Human ESR1 gene mutation detection kit for breast cancer endocrine resistance treatment guidance

Abstract

The invention discloses a human ESR1 gene mutation detection kit for breast cancer endocrine resistance treatment guidance, and belongs to the technical field of biology. In particular to a primer probe combination for ESR1 gene mutation detection and application thereof, the primer probe combination comprises an ESR1 gene mutation specific primer pair, an ESR1 gene specific probe and an amplification blocking probe, and further comprises an internal reference primer and an internal reference probe. According to the invention, sensitive and rapid detection of drug-resistant related mutation sites such as L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, D538G, E380Q, S463P and the like of the common ESR1 genes in breast cancer is realized based on ctDNA, the detection efficiency of the ESR1 genes is greatly improved, and the detection cost is reduced, so that subsequent endocrine treatment is guided.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a human ESR1 gene mutation detection kit that can be used to guide endocrine resistance treatment of breast cancer. Background Art

[0002] Breast cancer is currently the most common female malignant tumor in the world, of which estrogen receptor (ER)-positive breast cancer accounts for approximately 70%. After binding to estrogen, ER can promote the growth, proliferation and survival of breast tumor cells. Drug therapy targeting ER is called endocrine therapy, which is the basis of breast cancer treatment.

[0003] The ESR1 gene is located on human chromosome 6 and encodes estrogen receptor α protein (ERα). ER is a nuclear receptor that mediates estrogen signals in the body and regulates the growth, differentiation and various physiological functions of the reproductive system. Mutations in this gene may lead to abnormalities in the estrogen signaling pathway, thereby affecting the growth and metastasis of tumor cells. ESR1 mutations have important clinical significance in breast cancer. On the one hand, ESR1 mutations are one of the important mechanisms of endocrine therapy resistance. About 20% to 40% of patients with metastatic breast cancer will develop ESR1 mutations after endocrine therapy. On the other hand, ESR1 mutations can also be used as therapeutic targets. In addition, ESR1 mutations are also associated with other prognostic indicators, such as shorter progression-free survival and poor overall survival. Therefore, ESR1 mutation detection can be used to assess the prognostic risk of breast cancer patients and develop individualized treatment strategies.

[0004] The non-invasive detection method based on peripheral blood ctDNA can detect ESR1 mutations, and is highly consistent with the tumor tissue DNA detection results. The 2023ASCO guidelines recommend: Sequence detection of ESR1 mutations in ctDNA of patients with ER+ / HER2- metastatic breast cancer. However, ctDNA has some limitations in clinical application, such as short half-life, low concentration and content, especially for multi-site detection, which generally requires a higher total amount of ctDNA. Therefore, it is of great significance to develop a high-sensitivity method based on ctDNA to detect ESR1 mutations.

[0005] Currently reported methods for detecting ESR1 include first-generation sequencing and second-generation sequencing. First-generation sequencing technology is simple and low-cost, but requires a series of subsequent operations after PCR, which is prone to contamination and false positive results, and cannot meet the requirements of detecting trace nucleic acid mutations in the blood. Second-generation sequencing is time-consuming and costly. Summary of the invention

[0006] The present invention provides an allele-specific PCR (AS-PCR) technology based on a qPCR platform, designs a primer-probe combination for detecting ESR1 gene mutations, combines locked nucleic acid (LNA) modification and blocking probe technology, further improves the sensitivity of detection, and optimizes the reaction system to establish a detection kit. It realizes sensitive and rapid detection of drug resistance-related mutation sites such as L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, D538G, E380Q, S463P, etc., of the ESR1 gene commonly found in breast cancer, based on ctDNA.

[0007] In order to achieve sensitive and rapid detection of ESR1 gene mutation in breast cancer based on ctDNA, a primer, probe combination and detection kit of allele-specific PCR technology based on qPCR platform are provided.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a primer-probe combination for detecting ESR1 gene mutations, the primer-probe combination comprising a primer pair specific for detecting ESR1 gene mutations, an ESR1 gene-specific probe, and an amplification blocking probe;

[0010] The nucleotide sequences of the specific primer pairs for detecting ESR1 gene mutations are shown in SEQ ID NO.1-8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.18;

[0011] The nucleotide sequences of the ESR1 gene mutation detection specific probes are shown in SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.19;

[0012] The nucleotide sequences of the ESR1 gene mutation detection amplification blocking probes are shown in SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.17.

[0013] Furthermore, the nucleotide at the mutation site corresponding to the amplification blocking probe is modified by LNA.

[0014] Furthermore, the primer-probe combination also includes a primer pair and a probe that specifically bind to the internal control gene.

[0015] Furthermore, the nucleotide sequence of the primer pair is shown in SEQ ID NO.20-21.

[0016] Furthermore, the nucleotide sequence of the probe is shown in SEQ ID NO.22.

[0017] A kit for detecting ESR1 gene mutation, comprising the primer-probe combination described above.

[0018] Furthermore, the kit also includes a reaction tube 1 and a reaction tube 2.

[0019] Furthermore, the components of the reaction solution in the reaction tube 1 are as follows:

[0020]

[0021]

[0022] Furthermore, the components of the reaction solution in the reaction tube 2 are as follows:

[0023]

[0024]

[0025] In addition, the present invention also provides the use of the primer-probe combination in preparing a reagent for detecting ESR1 gene mutation.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. One kit can simultaneously detect 11 common drug resistance-related mutation sites in the ESR1 gene of breast cancer patients, greatly improving the efficiency of ESR1 gene detection and reducing detection costs, so as to guide subsequent endocrine therapy.

[0028] 2. Allele-specific PCR (AS-PCR) technology was used for primer design, and blocking probes were added to improve the specificity of PCR amplification. At the same time, a total of 9 mutation sites, including L536, Y537, and D538, could share a blocking probe, downstream primers, and probes, saving costs;

[0029] 3. The amplification length of the designed primers is within 120bp, and only a two-tube system is needed to detect mutations at 11 sites, with low nucleic acid usage and high template utilization. It is suitable for cfDNA detection extracted from peripheral blood and realizes liquid biopsy;

[0030] 4. Placing primer probes for internal control gene detection in each reaction tube can quality control the reaction system and the input DNA to avoid false negative results. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The schematic diagram of primer probe design and amplification using the E380Q site of the ESR1 gene as an example;

[0032] Figure 2This is a graph showing the results of the 0.5% mutation frequency reference test for the ESR1 gene L536Q, L536R, L536P, Y537C, Y537N, Y537D, E380Q, and S463P sites in Example 3; DETAILED DESCRIPTION

[0033] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0034] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0035] The technical solution of the present invention is to design a corresponding AS-PCR upstream amplification primer according to the mutation sequence of the detected mutation site. The base at the 3' end of the primer is consistent with the mutation site sequence and mismatches with the wild-type template sequence. In order to improve the specificity of the primer, a mismatch is introduced into the penultimate or third base at the 3' end of the primer. The downstream primer and fluorescent probe are designed according to the wild-type sequence. At the same time, in order to further improve the specificity of amplification, a blocking probe is designed for the wild-type sequence. The bases at the corresponding positions of the blocking probe mutation site are modified with LNA to improve the binding ability of the blocking probe to the wild-type template, so as to ensure that during PCR amplification, the blocking probe preferentially binds to the wild-type template and blocks the amplification of the wild-type template by the AS-PCR primer. The primer probe design scheme and the amplification schematic diagram are shown in the figure. Figure 1 After the designed primers and probes are screened, the reaction system is optimized to determine the most suitable dosage of the system components and establish a detection kit.

[0036] Example 1

[0037] According to the transcript NM_000125.4 sequence corresponding to ESR1, the mutation site was determined, and the sequences before and after the site were obtained in the reference genome for the design of primers and probes;

[0038] The designed primers and probes were screened to determine the sequences of the primers and probes used in the present invention, as shown in Table 1 below.

[0039] Table 1 Primer and probe sequences

[0040]

[0041]

[0042] Note: In the table, bold bases indicate LNA modification; -C3 indicates C3 Spacer modification and closure at the 3' end; ACTB-E4-F, ACTB-E4-R and ACTB-E4-ROX-P are primer probes for internal control genes.

[0043] The dosage of the components of the PCR reaction system was optimized to determine the formula of different reaction tubes and the PCR amplification reaction program. At the same time, the corresponding positive and negative quality control products (the negative quality control product is the wild-type cell line) were prepared to complete the development of the kit for 2 reaction tubes. The formula of the 2 reaction tubes is shown in Table 2-3. When using, 0.3μL of TaKaRaTaq HS (5U / μl) DNA polymerase and 5μL of DNA need to be added to each reaction tube. The PCR amplification reaction program is shown in Table 4.

[0044] Table 2 Formula of ESR1 gene mutation detection reaction tube 1

[0045]

[0046]

[0047] Table 3 Formula of ESR1 gene mutation detection reaction tube 2

[0048]

[0049]

[0050] Table 4 PCR amplification reaction program

[0051]

[0052] Result interpretation: By testing positive and negative samples with known mutation information, the positive judgment Ct values ​​of the L536 site, Y537 site, D538G site, E380Q site, and S463P site of this kit were determined by the ROC curve method to be 25, 25, 25, 24, and 26, respectively. If the FAM or HEX signal amplification curve of the sample to be tested rises and the Ct value is less than the positive judgment value, the sample is positive; otherwise, it is negative. The internal control ROX signal of the sample to be tested should have an amplification curve rise, and the Ct value should be between 8-15. The negative control FAM / HEX signal should have no amplification curve rise.

[0053] Example 2

[0054] This example takes the detection of ESR1 gene D538G and Y537S mutations as an example to compare and verify the role of the blocking probe in the reaction system. Six clinical samples diagnosed as breast cancer by pathology were collected, of which two samples were positive for ESR1 gene D538G mutation, two samples were Y537S mutation, and two samples were negative for ESR1 mutation after NGS verification. The above method includes the following steps:

[0055] 1. Sample processing and template extraction quality control:

[0056] Peripheral blood was collected using a free DNA storage tube with a collection volume of no less than 10 mL. Peripheral blood plasma DNA was extracted using the nucleic acid extraction reagent [Plasma DNA (centrifugal column method)] of Xiamen Feishuo Biotechnology Co., Ltd. The specific steps were in accordance with the operating instructions of the kit. ctDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0) with an elution volume of 50 μL. The extraction quality was detected by ultraviolet spectrophotometer and the concentration was determined. 5 μL ctDNA was used as the DNA template for detection.

[0057] 2. Preparation of reaction solution

[0058] The reaction solution with the addition of blocking probe is prepared according to Table 2-3, and the reaction tube formula without the addition of blocking probe is shown in Table 5-6. 0.3 μL of TaKaRa Taq HS (5U / μL) DNA polymerase is added to the two reaction solutions in the two reaction tubes, and 5 μL of the above ctDNA template is added to each reaction tube.

[0059] Table 5 ESR1 gene mutation detection reaction tube 1 formula (no blocking probe added)

[0060]

[0061] Table 6 ESR1 gene mutation detection reaction tube 2 formula (no blocking probe added)

[0062]

[0063]

[0064] 3. Fluorescence PCR Detection

[0065] Perform fluorescence PCR detection according to the PCR reaction program in Table 4.

[0066] 4. Statistical analysis of test results

[0067] The Ct values ​​of the six clinical samples detected in the two reaction tubes were statistically shown in Table 7.

[0068] Table 7 Comparison of Ct values ​​of mutation detection reaction solutions of 6 clinical samples with and without added blocking probes

[0069]

[0070] The above results show that the addition of blocking probes does not affect the efficiency of detection of ESR1 gene D538G and Y537S mutation-positive samples, nor does it affect the detection of negative samples. At the same time, it can also inhibit nonspecific amplification of the template and improve the specificity of mutation detection.

[0071] Example 3

[0072] In this example, three reference samples with mutation frequencies of 0.5% and six clinical negative plasma ctDNA samples were used to verify the detection sensitivity and specificity of the kit of the present invention. The above method includes the following steps:

[0073] 1. Sample processing and template extraction quality control:

[0074] Preparation of reference samples for three mutation sites with a mutation frequency of 0.5%: Use plasmids with positive mutations of ESR1-L536H, ESR1-Y537S, and ESR1-D538G, quantify by digital PCR, and then mix and dilute with negative plasma to a reference sample with a mutation frequency of 0.5%. The preparation method of reference samples for other sites is the same as the above three sites, and the test is repeated twice. The test results are attached Figure 2 .

[0075] Peripheral blood samples from 6 clinical cases were collected using free DNA storage tubes, with a collection volume of no less than 10 mL. Peripheral blood plasma DNA was extracted using the nucleic acid extraction reagent [PlasmaDNA (centrifugal column method)] of Xiamen Feishuo Biotechnology Co., Ltd. The specific steps were in accordance with the operating instructions of the kit. ctDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0) with an elution volume of 50 μL. The extraction quality was detected by ultraviolet spectrophotometer, and the concentration was determined. 5 μL ctDNA was used as the DNA template for detection.

[0076] 2. Preparation of reaction solution

[0077] Prepare the reagent kit reaction solution referring to Table 2-3, add 0.3 μL of TaKaRa Taq HS (5U / μL) DNA polymerase to each reaction tube, and add 5 μL of the above ctDNA template to each reaction tube, wherein the 0.5% mutation frequency reference of the 3 mutation sites is tested repeatedly 5 times.

[0078] 3. Fluorescence PCR Detection

[0079] Perform fluorescence PCR detection according to the PCR reaction program in Table 4.

[0080] 4. Statistical analysis of test results

[0081] The test results of the three mutation site references with a mutation frequency of 0.5% and six clinical negative samples corresponding to the two reaction tubes of the kit were statistically analyzed. The Ct value statistics are shown in Tables 8 and 9.

[0082] Table 8 Statistics of Ct values ​​of three reference kits

[0083]

[0084]

[0085] Table 9 Statistics of Ct values ​​of 6 clinical negative samples detected by kit

[0086]

[0087] The above results show that the Ct values ​​of the reference samples with 0.5% mutation frequency at the three mutation sites were all less than the positive judgment value after 5 repeated tests, and the FAM / HEX signals of the six negative clinical samples did not show an increase in the amplification curve. This shows that the kit can accurately detect 0.5% mutations, and there will be no false positives for negative samples. The kit has high detection sensitivity and specificity.

[0088] Example 4

[0089] This example uses the second generation sequencing methodology as a control method to verify the accuracy of the kit detection. 20 clinical samples diagnosed as breast cancer by pathology were collected. Each sample has a known second generation sequencing test result. The detection using the above method includes the following steps:

[0090] 1. Sample processing and template extraction quality control:

[0091] The ctDNA was extracted using the extraction kit described above, and ctDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0) in a volume of 50 μL. The extraction quality was detected by UV spectrophotometer, and the concentration was determined. 5 μL ctDNA was used as the DNA template for detection.

[0092] 2. Preparation of reaction solution

[0093] Prepare the reagent kit reaction solution according to Table 2-3, add 0.3 μL of TaKaRa Taq HS (5U / μL) DNA polymerase to each reaction tube, and add 5 μL of the above ctDNA template to each reaction tube.

[0094] 3. Fluorescence PCR Detection

[0095] Perform fluorescence PCR detection according to the PCR reaction program in Table 4.

[0096] 4. Statistical analysis of test results

[0097] The results of 20 samples were statistically analyzed and judged according to the positive judgment value. The consistency with the detection results of the second-generation sequencing is shown in Table 10. It can be seen from the table that the consistency rate between the results of the 20 clinical samples detected by this kit and the detection results of the second-generation sequencing is 100%, which further illustrates the accuracy of the primer probe combination and the kit of the present invention in detecting the ESR1 gene L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, D538G, E380Q, and S463P mutations.

[0098] Table 10 Analysis of consistency between the test results of 20 clinical samples tested by this kit and the results of second-generation sequencing

[0099]

[0100] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer-probe combination for detecting ESR1 gene mutation, characterized in that: The primer-probe combination includes a primer pair specific for detecting ESR1 gene mutation, an ESR1 gene-specific probe, and an amplification blocking probe; The nucleotide sequences of the ESR1 gene mutation detection specific primer pairs are shown in SEQ ID NO.1-8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.18; The nucleotide sequences of the ESR1 gene mutation detection specific probes are shown in SEQ ID NO.11, SEQ ID NO.15, and SEQ ID NO.19; The nucleotide sequences of the ESR1 gene mutation detection amplification blocking probes are shown in SEQ ID NO.9, SEQ ID NO.13, and SEQ ID NO.

17.

2. The primer-probe combination according to claim 1, characterized in that: The nucleotide at the mutation site corresponding to the amplification blocking probe is modified by LNA.

3. The primer-probe combination according to claim 2, characterized in that: The primer-probe combination also includes a primer pair and a probe that specifically bind to the internal control gene.

4. The primer-probe combination according to claim 3, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 20-21.

5. The primer-probe combination according to claim 4, characterized in that: The nucleotide sequence of the probe is shown in SEQ ID NO.

22.

6. A kit for detecting ESR1 gene mutation, characterized in that: The kit comprises the primer-probe combination according to any one of claims 3 to 5.

7. The kit according to claim 6, characterized in that The kit also includes a reaction tube 1 and a reaction tube 2.

8. The kit according to claim 7, characterized in that The components of the reaction solution in the reaction tube 1 are as follows:

9. The kit according to claim 7, characterized in that The components of the reaction solution in the reaction tube 2 are as follows:

10. Use of the primer-probe combination according to any one of claims 1 to 5 in preparing a reagent for detecting ESR1 gene mutation.

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