Primer probe set, kit and application for detecting ESR1 gene mutation

Abstract

The present invention discloses a primer probe set, a kit, and applications for detecting ESR1 gene mutations. The primer probe set includes primer probe sets designed for mutation sites such as E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G, as well as wild-type primers and probe sets. The primer probe set has good specificity and high amplification efficiency, and can be applied to digital PCR to detect trace nucleic acids of ESR1 gene mutations E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G, thereby quickly and accurately monitoring various mutations in the ESR1 gene of tumor patients, timely monitoring the occurrence of new gene mutations in patients, and providing a basis for the formulation and adjustment of clinical treatment plans.

Description

Technical Field

[0001] The present invention relates to the technical field of ESR1 gene mutation detection, and in particular to a primer probe set, a kit and applications for detecting ESR1 gene mutation. Background Art

[0002] Globally, China accounts for 12.2% of newly diagnosed breast cancer cases and 9.6% of breast cancer deaths. According to the International Agency for Research on Cancer (IARC) global cancer report, the incidence and mortality rates of breast cancer in Chinese women are relatively low among 184 countries and regions worldwide. However, the number of cases in China accounts for 11.19% of the global total, second only to the United States. Furthermore, the incidence and mortality rates have increased rapidly over the past 20 years, posing a serious challenge to prevention and control.

[0003] The human estrogen receptor alpha (ESRα) protein, encoded by the ESR1 (Estrogen Receptor 1) gene, activates a series of cellular responses upon binding to estrogen, promoting cell growth and proliferation. Activating mutations in the ESR1 gene are closely associated with the development and progression of breast cancer. The most common mutations in the ESR1 gene are E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, and D538G. These mutations alter the conformation of ESRα, allowing it to remain persistently activated even when not bound to estrogen.

[0004] Breast cancer is one of the most common gynecological cancers. Over 70% of primary breast cancers are estrogen receptor (ER)-positive, and many initially respond to endocrine therapy. Despite the continuous improvement in the development of new therapies for breast cancer, innate and acquired resistance to these drugs remains a challenge. The tumor microenvironment is considered a major factor in conferring innate resistance to cancer therapy, and a large proportion of patients develop resistance when receiving systemic antiestrogens such as tamoxifen or estrogen deprivation therapies such as aromatase inhibitors (AIs). Numerous mechanisms, including activation of cell survival, cellular stress, and cell signaling pathways, have been implicated as drivers of acquired resistance. Recent studies have implicated activating mutations in the estrogen receptor (ESR1) gene as a key driver of resistance. Key activating mutation hotspots identified include p.L536, p.Y537, and p.D538, which confer endoplasmic reticulum transcriptional activity independent of estrogen ligands and are considered resistance mutations. Due to tumor heterogeneity, acquired resistance can also occur. Cancer biomarkers vary by disease type and stage of progression, complicating early-stage cancer detection and identification. Circulating tumor DNA (ctDNA) is gaining prominence as a "liquid biopsy" for detecting and monitoring resistance to systemic therapies. Acquired resistance to hormonal therapy may be based on activating mutations in the estrogen receptor gene (ESR1). Advantages of using cfDNA for tumor mutation testing include i) noninvasive collection, ii) availability at any time during the disease course, and iii) real-time detection and dynamic monitoring, potentially reducing heterogeneity issues compared to tumor tissue testing. In vitro and preclinical data suggest that ESR1 mutations confer complete resistance to AIs and partial resistance to ER agonists and antagonists. Detection of ESR1 activating mutations may be helpful in guiding clinicians in endocrine and non-endocrine therapies. cfDNA fragments are relatively small, with a peak size of approximately 180 bp. The percentage of tumor-derived DNA in total cfDNA is individually variable and often too low to be detected. Therefore, it is of great significance to develop a cfDNA-based ESR1 mutation detection method with high sensitivity.

[0005] Therefore, the detection of ESR1 mutations has the potential to predict hormone resistance and guide therapy. Next-generation sequencing (NGS) is a common approach for such testing because it can detect many mutations simultaneously using a small sample. However, NGS is very laborious, lengthy, and expensive.

[0006] Now, droplet PCR (dPCR) is transforming the landscape of traditional PCR and redefining the detection limit for mutation detection. For many assays, dPCR is significantly more sensitive than traditional PCR analysis and, by counting more molecules individually, improves the accuracy and precision of the assay. The sensitivity of digital PCR will increase the limit of detection and redefine our understanding of disease onset, progression, and recurrence. A particularly attractive application of digital PCR is the quantitative detection of a small number of mutant DNA molecules among a large number of wild-type molecules, which is relevant to cancer research, especially for the detection of minor alleles.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The present invention provides a primer probe set, a kit, and an application for detecting ESR1 gene mutations, which can quickly and accurately monitor various mutations in the ESR1 gene of tumor patients, facilitate timely monitoring of the occurrence of new gene mutations in patients, and provide a basis for the formulation and adjustment of clinical treatment plans.

[0009] In view of this, the solution of the present invention is:

[0010] The first aspect of the present invention is to provide a primer-probe set for detecting ESR1 gene mutations, comprising a PCR amplification primer set and a probe set; wherein:

[0011] The primer set includes a wild-type primer set having a nucleotide sequence as shown in SEQ ID NO: 1-2, and an E380Q mutant primer set having a nucleotide sequence as shown in SEQ ID NO: 3-4;

[0012] The probe group includes: a Y537N mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 5-6; a Y537C mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 7-8, a Y537S mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 9-10, a D538G mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 11-12, an L536R mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 13-14, an L536P mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 15-16, an L536H mutation probe group with a nucleotide sequence as shown in SEQ ID NOs: 17-18, and a wild-type probe group with a nucleotide sequence as shown in SEQ ID NOs: 19-20.

[0013] Furthermore, in the Y537N mutation probe set, the Y537C mutation probe set, and the Y537S mutation probe set, at least one sequence of each probe set has several locked nucleic acid modifications;

[0014] And / or, in the Y537N mutation probe set, the Y537C mutation probe set, and the Y537S mutation probe set, at least one sequence of each probe set has been modified with several locked nucleic acids;

[0015] And / or, the sequences of the wild-type probe set all have several locked nucleic acid modifications.

[0016] Preferably, in the probe set, the locked nucleic acid is modified as follows:

[0017] The nucleotide sequence shown in SEQ ID NO: 5 is ， The nucleotides at positions 7-10 and 12-13 from the end are modified with locked nucleic acid;

[0018] The nucleotide sequence shown in SEQ ID NO: 6 is from 5 ， The 2nd, 5th to 7th, and 10th nucleotides from the end are modified with locked nucleic acid;

[0019] The nucleotide sequence shown in SEQ ID NO: 7 is from 5 ， The 3rd-4th, 6th-8th, and 10th nucleotides from the end are modified with locked nucleic acid;

[0020] The nucleotide sequence shown in SEQ ID NO: 8 is from 5 ， The 4th to 5th, 7th to 9th, and 11th nucleotides from the end were modified with locked nucleic acid;

[0021] The nucleotide sequence shown in SEQ ID NO: 10 is from 5 ， The 3rd, 5th to 7th, and 9th nucleotides from the end are modified with locked nucleic acid;

[0022] The nucleotide sequence shown in SEQ ID NO: 11 is from 5 ， The 10th nucleotide from the end is modified with locked nucleic acid;

[0023] The nucleotide sequence shown in SEQ ID NO: 12 is from 5 ， The 3rd-4th and 6th-10th nucleotides from the end are modified with locked nucleic acid;

[0024] The nucleotide sequence shown in SEQ ID NO: 13 is from 5 ， The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid;

[0025] The nucleotide sequence shown in SEQ ID NO: 15 is from 5 ， The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid;

[0026] The nucleotide sequence shown in SEQ ID NO: 16 is from 5 ， The 10th and 12th nucleotides from the end were modified with locked nucleic acid;

[0027] The nucleotide sequence shown in SEQ ID NO: 17 is from 5 ， The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid;

[0028] The nucleotide sequence shown in SEQ ID NO: 18 is from 5 ， The 9th-10th and 12th nucleotides from the end were modified with locked nucleic acid;

[0029] The nucleotide sequence shown in SEQ ID NO: 19 is from 5 ， The 9th and 12th nucleotides from the end were modified with locked nucleic acid;

[0030] The nucleotide sequence shown in SEQ ID NO: 20 is from 5 ， The 10th nucleotide from the end is modified with locked nucleic acid;

[0031] The nucleotide sequence shown in SEQ ID NO: 21 is from 5 ， The 9th to 10th nucleotides from the end are modified with locked nucleic acid; further, the probe group 5 shown in SEQ ID NO: 5-18 ， The end is labeled with a reporter group, 3 ， The end is labeled with a quencher group.

[0032] Preferably, the reporter group is selected from FAM, ROX, CY5, HEX, CY5.5.

[0033] The second aspect of the present invention is to propose the use of the primer probe set described in the first aspect in preparing an ESR1 gene mutation detection kit.

[0034] The third aspect of the present invention is to propose a kit for detecting ESR1 gene mutations, including tube A and / or tube B, and reaction reagents, positive quality control products, and negative quality control products; the tube A includes the wild primer group, Y537N mutation probe group, Y537C mutation probe group, Y537S mutation probe group, D538G mutation probe group and wild probe group in the primer probe group described in the first aspect; the tube B includes the wild primer group, E380Q mutation primer group, E380Q mutation probe group, L536H mutation probe group, L536R mutation probe group, L536P mutation probe group and wild probe group in the primer probe group described in the first aspect.

[0035] Furthermore, the concentration of each primer set is 500-700 nM, preferably 600 nM; the concentration of each mutation probe set is 200-400 nM, preferably 300 nM; and the concentration of the wild-type probe set is 100-300 nM, preferably 200 nM.

[0036] A fourth aspect of the present invention is to provide a method for detecting ESR1 gene mutations for non-disease diagnosis purposes, comprising the following steps:

[0037] Obtaining nucleic acid from a sample to be tested as a template;

[0038] Perform digital PCR on the template using the kit described in the third aspect;

[0039] The ESR1 gene mutation detection result of the sample is determined based on the digital PCR amplification result.

[0040] Furthermore, the sample to be tested is a cell, body fluid or tissue, and the body fluid includes serum, plasma or tissue fluid; the detection method is particularly suitable for the detection of trace nucleic acid samples.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The ESR1 gene mutation primer probe set provided by the present invention has good specificity and high amplification efficiency. It can be used for digital PCR detection to quickly and accurately monitor various mutations in the ESR1 gene of tumor patients based on trace amounts of ESR1 gene E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G mutations in samples. Therefore, the detection of this product can timely monitor the occurrence of new gene mutations in patients, providing a basis for the formulation and adjustment of clinical treatment plans.

[0043] The ESR1 gene mutation typing detection kit of the present invention uses standards prepared by combining nucleic acid from an ESR1-negative sample and enzyme-digested mutant plasmids inserted with ESR1 gene mutation fragments E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G, in a copy number ratio. Standards with different mutation frequencies play different roles. The use of cfDNA and plasmids as standards can maximize the characteristics of the test sample, providing a solid foundation for system optimization and playing a decisive role in system optimization.

[0044] The ESR1 gene mutation typing detection kit of the present invention determines the endpoint fluorescence signal value generated by each mutation probe at different concentrations through digital PCR detection results of a medium mutation frequency standard, making the data statistics more accurate. The ESR1 gene mutation detection system of the present invention determines the background threshold value of each mutation site of the detection system through digital PCR detection results of wild-type templates. When detecting the mutation copy number of a sample, the mutation copy number is equal to the test result minus the background threshold value, which can make the results more accurate.

[0045] The mutation detection system of the ESR1 gene mutation typing detection product of the present invention can determine the sensitivity of the detection system through the digital PCR detection results of low mutation frequency standards;

[0046] The optimization of the ESR1 gene mutation typing multiplex detection product of the present invention optimizes the concentration of each probe based on the traditional real-time fluorescence PCR detection results of high mutation frequency standards. The appropriate probe concentration is selected based on the difference in fluorescence intensity after reaction with different concentrations of probes for each mutation. This method has accurate results and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a diagram showing the PCR amplification results of the E380Q mutation site described in Example 4 of the present invention;

[0048] Figure 2 This is a diagram showing the PCR amplification results of the L536H mutation site described in Example 4 of the present invention;

[0049] Figure 3 This is a diagram showing the PCR amplification results of the L536R mutation site described in Example 4 of the present invention;

[0050] Figure 4 This is a diagram showing the PCR amplification results of the L536P mutation site described in Example 4 of the present invention;

[0051] Figure 5 This is a diagram showing the PCR amplification results of the Y537C mutation site described in Example 4 of the present invention;

[0052] Figure 6 This is a diagram showing the PCR amplification results of the Y537N mutation site described in Example 4 of the present invention;

[0053] Figure 7 This is a diagram showing the PCR amplification results of the Y537S mutation site described in Example 4 of the present invention;

[0054] Figure 8 This is a diagram showing the PCR amplification results of the D538G mutation site described in Example 4 of the present invention;

[0055] Figure 9This is the linear result of different mutation frequencies of the E380Q mutation site described in Example 5 of the present invention;

[0056] Figure 10 This is the linear result of different mutation frequencies of the L536H mutation site described in Example 5 of the present invention;

[0057] Figure 11 This is the linear result of different mutation frequencies of the L536R mutation site described in Example 5 of the present invention;

[0058] Figure 12 This is the linear result of different mutation frequencies of the L536P mutation site described in Example 5 of the present invention;

[0059] Figure 13 This is the linear result of different mutation frequencies of the Y537C mutation site described in Example 5 of the present invention;

[0060] Figure 14 This is the linear result of different mutation frequencies of the Y537N mutation site described in Example 5 of the present invention;

[0061] Figure 15 This is the linear result of different mutation frequencies of the Y537S mutation site described in Example 5 of the present invention;

[0062] Figure 16 This is the linear result of different mutation frequencies of the D538G mutation site described in Example 5 of the present invention; DETAILED DESCRIPTION

[0063] The technical solutions of the present invention will be clearly and completely described below in conjunction with preferred embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0064] In the following examples, unless otherwise stated, all reagents used are assumed to be analytically pure and commercially available. Experimental methods not explicitly described in this invention can generally be performed according to conventional experimental methods, such as the basic biochemical and molecular experimental methods disclosed in the book "Molecular Cloning Laboratory Manual" edited by J. Sambrook et al., published by Science Publishing in 2002, or according to the experimental methods clearly specified by the reagent supplier. With the exception of a few explicit definitions, all professional and scientific terms used herein have the same meanings as those generally recognized by those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied in the present invention.

[0065] Main reagents: All the reagents and consumables used in the experiments described in the examples were purchased from regular reagent and consumable manufacturers. The main reagents used are (a few common conventional reagents are not listed here): digital PCR system from Xinyi Biotechnology Co., Ltd., E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G and wild plasmid templates provided by Bioengineering, extraction reagents from Xinyi Biotechnology, etc.

[0066] Main instruments: vortex shaker, high-speed centrifuge, A300 PCR, micro-droplet preparation instrument (drop maker M1), micro-droplet sample detection instrument (chip reader R2) (manufacturer: Xinyi Manufacturing Technology (Beijing) Co., Ltd.), biological safety cabinet, water bath, pipette, etc.

[0067] In one embodiment, a rapid and efficient detection system is provided, which has high specificity and sensitivity, high utilization of free DNA, and high accuracy. A primer probe set for detecting ESR1 gene mutation typing is provided, which can detect mutations using a small amount of sample and provide the mutation frequency of the sample, as well as a kit using the primer probe set. The ESR1 gene mutation detection primer probe set includes an upstream primer, a downstream primer, and a probe for detecting ESR1 gene mutations E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G.

[0068] The nucleotide sequences of the upstream and downstream primers are shown in Table 1.

[0069] Table 1: Primer probe sequences

[0070]

[0071] In the sequences shown in Table 1, the base after "+" indicates a locked nucleic acid-modified base, such as "+T" representing locked nucleic acid modification of base T. It should be noted that in the event of any inconsistency between the sequence listing provided separately and the sequence shown in Table 1, the sequence in Table 1 shall prevail.

[0072] The primers and probes were used at concentrations of 600 nM and 300 nM, respectively, and the wild-type probe was used at a concentration of 200 nM. All probes used except the wild-type probe were modified MGB probes, with the 5' end of the probes modified with FAM, ROX, CY5, HEX, or CY5.5 fluorescent groups. Example 1

[0073] To determine the optimal primer and probe concentration, wild-type plasma template and mutant plasmid template were prepared at a 1:1 ratio to obtain a template with theoretical mutation frequency. The preparation method is as follows:

[0074] 1. Sample extraction

[0075] Plasma extraction was performed using the CFDNA extraction kit (11014) from Xinyi Biotechnology Co., Ltd. For specific steps, please refer to the product instructions of the kit;

[0076] Plasmids were extracted using a plasmid miniprep kit (D1100, Beijing Solebow Technology Co., Ltd.) according to the product instructions. For specific steps, please refer to the official instructions of the kit.

[0077] 2. Determination of cfDNA and mutant plasmid concentrations

[0078] The extracted cfDNA and plasmids were quantified using the Qubit™ dsDNA HS Assay Kit (Q32854). The specific experimental procedures were described in the instructions provided with the kit.

[0079] 3. Prepare the standard products required for the experiment

[0080] (1) Theoretically, the number of single gene copies per 1ng of human DNA is about 300. Plasma free DNA (cfDNA) is diluted step by step to about 2.0×10 5 copies / μl;

[0081] (2) Calculation of the plasmid copy number obtained after enzyme digestion: 9.1×10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800bp); dilute the obtained plasmid stepwise to 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 2 copies / μl, 20 copies / μl;

[0082] Adjust the working concentrations of primers and probes based on the development objectives and determine the final experimental concentrations. Primers were set at 800 nM, 600 nM, and 400 nM, respectively, and probes were set at 200 nM, 300 nM, and 400 nM, respectively. Amplification templates were prepared using a mutant plasmid template and a wild-type plasma template at a 1:1 copy number ratio. Prepare the reaction system according to the reaction system components shown in Table 2.

[0083] Table 2: Reaction system, primer concentration system / 30 μl

[0084]

[0085] The PCR amplification program is shown in Table 3.

[0086] Table 3:

[0087] The screening results of the optimal primer and probe concentrations are shown in Table 4.

[0088] Table 4:

[0089] Conclusion: In each system, there is only one pair of primer probes and the template amount is uniform. The optimal concentrations of primers and probes can be screened out respectively.

[0090] Example 2

[0091] To demonstrate no difference between the mixed and individual plasmids, digital PCR was used to quantify each plasmid using a consensus sequence as a probe. Both the mutant and wild-type plasmids were quantified to 1000 copies / μl. Based on the development objectives, the working concentrations of primers and probes were adjusted to determine the final experimental concentrations. Amplification templates were prepared using the mutant plasmid template and the wild-type plasma template at 1000 copies. The reaction system was prepared according to the reaction system components shown in Table 5.

[0092] Table 5:

[0093] Perform the PCR amplification program according to Table 3.

[0094] The results of each reaction system are shown in Table 6.

[0095] Table 6:

[0096] Conclusion: The difference between the detected copy numbers of the mixed system and the single system is within the normal range, proving that there is no significant difference in detection between the mixed system and the single system.

[0097] Example 3

[0098] To fully simulate real-world samples, we used gene-edited cell lines harboring ESR1 gene mutations E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G, and negative plasma to extract cell line DNA and ctDNA, respectively. After digital PCR quantification, the mixtures were mixed in a specific ratio and tested for detection. The reaction system was prepared according to the reaction system components shown in Table 7.

[0099] Table 7:

[0100] Amplification was performed according to the PCR amplification program shown in Table 3.

[0101] The test results are shown in Table 8. Table 8

[0102] Conclusion: This reagent can distinguish different mutation types using different fluorescence channels.

[0103] Example 4

[0104] To test the specificity of this reagent and whether it will react non-specifically with the template, simulate the plasma sample by mixing the template extracted from wild type (4 ml) plasma and the mutant plasmid template in a certain ratio to prepare a template with theoretical mutation frequency (quantified by digital PCR). The preparation method is as follows:

[0105] Sample extraction Plasma extraction Free DNA was extracted using the Aide sample preparation kit. For specific operation steps, please refer to the product instructions of the kit.

[0106] 2. Determination of cfDNA and mutant plasmid concentrations

[0107] The extracted cfDNA and plasmid were quantified using the QubitTM dsDNA HS Assay Kit (Q32854). The specific experimental steps were referred to the instructions provided in the kit.

[0108] 3. Prepare the standard products required for the experiment

[0109] Theoretically, the number of copies of a single gene per 1ng of human DNA is about 300. If plasma free DNA (cfDNA) is diluted to about 1.0×10 3 copies / μl.

[0110] 1) Calculation of the plasmid copy number after enzyme digestion: 9.1×10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800bp); dilute the obtained plasmid stepwise to 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 2 copies / μl, 20 copies / μl.

[0111] 2) Based on the intended purpose, use the mutant plasmid template and the wild-type plasma template in a copy number ratio to prepare an amplification template. Prepare the reaction system according to the reaction system component table shown in Table 9.

[0112] Table 9:

[0113] Perform the PCR amplification program according to Table 3.

[0114] Tables 10-13 show the test results.

[0115] Table 10:

[0116] Table 11:

[0117] Table 12:

[0118] Table 13:

[0119] The specific PCR amplification results of each mutation site E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G are as follows: Figure 1-8 shown.

[0120] Conclusion: Y537C, Y537N, Y537S, and D538G in system 1 do not nonspecifically bind to L536H, L536R, L536P, E380Q, or the wild-type template, and neither 537CNS nor 538 bind to each other. In system 2, 536RPH and 380 do not nonspecifically bind to 537CNS, 538, or the wild-type template, and neither 536RPH nor 380 bind to each other.

[0121] Example 5

[0122] To test the detection limit of this reagent, we used a wild-type template (negative sample) with a background of approximately 5000 copies of cell-free DNA in plasma, as this was a low-level background. The wild-type template (negative sample) was mixed with the mutant plasmid to simulate a real sample. The assay was performed at mutation ratios of 50%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, and 0.1%.

[0123] According to the reaction system component table shown in Table 14, the reaction system was prepared.

[0124] Table 14:

[0125] Amplification was performed according to the PCR amplification program shown in Table 3. The linear results of different mutation frequencies of E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutation sites under the wild type of 5000 copies were as follows: Figure 9-16 shown.

[0126] Conclusion: The linear expression of different mutation frequencies of E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutation sites under a wild-type population of 5000 copies is all above R-square 0.999, and they can all be detected normally at a mutation frequency of 0.1%.

[0127] Example 6

[0128] For an accuracy comparison experiment, 10 samples confirmed positive by NGS were used to test whether the typing was consistent with this kit. Prepare the reaction system according to the reaction system components shown in Table 15.

[0129] Table 15:

[0130] The PCR amplification program shown in Table 3 was performed. The test results are shown in Table 16.

[0131] Table 16:

[0132] Conclusion: The detection results of this kit are consistent with the NGS typing results, but the time is shorter and the cost is lower.

[0133] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A primer probe set for detecting ESR1 gene mutation, characterized in that: The invention comprises a PCR amplification primer set and a probe set placed in tube A and tube B respectively; wherein: The A tube includes a wild-type primer set with nucleotide sequences as shown in SEQ ID NOs: 1-2, a Y537N mutation probe set with nucleotide sequences as shown in SEQ ID NOs: 5-6, a Y537C mutation probe set with nucleotide sequences as shown in SEQ ID NOs: 7-8, a Y537S mutation probe set with nucleotide sequences as shown in SEQ ID NOs: 9-10, a D538G mutation probe set with nucleotide sequences as shown in SEQ ID NOs: 11-12, and a wild-type probe set with nucleotide sequences as shown in SEQ ID NOs: 21-22; The B tube includes a wild-type primer group whose nucleotide sequence is shown in SEQ ID NO: 1-2, an E380Q mutation primer group whose nucleotide sequence is shown in SEQ ID NO: 3-4, an L536R mutation probe group whose nucleotide sequence is shown in SEQ ID NO: 13-14, an L536P mutation probe group whose nucleotide sequence is shown in SEQ ID NO: 15-16, an L536H mutation probe group whose nucleotide sequence is shown in SEQ ID NO: 17-18, an E380Q mutation probe group whose nucleotide sequence is shown in SEQ ID NO: 19-20, and a wild-type probe group whose nucleotide sequence is shown in SEQ ID NO: 21-22.

2. The primer probe set according to claim 1, characterized in that In the Y537N mutation probe set, the Y537C mutation probe set, and the Y537S mutation probe set, at least one sequence of each probe set has several locked nucleic acid modifications; And / or, in the Y537N mutation probe set, the Y537C mutation probe set, and the Y537S mutation probe set, at least one sequence of each probe set has been modified with several locked nucleic acids; And / or, the sequences of the wild-type probe set all have several locked nucleic acid modifications.

3. The primer probe set according to claim 2, characterized in that In the probe set, the locked nucleic acid is modified as follows: The nucleotide sequence shown in SEQ ID NO: 5 is ’ The nucleotides at positions 7-10 and 12-13 from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 6 is from 5 ’ The 2nd, 5th to 7th, and 10th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 7 is from 5 ’ The 3rd-4th, 6th-8th, and 10th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 8 is from 5 ’ The 4th to 5th, 7th to 9th, and 11th nucleotides from the end were modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 10 is from 5 ’ The 3rd, 5th to 7th, and 9th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 11 is from 5 ’ The 10th nucleotide from the end is modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 12 is from 5 ’ The 3rd-4th and 6th-10th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 13 is from 5 ’ The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 15 is from 5 ’ The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 16 is from 5 ’ The 10th and 12th nucleotides from the end were modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 17 is from 5 ’ The 2nd to 5th and 7th to 8th nucleotides from the end are modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 18 is from 5 ’ The 9th-10th and 12th nucleotides from the end were modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 19 is from 5 ’ The 9th and 12th nucleotides from the end were modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 20 is from 5 ’ The 10th nucleotide from the end is modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 21 is from 5 ’ The 9th to 10th nucleotides from the end were modified with locked nucleic acid; The nucleotide sequence shown in SEQ ID NO: 22 is from 5 ’ Nucleotides 3-5 and 7-9 from the end were modified with locked nucleic acid.

4. The primer probe set according to claim 1, characterized in that Probe set 5 as shown in SEQ ID NO: 5-18 ’ The end is labeled with a reporter group, 3 ’ The end is labeled with a quencher group.

5. The primer probe set according to claim 4, characterized in that The reporter group is selected from FAM, ROX, CY5, HEX or CY5.

5.

6. Use of the primer probe set according to any one of claims 1 to 5 in preparing a kit for detecting ESR1 gene mutations.

7. A kit for detecting ESR1 gene mutation, characterized in that: The method comprises the probe primer set according to claim 1, reaction reagents, positive quality control products, and negative quality control products.

8. The kit according to claim 7, characterized in that The concentration of each primer set was 500–700 nM; and / or, the concentration of each mutation probe set is 200-400 nM; And / or, the wild-type probe group concentration is 100-300 nM.

9. A method for detecting ESR1 gene mutation for non-disease diagnosis purposes, characterized in that the steps include: Obtaining nucleic acid from a sample to be tested as a template; Performing digital PCR on the template using the kit according to claim 7; The ESR1 gene mutation detection result of the sample is determined based on the digital PCR amplification result.

10. The detection method according to claim 9, characterized in that: The sample to be tested is a cell, body fluid or tissue; the body fluid includes serum, plasma or tissue fluid.

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