the probe can specifically bind with the ACTB.
3. In order to quantitatively detect the amplification proportion of HER2, a standard substance is designed, and fluorescent quantitative PCR detection of HER2 and an internal reference gene is carried out on a sample to be detected and the standard substance.
4. Obtaining a standard curve for quantification from the detection result of the standard substance, calculating the copy numbers of the HER2 gene and the ACTB gene of the nucleic acid of the sample to be detected from the standard curve, carrying out repeated test on the sample to be detected, and finally carrying out result interpretation according to the average value of the copy numbers of the HER2 gene and the ACTB gene.
5. The primers and probes are shown in Table 1.
TABLE 1 primer probe sequences
6. Preparation of Positive standards target fragment clone PUC57 vector synthesis was purchased from biological engineering (Shanghai) Co.
Post-synthesis plasmid solubilization dilution to calculate copy number, copy number = mass/molecular weight x 6.02 x 10 23 10-fold dilutions of the standard were made according to the selected mother liquor, 4 gradients total: the method comprises the steps of taking 4 PCR reaction tubes to be respectively marked with 1, 2, 3 and 4, respectively, adding 18ul of nucleic-free Water into each PCR reaction tube, taking 2ul of standard stock solution into a tube 1, taking 2ul of standard stock solution from the tube 1 after full shaking and uniform mixing, and adding the standard stock solution into the tube 2, and sequentially diluting to the required copy number.
Table 2 standard preparation table
Example 2
Specific flow for quantitative detection of HER2 gene amplification
1. The main composition is shown in Table 3.
TABLE 3 component Table of kit
Wherein the relevant ingredients in table 3 were formulated according to table 4.
TABLE 4 preparation of the relative composition of the kit
2. Storage conditions and expiration dates
The kit is stored at the temperature of minus 20 plus or minus 5 ℃ and the effective period is 12 months; the effective period after unsealing is 6 months; repeatedly freezing and thawing for 3 times.
3. Suitable instrument
ABI 7500 fluorescent PCR instrument
4. Sample requirement
1. Sample type: a plasma sample.
2. And (3) sample collection:
blood was collected 10mL using K2EDTA or free nucleic acid blood collection tubes, as recommended by the manufacturer, and the blood sample should be immediately processed. Before plasma preparation, K2EDTA blood collection tube is kept at 2-8deg.C for no more than 24 hr without freezing blood sample. The free nucleic acid sample tube can be stored at 15-25deg.C for no more than 72 hr before plasma preparation, without freezing blood sample.
3. Preparation and preservation of plasma:
putting the blood collection tube into a centrifugal machine for centrifugation, wherein the rotation speed is 1350+/-150 rcf, and the centrifugation is carried out for 12 minutes (the braking function of the centrifugal machine is forbidden to be used so as to prevent the damage of a blood cell layer); the blood collection tube is taken out from the centrifuge, the blood plasma is transferred to a centrifuge tube made of polypropylene and 15mL with a conical bottom by a new disposable pipette, and the blood collection tube is centrifuged again for 12 minutes at 1350+/-150 rcf. 3.5mL of plasma was added to a new centrifuge tube using a new disposable pipette or a serum pipette, and the sample number was labeled. The plasma sample can be used for detection immediately, or can be stored at 2-8deg.C (not more than 24 h), and can be stored for one month at 15-25deg.C and for six months at below-70deg.C. The rpm was converted to rcf by reference to the centrifuge operating manual.
4. Sample usage: 3.5mL.
5. Inspection method
1. Reagent preparation
1.1 And taking out the PCR reaction liquid from the kit, thawing at room temperature, gently shaking and uniformly mixing after full thawing, and performing short-term centrifugation for later use.
1.2 According to the number (n) of the samples to be detected, the required reaction number is n multiplied by 3 (three times of parallel samples to be detected) +3 (negative quality control, positive quality control and template-free control), and 18 mu l of each PCR reaction liquid is respectively split-packed in each reaction.
2. Sample processing
2.1 Thawing of plasma
In the case of frozen plasma samples, which are thawed for about 30 minutes at room temperature (15-30 ℃), the sample must undergo a lysis procedure within 60 minutes of thawing.
2.2 Nucleic acid extraction
The nucleic acid extraction or purification kit is operated with reference to the commercial kit instructions purchased for plasma free nucleic acid extraction.
3. Sample addition
Sample adding: the sample DNA to be tested was added to each of the PCR reaction tubes prepared with the reagents, as shown in Table 5. 2. Mu.L of DNA was added to each PCR reaction, and after the tube cap was closed, the mixture was centrifuged at a low speed. Note that: the sealed PCR plate can be left at 2-8℃for up to 4 hours.
TABLE 5 sample DNA addition System Table
4. PCR amplification
4.1 sample setup: sample numbers are set according to sample types, and the 8-row sample adding layout of the PCR instrument is shown in Table 6. In the table, PC represents Positive Control, NC represents Negative Control, NTC represents no-template Control (No Template Control), and S represents sample.
Table 6 PCR instrument 8 row sample addition layout table
4.2 fluorescence channel selection: the HER2 gene selected FAM channel per sample and ACTB gene selected JOE channel per sample, and the reference fluorescence (Passive Reference) was set to none.
4.3 reaction condition settings (reaction volume was set to 20. Mu.L) are given in Table 7.
TABLE 7 PCR amplification reaction conditions Table
4.4 storing the file and running the program.
5. Analysis of results
Automatically storing the result after the reaction is finished, automatically analyzing the result by using instrument matched software, adjusting the Start value, end value and Threshold value of Baserine according to the analyzed image (a user can adjust the Start value at 2-8 and the End value at 10-20 according to actual conditions), setting a fluorescence Threshold (Threshold) to be just higher than the highest point of an amplification curve (an irregular noise line) of a negative quality control product by a Threshold line, displaying the Ct value as undet), and clicking Analysis to automatically obtain the Analysis result.
6. Result determination
The interpretation of the PCR reaction results is shown in Table 8. If the internal reference ACTB shows that the amount of DNA added to the single sample is sufficient (ACTB Ct values are shown in the table), the HER2 gene and ACTB gene copy numbers are calculated according to the standard, the average value is calculated for three parallel results of the copy numbers of each gene, and finally the ratio of the HER2 gene copy number to the ACTB gene copy number is calculated and taken as the result of the PCR reaction. If the Ct value of ACTB is greater than the threshold set in the table, then the PCR reaction is defined as "invalid".
TABLE 8 interpretation of PCR reaction results
6. Positive judgment value or reference interval
The sensitivity of the kit for detecting HER2 gene amplification is 95.46%, the specificity is 92.65%, and the kit can realize high sensitivity and high specificity detection of HER2 gene amplification.
7. The results of the test are explained in Table 9.
Table 9 test result interpretation table
The amplification curve of the HER2 plasmid standard product is shown in figure 1, the amplification curve of the ACTB plasmid standard product is shown in figure 2, the positive amplification curve of the HER2 gene of the sample is shown in figure 3, and the negative amplification curve of the HER2 gene of the sample is shown in figure 4.
1) If the Ct value of the reference gene ACTB is less than or equal to 35, the Ct value of HER2 gene is less than or equal to 45, the amplification curve is of an S type, the copy numbers of the HER2 gene and the ACTB gene are calculated through a standard substance, the average value is calculated according to three parallel results of the copy numbers of each gene, and the average value ratio of the copy numbers of the HER2 gene to the ACTB gene is more than or equal to 2, so that the detection result is positive;
2) If the Ct value of the reference gene ACTB is less than or equal to 35, the Ct value of HER2 gene is less than or equal to 45, the amplification curve is of an S type, the copy numbers of the HER2 gene and the ACTB gene are calculated through a standard substance, the average value is calculated aiming at three parallel results of the copy numbers of each gene, and the average value ratio of the copy numbers of the HER2 gene to the ACTB gene is less than 2, so that the detection result is negative;
3) If the Ct value of the reference gene ACTB is more than 35 or no amplification exists, the experiment is ineffective, and the added DNA contains a PCR inhibitor, so that the DNA detection needs to be extracted again.
8. Corresponding to the flow, a system for quantitative detection of HER2 gene amplification can be designed, and comprises an acquisition module, an extraction module, an amplification module, a detection module and an analysis module;
the collection module is used for collecting a plasma sample;
an extraction module for extracting a DNA sample from a plasma sample;
the amplification module is used for carrying out PCR amplification on the DNA sample by adopting a primer probe set for quantitative detection of HER2 gene amplification or a kit for quantitative detection of HER2 gene amplification;
the detection module is used for carrying out fluorescence detection on the amplified product;
the analysis module is used for analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, the detection result is positive; if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is less than 2, and the detection result is negative.
Example 3
The samples to be detected in the embodiment are plasma samples, and the sample numbers are S1-S22 respectively; cfDNA in the plasma samples was extracted using the kit, specific operations are described in QIAamp Circulating Nuleacid Kit kit instructions by QIAGEN corporation, and the extracted cfDNA was used as a template for fluorescent quantitative PCR detection, wherein 13 cases were HER2 positive samples, 9 cases were HER2 negative samples, and the detection results are shown in table 10.
Table 10 comparison of the results of fluorescent quantitative PCR detection and IHC detection of different samples
IHC is the detection of HER2 protein overexpression at the protein level by formalin-fixed paraffin section immunochromogenic reaction, while FISH is the detection of gene amplification on the chromosome by hybridization of a fluorescently labeled nucleic acid probe with the DNA target sequence of the HER2 gene in the nuclei fixed on paraffin sections. Further FISH assay confirmation is required when IHC assay results are 2+.
According to the judgment basis of the invention, and by combining with the qPCR detection result, the invention provides a quantitative detection method of HER2 genes, wherein the copy number ratio of the two genes is more than or equal to 2, the two genes are HER2 positive, and 13 cases of HER2 positive samples are detected; the copy number ratio of both genes was < 2, HER2 negative, and 9 samples were detected as HER2 negative samples. The results show that the detection results of the invention are completely consistent with the results of FISH and IHC. The kit can detect not only HER2 amplification, but also the copy number of HER 2.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be understood that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A primer probe group for quantitative detection of HER2 gene amplification is characterized by comprising primers and probes with nucleotide sequences shown as SEQ ID NO.1-SEQ ID NO. 3.
2. The primer probe set for quantitative detection of HER2 gene amplification according to claim 1, further comprising a primer and a probe with nucleotide sequences shown as SEQ ID NO.4-SEQ ID NO. 6.
3. A kit for quantitative detection of HER2 gene amplification, comprising the primer probe set for quantitative detection of HER2 gene amplification according to claim 1 or 2.
4. The kit for quantitative detection of HER2 gene amplification according to claim 3, comprising 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.1, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.2, 0.5 part by volume of the probe of the nucleotide sequence shown in SEQ ID NO.3, 5.5 parts by volume of the non-nucleic acid water and 10 parts by volume of the PCR premix;
or, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.4, 1 part by volume of the primer of the nucleotide sequence shown in SEQ ID NO.5, 0.5 part by volume of the probe of the nucleotide sequence shown in SEQ ID NO.6, 5.5 parts by volume of the non-nucleic acid water and 10 parts by volume of the PCR premix solution are also included.
5. The HER2 gene amplification quantitative detection system is characterized by comprising an acquisition module, an extraction module, an amplification module, a detection module and an analysis module;
the collection module is used for collecting a plasma sample;
the extraction module is used for extracting a DNA sample from the plasma sample;
the amplification module is used for carrying out PCR amplification on the DNA sample by adopting the primer probe set for the quantitative detection of the HER2 gene amplification according to claim 1 or 2 or the kit for the quantitative detection of the HER2 gene amplification according to claim 3 or 4;
the detection module is used for carrying out fluorescence detection on the amplified product;
the analysis module is used for analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is S, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, the detection result is positive; if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is less than 2, and the detection result is negative.
6. The system for quantitative detection of HER2 gene amplification according to claim 5, wherein the procedure of PCR amplification in the amplification module comprises: activating at 95 ℃ for 300s; cycling for 45 times at 95 ℃, 15s,56 ℃ and 35 s; the temperature was kept at 25℃for 10s.
7. The system for quantitative detection of HER2 gene amplification according to claim 5, wherein the detection is not effective in the analysis module if the Ct value of the reference gene is > 35 or no amplification.
8. A method for non-disease diagnostic purposes of HER2 gene amplification quantitative detection comprising:
collecting a plasma sample;
extracting a DNA sample from the plasma sample;
performing PCR amplification on the DNA sample using the primer probe set for HER2 gene amplification quantitative detection of claim 1 or 2 or the kit for HER2 gene amplification quantitative detection of claim 3 or 4;
carrying out fluorescence detection on the amplified product;
analyzing the fluorescence detection result, and if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, and the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is more than or equal to 2, determining that the detection result is positive; if the Ct value of the reference gene is less than or equal to 35, the Ct value of the HER2 gene is less than or equal to 45, the amplification curve is of an S type, the average ratio of the copy number of the HER2 gene to the copy number of the reference gene is less than 2, and the detection result is negative.
9. Use of the primer probe set for quantitative detection of HER2 gene amplification according to claim 1 or 2 for preparing a reagent for quantitative detection of HER2 gene amplification.
10. Use of the kit for quantitative detection of HER2 gene amplification according to claim 3 or 4 for the preparation of a reagent for quantitative detection of HER2 gene amplification.
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