阅读说明：本技术 一种用于检测乳腺癌her2拷贝数变异的数字pcr方法及检测试剂盒 (Digital PCR method and detection kit for detecting breast cancer HER2 copy number variation ) 是由 董莲华 王霞 王晶 于 2019-09-29 设计创作，主要内容包括：本发明公开了一种用于检测乳腺癌HER2拷贝数变异的数字PCR方法及检测试剂盒,所述试剂盒包括用于检测HER2基因拷贝数变异的至少一对引物及其相配套的一条探针,和用于检测的内参基因的一对引物及其相配套的一条探针。本发明的方法采用三组HER2基因的特异性引物探针和一组内参基因的方式进行,从而有效地排除了单一引物探针扩增导致的假阴性结果,因此相较于现有技术中具有更高的准确性,而且将现有方法的不确定区间1.8-2.2降低至1.0-1.2,具有更高的灵敏度。(The invention discloses a digital PCR method and a detection kit for detecting breast cancer HER2 copy number variation, wherein the kit comprises at least one pair of primers for detecting HER2 gene copy number variation and a probe matched with the primers, and a pair of primers for detecting reference genes and a probe matched with the primers. The method of the invention is carried out by adopting a mode of three groups of specific primer probes of HER2 genes and one group of reference genes, thereby effectively eliminating false negative results caused by single primer probe amplification, having higher accuracy compared with the prior art, reducing the uncertain interval 1.8-2.2 of the prior method to 1.0-1.2 and having higher sensitivity.)

1. A test kit for detecting HER2 copy number variation in breast cancer, comprising: comprises at least one pair of primers for detecting HER2 gene copy number variation and a probe matched with the primers, and a pair of primers for detecting reference genes and a probe matched with the primers.

2. The detection kit according to claim 1, characterized in that: the internal reference gene is RPPH1 gene, the primer of the internal reference gene RPPH1 for detection is P1, the sequences are shown as SEQ ID NO 10 and SEQ ID NO 11, and the sequence of the probe is shown as SEQ ID N0: 12.

3. The detection kit according to claim 2, characterized in that: comprises three pairs of primers H1, H2 and H3 for detecting HER2 gene copy number variation and three matched probes, wherein the sequence of H1 is shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of H2 is shown in SEQ ID NO 3 and SEQ ID N0: 4; the sequence of H3 is shown in SEQ ID NO. 5 and SEQ ID NO. 6; the sequence of the probe is shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9.

4. The test kit of claim 3, wherein the kit comprises:

(a) a first container, and a specific primer pair and probe for specifically amplifying a HER 2-containing gene located within said container;

(b) a second container, and a specific primer pair and probe for specifically amplifying a HER 2-containing gene located within said container;

(c) a third container, and a specific primer pair and a probe for specifically amplifying a HER 2-containing gene located within said container;

(d) a fourth container, and a specific primer pair and a probe which are positioned in the container and are used for specifically amplifying the RPPH 1-containing gene.

5. A method for positively detecting a HER2 mutation in a sample, the method comprising:

(1) providing a detection sample, wherein the detection sample is a tumor tissue sample or cells over-expressed by HER2 and cells normally expressed by HER 2;

(2) performing DNA extraction on the detection sample to obtain a DNA extract;

(3) carrying out micro-droplet digital PCR (ddPCR) amplification by taking the DNA extract obtained in the step (2) as a template to obtain an amplification product containing HER2 gene and reference gene;

(4) analyzing the amplification product to obtain the ratio of HER2 gene and reference gene;

(5) using any pair of primers and probes for the HER2 gene of claim 4;

wherein, the reference gene is RPPH 1.

6. A HER2 mutation positive detection method according to claim 5, wherein: in ddPCR, the reaction system also contains probes for detecting the HER2 gene and an internal reference gene.

7. A HER2 mutation positive detection method according to claim 5, wherein: the step (4) comprises the following steps: analyzing the copy number of amplified products of the HER2 primer pair H1, H2 and H3 and the internal reference gene RPPH1 primer pair P1, and removing the copy number of the internal reference gene by using the copy number of the HER2 gene to obtain three groups of ratios, namely H1/P1, H2/P1 and H3/P1, wherein all three ratios are less than or equal to 1.0, and judging that the HER2 is negative, and if 2 of the three ratios are more than 1.2, judging that the HER2 is positive.

Technical Field

The invention relates to the technical field of gene detection related to tumor targeted therapy, in particular to a digital PCR method and a detection kit for detecting breast cancer HER2 copy number variation.

Background

Breast cancer is one of the most common malignant tumors in women, with over 100 million new cases each year, and is a disease that seriously threatens the health and even life of patients. The therapeutic effect and prognosis of clinical drugs may also vary greatly from patient to patient.

The HER2 gene is a protooncogene located on the long arm of human chromosome 17, encodes human epidermaL growth factor receptor-2 (HER 2) transmembrane glycoprotein with receptor tyrosine kinase (PTK) activity, is a member of epidermaL growth factor receptor family, and needs to be combined with other receptors of the family to form heterodimers to play a signal transduction function. Overexpression or amplification of the HER2 gene, which results in excessive signaling, will stimulate cancer cell growth metastasis. HER-2 positive breast cancer accounts for about 20-30% of invasive breast cancer, and the classified tumor has high invasiveness, high recurrence risk, rapid progression and poor prognosis. Determination of the expression level of the HER2 gene in tumor cells of breast cancer patients facilitates selection of treatment regimens and prognosis. The HER2 molecule is one of important molecular biological markers of breast cancer and is an important target site for gene therapy of breast cancer.

Trastuzumab, also known as Herceptin (Herceptin), is a targeted drug for HER2 positive breast cancer, and has been approved by the U.S. FDA for the treatment of HER2 positive breast cancer. The herceptin can be specifically combined with the 4 th functional domain outside the cell membrane of the HER-2 protein, and interferes with the formation of a heterodimer between HER2 and other ERBB family members, thereby effectively inhibiting the proliferation of tumor cells.

Currently, clinically routine HER2 detection means include Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH). The IHC detects the expression condition of HER2 protein on cell membrane, the detection result is 3+, namely, the HER2 is judged to be positive, and HER2 targeted drug can be selected for treatment; the detection result is 0 or 1+, the HER2 negativity can be judged, and the HER2 targeted drug is not suitable for treatment; when the detection result is 2+, the comprehensive judgment needs to be carried out by combining the FISH result. FISH uses the chromosome 17 centromere probe as an internal reference, and judges whether the HER2 gene is amplified or not by calculating the ratio of the hybridization signals of the HER2 gene probe and the chromosome 17 centromere probe (HER2/CEP 17). The HER2 positive result is judged if the FISH detection result is more than 2.2, the HER2 negative result is judged if the FISH detection result is less than 1.8, and the HER2 negative result cannot be accurately judged if the FISH detection result is between 1.8 and 2.2. For HER2 positive patients, HER2 targeted drugs, such as herceptin and the like; HER2 negative patients do not recommend the use of such drugs.

However, at present, the two detection methods have defects, both gray areas have results which cannot be accurately judged, and in recent years, even if patients judged to be positive have poor effect on HER2 targeted therapy, some suspected positive patients and some negative patients are sensitive to HER2 targeted drugs. Therefore, the medication instruction according to the detection result of IHC or FISH method is not very accurate, so a more sensitive and accurate detection method of HER2 positive breast cancer is needed to perform the instruction of HER2 targeted drugs, so that more patients can be treated accurately.

CN105986028A discloses a method for detecting breast cancer HER2 positivity by ddPCR technology, and the kit comprises two pairs of primers for detecting HER2 gene, one pair of primers for detecting CEP17 gene and 2 pairs of primers for detecting EFTUD2 gene. However, the technical solution of the patent application can only solve the problem that the conventional method determines whether the sample suspected to be positive has HER2 positive, but cannot solve the problem that the conventional method determines to be negative and actually a false negative sample, and since the sample is chromosome multiplication, the conventional method and the method of the patent application cannot completely and accurately determine HER2 positive.

In view of the above, there is a lack in the art of a method and a kit capable of accurately and effectively detecting HER2 copy number variation.

Disclosure of Invention

The invention aims to provide a digital PCR method and a detection kit for detecting breast cancer HER2 copy number variation. The invention adopts ddPCR amplification technology, designs specific HER2 gene copy number detection primers and probes, optimizes stably expressed reference genes, and combines RPPH1 as the reference genes, thereby accurately detecting the HER2 expression state, eliminating the false negative and false positive conditions to a great extent, and more accurately judging whether the HER2 is positive.

(1) The invention designs three pairs of primers and probes for specifically amplifying HER2 gene

Three pairs of specific primers and probes were designed using primer express3.0 for the sequences of exons 7, 16, 24, respectively, based on the total length and exon distribution of the HER2 gene. And the human HER2 gene is high in specificity and has no potential non-specific amplification region by adopting Blast for comparison. The specific sequence is shown in SEQ ID NO. 1-SEQ ID NO. 9.

(2) Primer and probe amplification conditions with optimized specificity of three pairs of HER2 genes

The final concentrations of primers for exons 7 and 24 of HER2 were fixed at 300nM, the probe concentration was fixed at 300nM (see FIGS. 1 and 2), the final concentration of primers for exon 16 of HER2 was fixed at 300nM, and the probe concentration was fixed at 400nM for annealing temperature optimization, which was set at six annealing temperatures of 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃ (see FIG. 3). From the one-dimensional scatter diagram of amplification, the better amplification is obtained at six annealing temperatures, two clusters of microdroplets of positive microdroplets and negative microdroplets are dispersed best at 57 ℃, but from the copy number concentration of a quantitative result, the difference between the copy number concentration under the condition of 60 ℃ and the internal reference gene is minimum, and therefore, the optimal annealing temperature of 60 ℃ is determined. Then, the primer and probe concentrations were optimized, with primer concentrations of exon No. 7 and exon No. 24 of HER2 set at 200nM, 300nM, and 400nM, respectively, probe concentrations set at 200nM, 300nM, and 400nM, respectively, primer concentrations of exon No. 16 of HER2 set at 200nM, 300nM, and 400nM, respectively, and probe concentrations set at 200nM, 300nM, 400nM, and 500nM, respectively, for optimization (see FIGS. 4-6).

When the concentration of the primer is unchanged, the probe concentration of the exon No. 7 and 24 of HER2 is increased from 200nM to 400nM, and the probe concentration of the exon No. 16 of HER2 is increased from 200nM to 500nM, the fluorescence intensity of the negative droplet and the fluorescence intensity of the positive droplet are increased, and the separation degree of the negative droplet and the positive droplet is increased; when the probe concentration of the exons 7, 16 and 24 of HER2 is unchanged, the fluorescence intensity of the negative droplet is basically unchanged when the probe concentration of the exons 7, 16 and 24 of HER2 is increased from 200nM to 400nM, while the fluorescence intensity of the positive droplet is increased, so that the separation degree of the negative droplet and the positive droplet is increased, but the dispersion range of the positive droplet is larger and larger as the probe concentration is increased, for example, the distribution width of the positive droplet is obviously larger than that of the probe at 200nM (G01, D04 and B07) when the probe is at 300nM (B02, E04 and D07), and the primer concentration of the exons 7, 16 and 24 of HER2 is determined to be 300nM when the repeatability and copy number concentration factors of the quantitative result are considered together, and the concentration of the probe is 300 nM.

The final system for the microdroplet digital PCR reaction was: 2 XDddPCR mastermix 10. mu.L, bipartite primer (5. mu.M) 1.2. mu.L, probe (10. mu.M) 0.6. mu.L, DNA template 4. mu.L, ddH2O to make up 20. mu.L. PCR reaction procedure: 95 ℃ for 10min, 94 ℃ for 30s, 60 ℃ for 1min, 40cycles, 98 ℃ for 10 min.

(3) A pair of specific reference genes RPPH1 are designed

According to NCBI database search, the RPPH1 gene is found to be a relatively stable single copy gene, so that the gene is selected as an internal reference gene to carry out the design of primers and probes for specific amplification. A specific pair of primers and probes was designed using PrimerExpress3.0. The comparison is carried out by using Blast, and the human RPPH1 gene binding specificity is high, and no potential non-specific amplification region exists. The specific sequence is shown in SEQ ID N0:10-SEQ ID N0: 12.

(4) Optimizing the amplification condition of the RPPH1 reference gene

Firstly, optimizing annealing temperature, namely fixing the final concentration of a primer at 250nM, fixing the concentration of a probe at 100nM, and optimizing the annealing temperature, wherein the annealing temperature is set to be four annealing temperatures of 56 ℃, 58 ℃, 60 ℃ and 61 ℃, and from a one-dimensional scatter diagram of an amplification result (figure 3), the four annealing temperatures are all better amplified, two clusters of microdroplets of a positive microdroplet and a negative microdroplet are best dispersed at 56 ℃, but from the repeatability of a quantitative result, the repeatability at 60 ℃ is best, so that the 60 ℃ is determined to be the best annealing temperature. Then, the concentration of the primers and the concentration of the probe were optimized, with the primer concentrations set to 150nM, 250nM and 350nM, respectively, and the probe concentrations set to 50nM, 100nM and 200nM, respectively, and optimized (see FIG. 4).

When the concentration of the primer is unchanged and the concentration of the probe is increased from 50nM to 200nM, the fluorescence intensity of the negative droplet and the fluorescence intensity of the positive droplet are both increased, and the separation degree of the negative droplet and the separation degree of the positive droplet are increased, when the concentration of the probe is unchanged and the fluorescence intensity of the primer is increased from 150nM to 350nM, the fluorescence intensity of the negative droplet is not changed, and the fluorescence intensity of the positive droplet is increased, so that the separation degree of the negative droplet and the positive droplet is increased, but the dispersion range of the positive droplet is larger and larger along with the increase of the concentration of the probe, for example, the distribution width of the positive droplet at 200nM (F05, F06 and F07) is obviously larger than that of the probe at 100nM (D05, C06 and D07), and the concentration of the probe is determined to be 100nM after the comprehensive consideration of repeatability factors of the. The final system for the microdroplet digital PCR reaction was: 2 XDddPCR mastermix 10. mu.L, bipartite primer (5. mu.M) 1. mu.L, probe (10. mu.M) 0.2. mu.L DNA template 4. mu.L, ddH2O to make up 20. mu.L. PCR reaction procedure: 5min at 95 ℃, 20s at 95 ℃, 40s at 60 ℃, 45cycles and 10min at 98 ℃.

(5) Quantification of HER2 Gene copy number in different cell lines

In order to prove that the established HER2 gene specific primer probe can realize accurate detection of HER2 gene copy number, 6 strains including normal human cells and breast cancer cell strains are selected, and the HER2 gene copy number is measured. The measurement results show that the ratio of the HER2 gene copy number to the internal reference gene copy number in the DNA of the normal human cells is less than 1.0, and the measurement results of the three pairs of primer probes are consistent. And the ratio of the HER2 gene copy number to the internal reference gene copy number of the two breast cancer cell lines is 22.4 and 34.6 respectively, so that the two cells can be determined to be positive for HER2 copy number variation.

(6) The sensitivity and linearity of the method for detecting HER2 copy number variation are studied

The method comprises the steps of mixing normal human genome DNA and breast cancer cell genome DNA according to a certain proportion to prepare 4 DNA samples with different mutation levels, amplifying by using three pairs of established HER2 specific primers and probes, amplifying internal reference gene RPPH1, analyzing the copy number ratio of the primers and probes of each pair of HER2 specific genes to the internal reference gene, and performing linear fitting by using the prepared copy number ratio of the HER2 gene to the RPPH1 gene as a horizontal coordinate and the ratio determined by ddPCR as a vertical coordinate (as shown in figures 5-7). The preparation value is consistent with the result of ddPCR measurement, and the linear correlation coefficients are both larger than 0.995, which indicates that the established ddPCR method is accurate and reliable in detecting HER2 gene copy number variation, and can detect variation as low as one copy number at least.

Based on this, the technical scheme provided by the invention is as follows:

a test kit for detecting HER2 copy number variation in breast cancer, the kit comprising: at least one pair of primers and a matched probe for detecting HER2 gene, and a pair of primers and a probe for detecting single-copy reference gene RPPH 1.

The kit comprises a pair of primers and a probe for detecting the RPPH1 gene.

The kit comprises three pairs of primers and three probes for detecting HER2 gene.

Specifically, the kit comprises: comprises three pairs of primers H1, H2 and H3 for detecting HER2 gene copy number variation and three matched probes, wherein the sequence of H1 is shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of H2 is shown in SEQ ID NO 3 and SEQ ID N0: 4; the sequence of H3 is shown in SEQ ID NO. 5 and SEQ ID NO. 6; the sequence of the probe is shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9. The probe is a VIC probe or a FAM probe.

The sequence of the probe for detecting the HER2 gene is shown as SEQ ID N0:7 or SEQ ID N0:8 or SEQ ID N0: 9; preferably, the probe is a FAM probe.

The sequence of the primer pair P1 for detecting the RPPH1 gene is shown as SEQ ID NO. 10 and SEQ ID N0: 11. The sequence of the probe for detecting the RPPH1 gene is shown as SEQ ID N0: 12: preferably, the probe is a VIC probe.

Further, the kit comprises:

(a) a first container, and a specific primer pair and corresponding probe for specifically amplifying a HER 2-containing gene located within said container;

(b) a second container, and a specific primer pair and corresponding probe for specifically amplifying a HER 2-containing gene located within said container;

(c) a third container, and a specific primer pair and corresponding probe for specifically amplifying a HER 2-containing gene located within said container;

(d) a fourth container, and a specific primer pair and a corresponding probe for specific amplification containing RPPH1 gene positioned in the fourth container;

(e) instructions for use, wherein the instructions describe a method of use.

The using method comprises the following steps:

(1) providing a test sample, wherein the test sample is a tumor tissue sample;

(2) performing DNA extraction on the detection sample to obtain a DNA extract;

(3) carrying out micro-droplet digital PCR (ddPCR) amplification by taking the DNA extract obtained in the step (2) as a template so as to obtain an amplification product containing the HER2 gene and the reference gene;

(4) analyzing the amplification products to obtain the ratio of HER2 gene and reference gene RPPH 1; in the step (2), for 2ml of sample, the concentration of the DNA extract is 20-100 μ L and is not lower than 15ng/μ L of DNA extract.

In the step (3), the amount of the DNA template used for ddPCR amplification is 30-100 ng.

The methods are non-diagnostic and non-therapeutic.

The method further comprises the following steps: three sets of amplifications were performed on the HER2 gene using different primer and probe pairs.

The method further comprises the following steps: the RPPH1 gene was amplified.

A method for positive detection of a HER2 mutation in a sample, the method comprising;

(1) providing a detection sample, wherein the detection sample is a tumor tissue sample or 4 horizontal DNA solutions prepared after genomic DNA is respectively extracted from cells normally expressed by HER2 and cells overexpressed by HER 2;

(2) carrying out DNA extraction treatment on the detection sample so as to obtain a DNA extract;

(3) carrying out microdroplet digital PCR (ddPCR) by using the DNA extract obtained in the step (2) as a template so as to obtain an amplification product containing the HER2 gene and an internal reference gene;

(4) analyzing the amplification products to obtain the ratio of HER2 gene and reference gene RPPH 1; the steps of (A), (B), (C2) Middle, 10^ s⁶-10^⁷For each cell detection sample, the DNA extract contains 5-30. mu.g of DNA extract. In the step (3), the amount of the DNA template used for ddPCR amplification is 30-100 ng.

The method further comprises the following steps: three sets of amplifications were performed on the HER2 gene using different primer pairs. The method further comprises the following steps: the RPPH1 gene was amplified. In ddPCR, the reaction system also contains probes for detecting the HER2 gene and an internal reference gene. The probe is a FAM probe or a VIC probe. The sequences of the three pairs of primer pairs for detecting the HER2 gene are shown as SEQ ID N0:1 and SEQ ID N0:2, or SEQ ID NO 3 and SEQ ID N0:4, or SEQ ID NO 5 and SEQ ID N0: 6. The sequences of the primer pair for detecting the RPPH1 gene are shown as SEQ ID NO. 10 and SEQ ID N0: 11. The sequence of the probe for detecting the HER2 gene is shown as SEQ ID N0:7 or SEQ ID N0:8 or SEQ ID N0: 9; preferably, the probe is a FAM probe. The sequence of the probe for detecting the RPPH1 gene is shown as SEQ ID N0:12, preferably, the probe is a VIC probe.

The reaction system of the PCR reaction comprises: the genome to be tested: 30-100 ng of 2 XddPCR Supermix for Probes (Bio-Rad Laboratories) 10. mu.1, 1.2. mu.L of upstream and downstream of HER2 gene primer, 0.6. mu.L of probe, 1. mu.L of upstream and downstream of internal reference gene primer, 0.2. mu.L of probe and 0.1 of TE to 20. mu.L; wherein, the primer pair is selected from the following group: HER2, 3 primer pairs H1, H2 and H3, and reference gene RPPH1 primer pair P1.

The PCR reaction conditions in the step (3) are as follows: denaturation at 95 deg.C for 10 min; followed by denaturation at 94 ℃ for 30sec and annealing at 60 ℃ for 60sec for 40 cycles; finally, the extension is carried out for 10min at 98 ℃, and the product is stored at 4 ℃.

The step (4) comprises the following steps: and analyzing the copy numbers of amplified products of the HER2 primer pair H1, H2 and H3 and the internal reference gene RPPH1 primer pair P1, and removing the copy number of the internal reference gene by using the copy number of the HER2 gene to obtain three groups of ratios, namely H1/P1, H2/P1 and H3/P1, judging that HER2 is negative if all the three ratios are less than or equal to 1.0, and judging that HER2 is positive if 2 of the three ratios are more than 1.2.

Compared with the prior art, the invention has the outstanding effects that:

(1) the method can not only determine whether the HER2 positive exists in the suspected positive sample by the conventional method, but also solve the problem that the HER2 positive sample is actually false negative when the HER2 positive sample is determined to be negative by the conventional method. Whether HER2 is increased is clearly indicated by designing three pairs of primers to bind to HER2, and the other pair of primers binds at a constant single copy gene position to indicate whether chromosome doubling has occurred. This makes it possible to obtain HER2 amplification information and chromosome doubling information very clearly. Therefore, HER2 positive can be accurately judged.

(2) The detection method has higher sensitivity, and can detect and increase the variation of HER2 gene by one copy at least.

(3) The method disclosed by the invention is carried out in a mode of adopting three groups of specific primer probes of HER2 genes and one group of reference genes, so that a false negative result caused by amplification of a single primer probe is effectively eliminated, and the method has higher accuracy compared with the prior art.

(4) The method provided by the invention is carried out by adopting a mode of three groups of specific primer probes of HER2 genes and one group of reference genes, and the three groups of primers are respectively combined in different exon regions, so that false positive results caused by amplification of a single primer probe are effectively eliminated, and the method has higher reliability compared with the prior art.

(5) In the field, the method has high sensitivity and repeatability, and can determine the uncertain area of the result of the conventional detection method.

(6) The invention adopts a newly designed internal reference gene, and the internal reference gene can be used as a stable internal reference gene to represent whether the HER2 gene copy number has variation or not through the optimization and identification of a plurality of cell line samples.

The digital PCR method and the detection kit for detecting breast cancer HER2 copy number variation according to the present invention are further described with reference to the accompanying drawings and the specific examples.

Drawings

FIG. 1 is a graph of the results of the annealing temperature optimization of HER2-H1 (C02: 57 ℃; C04: 58 ℃; C06: 59 ℃; C08: 60 ℃; C10: 61 ℃; C12: 62 ℃);

FIG. 2 is a graph showing the results of the annealing temperature optimization of HER2-H2 (F01: 57 ℃; F03: 58 ℃; F05: 59 ℃; F07: 60 ℃; F09: 61 ℃; F11: 62 ℃);

FIG. 3 is a graph showing the results of the annealing temperature optimization of HER2-H3 (F02: 57 ℃; F04: 58 ℃; F6: 59 ℃; F08: 60 ℃; F10: 61 ℃; F12: 62 ℃);

FIG. 4 is a graph showing the results of the optimization of primers and probes concentrations of HER2-H1, (A01: primer concentration 200nM + probe concentration 200nM, C01: primer concentration 200nM + probe concentration 300nM, E01: primer concentration 200nM + probe concentration 400nM, G01: primer concentration 300nM + probe concentration 200nM, B02: primer concentration 300nM + probe concentration 300nM, D02: primer concentration 300nM + probe concentration 400nM, F02: primer concentration 400nM + probe concentration 200nM, H02: primer concentration 400nM + probe concentration 300nM, B03: primer concentration 400nM + probe concentration 400 nM);

FIG. 5 is a graph showing the results of optimization of primers and probes from HER2-H2, (D03: primer concentration 200nM + probe concentration 200nM, F03: primer concentration 200nM + probe concentration 300nM, H03: primer concentration 200nM + probe concentration 400nM, B04: primer concentration 200nM + probe concentration 500nM, D04: primer concentration 300nM + probe concentration 200nM, E04: primer concentration 300nM + probe concentration 300nM, G04: primer concentration 300nM + probe concentration 400nM, B05: primer concentration 300nM + probe concentration 500nM, D05: primer concentration 400nM + probe concentration 200nM, F05: primer concentration 400nM + probe concentration 300nM, H05: primer concentration 400nM + probe concentration 400nM, B06: primer concentration 400nM + probe concentration 500 nM);

FIG. 6 is a graph showing the results of the optimization of primers and probes concentrations of HER2-H3, (D06: primer concentration 200nM + probe concentration 200nM, F06: primer concentration 200nM + probe concentration 300nM, H06: primer concentration 200nM + probe concentration 400nM, B07: primer concentration 300nM + probe concentration 200nM, D07: primer concentration 300nM + probe concentration 300nM, F07: primer concentration 300nM + probe concentration 400nM, H07: primer concentration 400nM + probe concentration 200nM, B08: primer concentration 400nM + probe concentration 300nM, D08: primer concentration 400nM + probe concentration 400 nM);

FIG. 7 shows the temperature optimization of RPPH1 gene amplification annealing (C01: 56 ℃; C03: 58 ℃; C6: 60 ℃; C09: 61 ℃);

FIG. 8 is a graph showing the results of optimization of the concentration of the primers and probes in RPPH1 gene, (B05: the concentration of primers 150nM + the concentration of probes 50nM, D05: the concentration of primers 150nM + the concentration of probes 100nM, F05: the concentration of primers 150nM + the concentration of probes 200nM, H05: the concentration of primers 250nM + the concentration of probes 50nM, C06: the concentration of primers 250nM + the concentration of probes 100nM, F06: the concentration of primers 250nM + the concentration of probes 200nM, B07: the concentration of primers 350nM + the concentration of probes 50nM, D07: the concentration of primers 350nM + the concentration of probes 100nM, and F07: the concentration of primers 350nM + the concentration of probes 200 nM);

FIG. 9 is a linear correlation of theoretical and measured values for HER2-H1 for 5 different level samples;

FIG. 10 is a linear correlation of theoretical and measured values for HER2-H2 for 5 different level samples;

figure 11 is a linear correlation of theoretical and measured values for HER2-H3 determined for 5 different level samples.

The primer and probe sequences adopted by the invention are as follows:

SEQ ID NO:1 H1-F：CCAGTAGAATGGCCAGGACAA

SEQ ID NO:2 H1-R：TGGCTGCCAGGGTCTGA

SEQ ID NO:7 H1-FAM：CGCAGTGCAGCACAG

SEQ ID NO:3 H2-F：ATAACACCCACCTCTGCTTCGT

SEQ ID NO:4 H2-R：GGTGCGGGTTCCGAAAG

SEQ ID NO:8 H2-FAM：CACACGGTGCCCTGG

SEQ ID NO:5 H3-F：TGGGCATCTGCCTGACATC

SEQ ID NO:6 H3-R：AGCCATAGGGCATAAGCTGTGT

SEQ ID NO:9 H3-FAM：ACGGTGCAGCTGGT

SEQ ID NO:10 P1-F：GAGGGAAGCTCATCAGTGG

SEQ ID NO:11 P1-R：CCCTAGTCTCAGACCTTCC

SEQ ID NO:12 P1-VIC：CCACGAGCTGAGTGC

Detailed Description