Primer probe set, method, agent and kit for detecting ESR1 gene mutation on basis of cfdna

Abstract

Provided are a primer probe set, a method, an agent and a kit which are used for detecting an ESR1 gene mutation on the basis of cfDNA. The primer probe set comprises a primer probe set in a first container and a primer probe set in a second container. The primer probe set in the first container comprises a primer pair specific for ESR1 gene E380Q. The primer probe set in the second container comprises a primer pair specific for ESR1 gene L536H. The primer probe set in the first container further comprises a first specific probe, a second specific probe, and a first blocking probe, and the primer probe set in the second container further comprises a third specific probe and a second blocking probe.

Description

A primer probe set, method, reagent and kit for detecting ESR1 gene mutation based on cfDNA CROSS-REFERENCE

[0001] This application claims priority to the Chinese patent application No. 202411074046.8, filed on August 06, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present specification relates to the field of biomedicine, in particular to a primer probe set, method, reagent and kit for detecting ESR1 gene mutation based on cfDNA. BACKGROUND

[0003] ESR1 (Estrogen Receptor 1) gene encodes human estrogen receptor alpha protein (ESRα), which, after binding with estrogen, activates a series of periodic reactions in cells, and then promotes cell growth and proliferation. The activating mutation of ESR1 gene is closely related to the occurrence and development of breast cancer. Among them, the most common mutation types of ESR1 gene include L536H, Y537S, Y537C, Y537N and D538G. These mutations will cause the conformation of ESRα to change, so that it can present a persistent activated state without binding with estrogen.

[0004] Breast cancer is one of the most common gynecological tumors. More than 70% of primary breast cancer is estrogen receptor (ER) positive, and many breast cancers are initially responsive to endocrine therapy. Although new therapies for breast cancer are being developed and improved, the innate and acquired resistance to these drugs remains a major challenge. The tumor microenvironment is considered to be the main factor that confers innate resistance to cancer treatment, and a large proportion of patients develop resistance when receiving systemic anti-estrogens such as tamoxifen or estrogen deprivation therapy such as aromatase inhibitors (AIs). Numerous mechanisms, including activation of cell survival, cell stress, and cell signaling pathways, have been identified as drivers of acquired resistance. Recent studies have shown that activating mutations in the ESR1 gene play an important role in driving resistance. These mutations produce endoplasmic reticulum transcriptional activity independent of estrogen ligands, which are considered to be resistance mutations. Due to the heterogeneity of the tumor itself, there is secondary resistance. Cancer biomarkers differ in disease type and disease progression stage, which complicates early-stage cancer detection and identification. The "liquid biopsy" approach to circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA) is increasingly prominent and can be used to detect and monitor resistance to systemic therapy. Acquired resistance to hormone therapy can be based on activating mutations in the ESR1 gene. In vitro and preclinical data show that ESR1 mutations lead to complete resistance to AIs and partial resistance to ER agonists and antagonists. Detection of ESR1 activating mutations can guide clinicians for endocrine and non-endocrine therapy. cfDNA fragments are relatively small, with a peak size of about 180 bp. The percentage of tumor-derived ctDNA in total cfDNA is individually variable, often too low to detect.

[0005] Therefore, it is desirable to provide a cfDNA-based ESR1 mutation detection method with high sensitivity. SUMMARY

[0006] One or more embodiments of the present specification provide a primer probe set for detecting ESR1 gene mutations based on cfDNA, wherein the primer probe set comprises a primer probe set in a first container and a primer probe set in a second container; the primer probe set in the first container comprises a primer pair specific to ESR1 gene E380Q; the primer probe set in the second container comprises a primer pair specific to ESR1 gene L536H; the primer probe set in the first container further comprises a first specific probe, a second specific probe and a first blocking probe, and the primer probe set in the second container further comprises a third specific probe and a second blocking probe.

[0007] One or more embodiments of the present specification provide a reagent for detecting ESR1 gene mutations based on cfDNA, comprising the primer set described above.

[0008] One or more embodiments of the present specification provide an application for estimating the mutation frequency of ESR1 gene in a sample based on cfDNA detection of ESR1 gene mutation, comprising the primer set described above.

[0009] One or more embodiments of the present specification provide a kit for detecting ESR1 gene mutation based on cfDNA, comprising a first container and a second container; the first container comprises a primer pair specific to ESR1 gene E380Q, the sequence of which is shown as SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequence of which is shown as SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequence of which is shown as SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene Y537S, the sequence of which is shown as SEQ ID NO: 23 and SEQ ID NO: 82; the second container comprises a primer pair specific to ESR1 gene L536H, the sequence of which is shown as SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which is shown as SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which is shown as SEQ ID NO: 19 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene D538G, the sequence of which is shown as SEQ ID NO: 10 and SEQ ID NO: 82; the first container further comprises a first specific probe capable of specifically binding to the sequence of ESR1 gene E380Q, and a second specific probe capable of specifically binding to the sequences of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; the second container further comprises a third specific probe capable of specifically binding to the sequences of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; and the first container and the second container further comprise a reference probe and a reference primer pair, respectively.

[0010] One or more embodiments of the present specification provide a method for detecting ESR1 gene mutations based on cfDNA, the method comprising: (1) obtaining a biological sample of an individual and extracting a cfDNA sample therefrom; and (2) taking two equal amounts of cfDNA samples from the cfDNA sample, respectively adding them into a first container and a second container for PCR amplification, and detecting whether the ESR1 gene in the biological sample is mutated; wherein the first container comprises a primer pair specific to ESR1 gene E380Q, the sequence of which has at least 90% similarity with SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequence of which has at least 90% similarity with SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequence of which has at least 90% similarity with SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene Y537S, the sequence of which has at least 90% similarity with SEQ ID NO: 23 and SEQ ID NO: 82; the second container comprises a primer pair specific to ESR1 gene L536H, the sequence of which has at least 90% similarity with SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which has at least 90% similarity with SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which has at least 90% similarity with SEQ ID NO: 19 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene D538G, the sequence of which has at least 90% similarity with SEQ ID NO: 10 and SEQ ID NO: 82.

[0011] One or more embodiments of the present specification provide a method for determining whether two or more ESR1 mutations are present in a sample from an individual, the method comprising: (1) obtaining a cfDNA sample from the individual; and (2) performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P, and D538G.

[0012] One or more embodiments of the present specification provide a method of treating an individual afflicted with breast cancer or resistant to aromatase inhibitor (AI) therapy, the method comprising: (1) obtaining a cfDNA sample from the individual; (2) performing PCR in two vessels to determine the presence or absence of two or more ESR1 mutations in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P, and D538G; and (3) providing a treatment to the individual if the presence of an ESR1 mutation is determined. DETAILED DESCRIPTION

[0013] As used in the specification and claims, the words "a," "an," and "the" do not exclude the plural. The word "or" means either or both. The words "comprise," "comprises," and "comprising" are used herein to mean including, but not limited to.

[0014] The terms "about" and "approximately" can describe within a certain range of a value, such as plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value. For example, the term "about 10 mL" can include 9 mL to 11 mL.

[0015] As used herein, the terms "estrogen receptor" and "ER" refer primarily to the estrogen receptor protein (especially ERa); "ESR1" is used to denote the gene encoding this protein.

[0016] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides) and include naturally occurring (adenine, guanine, cytosine, uracil, and thymine), non-naturally occurring, and modified nucleic acids. The terms are not limited by the length of the polymer (e.g., the number of monomers) nor by the date of the polymer (e.g., single- or double-stranded). Nucleic acids can be DNA or RNA, and typically contain 5'-3' phosphodiester bonds, although in some cases nucleotide analogs can have other linkages. Monomers are often referred to as nucleotides.

[0017] The term "sample" or "biological sample" refers to any composition containing or presumed to contain nucleic acids, including tissue samples, fluid samples, such as isolated cells, tissues, or blood, etc. In some embodiments, the sample can be a plasma sample.

[0018] As used herein, the term "primer" refers to a short nucleic acid (oligonucleotide) that under suitable conditions serves as a starting point for the synthesis of a polynucleotide strand by a nucleic acid polymerase. A primer typically includes at least one region that hybridizes to a target, which is at least substantially complementary (e.g., has 0, 1, 2, or 3 mismatches) to the target sequence. The region typically has a length of about 8 to about 40 nucleotides, e.g., 12-25 nucleotides. A "primer pair" refers to a forward and reverse primer that are oriented in opposite directions with respect to a target sequence and that produce an amplification product under amplification conditions.

[0019] As used herein, the term "probe" refers to any molecule that is capable of selectively binding to a specifically intended target biomolecule (e.g., a target nucleic acid sequence to which the probe hybridizes). The probe is detectably labeled with at least one non-nucleotide moiety. In some embodiments, the probe has a fluorescent group and a fluorescent quenching group at its two ends, respectively.

[0020] As used herein, the term "complementary" refers to the ability of one nucleic acid in a polynucleotide to form base pairs with another nucleic acid in a second polynucleotide.

[0021] As used herein, the term "container" refers to a container that can hold a reagent or an assay. If the container is in a kit and holds a reagent or is used in an amplification reaction, it can be closed or sealed to avoid contamination or evaporation. If the container is used in an assay, it can be open or accessible.

[0022] As used herein, the term "amplification" refers to a nucleic acid amplification reaction that allows for the hybridization and template-dependent extension of primers. For example, PCR amplification.

[0023] As used herein, the term "cell-free DNA" (cfDNA), i.e., "cell-free DNA", refers to DNA fragments that are free from cells in a body fluid (such as blood, urine, cerebrospinal fluid, etc.).

[0024] One aspect of the present specification provides a primer probe set for detecting ESR1 gene mutation based on cfDNA, wherein the primer probe set comprises a primer probe set in a first container and a primer probe set in a second container; the primer probe set in the first container comprises a primer pair specific to ESR1 gene E380Q; the primer probe set in the second container comprises a primer pair specific to ESR1 gene L536H; the primer probe set in the first container further comprises a first specific probe, a second specific probe, and a first blocking probe, and the primer probe set in the second container further comprises a third specific probe and a second blocking probe.

[0025] In some embodiments, the cfDNA sample is loaded at about 20 ng.

[0026] In some embodiments, the sequence of the primer pair specific to ESR1 gene E380Q in the primer probe set in the first container is set forth in SEQ ID NO: 76 and SEQ ID NO: 83, respectively.

[0027] In some embodiments, the primer probe set in the first container further comprises a primer pair specific to ESR1 gene Y537C; a primer pair specific to ESR1 gene Y537N; and a primer pair specific to ESR1 gene Y537S.

[0028] In some embodiments, the sequence of the primer pair specific to ESR1 gene Y537C is set forth in SEQ ID NO: 66 and SEQ ID NO: 82, respectively; the sequence of the primer pair specific to ESR1 gene Y537N is set forth in SEQ ID NO: 34 and SEQ ID NO: 82, respectively; and the sequence of the primer pair specific to ESR1 gene Y537S is set forth in SEQ ID NO: 23 and SEQ ID NO: 82, respectively.

[0029] In some embodiments, the primer pair specific to ESR1 gene E380Q can further comprise a downstream primer set forth in SEQ ID NO: 83 and an upstream primer set forth in any one or more of SEQ ID NOs: 71-80.

[0030] In some embodiments, the primer pair specific to ESR1 gene Y537C can further comprise a downstream primer set forth in SEQ ID NO: 82 and an upstream primer set forth in any one or more of SEQ ID NOs: 61-70.

[0031] In some embodiments, the primer pair specific to ESR1 gene Y537N can further comprise a downstream primer set forth in SEQ ID NO: 82 and an upstream primer set forth in any one or more of SEQ ID NOs: 31-40.

[0032] In some embodiments, the primer pair specific to ESR1 gene Y537S can further comprise a downstream primer set forth in SEQ ID NO: 82 and an upstream primer set forth in any one or more of SEQ ID NOs: 21-30.

[0033] In some embodiments, the sequence of the primer pair specific to ESR1 gene L536H in the primer probe set in the second container is set forth in SEQ ID NO: 45 and SEQ ID NO: 82.

[0034] In some embodiments, the primer probe set in the second container further comprises a primer pair specific to the L536R of the ESR1 gene; a primer pair specific to the L536P of the ESR1 gene; and a primer pair specific to the D538G of the ESR1 gene.

[0035] In some embodiments, the primer pair specific to the L536R of the ESR1 gene has sequences as set forth in SEQ ID NO: 57 and SEQ ID NO: 82, respectively; the primer pair specific to the L536P of the ESR1 gene has sequences as set forth in SEQ ID NO: 19 and SEQ ID NO: 82, respectively; and the primer pair specific to the D538G of the ESR1 gene has sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 82, respectively.

[0036] In some embodiments, the primer pair specific to the L536H of the ESR1 gene can further comprise a downstream primer as set forth in SEQ ID NO: 82 and an upstream primer as set forth in any one or more of SEQ ID NOs: 41-50.

[0037] In some embodiments, the primer pair specific to the L536R of the ESR1 gene can further comprise a downstream primer as set forth in SEQ ID NO: 82 and an upstream primer as set forth in any one or more of SEQ ID NOs: 51-60.

[0038] In some embodiments, the primer pair specific to the L536P of the ESR1 gene can further comprise a downstream primer as set forth in SEQ ID NO: 82 and an upstream primer as set forth in any one or more of SEQ ID NOs: 11-20.

[0039] In some embodiments, the primer pair specific to the D538G of the ESR1 gene can further comprise a downstream primer as set forth in SEQ ID NO: 82 and an upstream primer as set forth in any one or more of SEQ ID NOs: 1-10.

[0040] In some embodiments, the concentration of the primer pair is in the range of 400-800 nM, preferably 600 nM. In some embodiments, the concentration of the primer pair can be 400 nM, 600 nM, or 800 nM.

[0041] In some embodiments, the first specific probe has a fluorescent group and a fluorescent quenching group at two ends, respectively, and can specifically bind to the sequence of the E380Q of the ESR1 gene, and the sequence of the first specific probe is as set forth in SEQ ID NO: 86.

[0042] In some embodiments, the second specific probe and the third specific probe have the same sequence as shown in SEQ ID NO: 85, and the second specific probe and the third specific probe are respectively provided with a fluorescent group and a fluorescent quenching group at both ends, and can be specifically combined with the sequence of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G.

[0043] In some embodiments, the concentration of the specific probe is in the range of 200-300nM, preferably 300nM. In some embodiments, the concentration of the specific probe can be 200nM, 250nM or 300nM.

[0044] In some embodiments, the first blocking probe and the second blocking probe have the same sequence as shown in SEQ ID NO: 88.

[0045] In some embodiments, the blocking probe is used to specifically combine with the non-mutated template in the sample, so as to reduce the probability of primer combination with the non-mutated template.

[0046] In some embodiments, the concentration of the blocking probe is in the range of 100-300nM, preferably 100nM. In some embodiments, the concentration of the blocking probe is 100nM, 200nM or 300nM.

[0047] In some embodiments, the primer probe group in the first container and the primer probe group in the second container further comprise an internal reference probe and an internal reference primer pair, the sequence of the internal reference probe is shown in SEQ ID NO: 87, and the sequences of the internal reference primer pair are shown in SEQ ID NO: 81 and SEQ ID NO: 84, and the internal reference probe and the internal reference primer pair are used for sample quality evaluation.

[0048] In some embodiments, the primer probe group in the first container and the primer probe group in the second container further comprise a probe of wild sequence of ESR1 as shown in SEQ ID NO: 91 and a primer pair of wild sequence of ESR1 as shown in SEQ ID NO: 92 and SEQ ID NO: 93, and the probe of wild sequence and the primer pair are used for estimating the mutation frequency range of ESR1 in the sample.

[0049] Another aspect of the present specification provides a primer set for detecting ESR1 gene mutation, comprising any one or more of the following primer combinations: primer combination 1, primer combination 2; wherein primer combination 1 comprises a downstream primer as shown in SEQ ID NO: 83, a specific probe as shown in SEQ ID NO: 86 and SEQ ID NO: 85, and an upstream primer selected from any one or more of SEQ ID NO: 21-30, SEQ ID NO: 31-40, SEQ ID NO: 61-70, SEQ ID NO: 71-80; primer combination 2 comprises a downstream primer as shown in SEQ ID NO: 82, a specific probe as shown in SEQ ID NO: 85, and an upstream primer selected from any one or more of SEQ ID NO: 1-10, SEQ ID NO: 11-20, SEQ ID NO: 41-50, SEQ ID NO: 51-60.

[0050] In some embodiments, primer combination 1 can detect E380Q, Y537C, Y537N, Y537S mutation sites.

[0051] In some embodiments, primer combination 1-a with sequences of SEQ ID NO: 76, SEQ ID NO: 83 and SEQ ID NO: 86 can detect E380Q.

[0052] In some embodiments, primer combination 1-b with sequences of SEQ ID NO: 66, SEQ ID NO: 82 and SEQ ID NO: 85 can detect Y537C.

[0053] In some embodiments, primer combination 1-c with sequences of SEQ ID NO: 34, SEQ ID NO: 82 and SEQ ID NO: 85 can detect Y537N.

[0054] In some embodiments, primer combination 1-d with sequences of SEQ ID NO: 23, SEQ ID NO: 82 and SEQ ID NO: 85 can detect Y537S.

[0055] In some embodiments, primer combination 2 can detect L536H, L536R, L536P, D538G mutation sites.

[0056] In some embodiments, primer combination 2-a with sequences of SEQ ID NO: 45, SEQ ID NO: 82 and SEQ ID NO: 85 can detect L536H.

[0057] In some embodiments, the primer combination 2-b of sequences SEQ ID NO: 57, SEQ ID NO: 82 and SEQ ID NO: 85 can detect L536R.

[0058] In some embodiments, the primer combination 2-c of sequences SEQ ID NO: 19, SEQ ID NO: 82 and SEQ ID NO: 85 can detect L536P.

[0059] In some embodiments, the primer combination 2-d of sequences SEQ ID NO: 10, SEQ ID NO: 82 and SEQ ID NO: 85 can detect D538G.

[0060] In some embodiments, the specific probe as shown in SEQ ID NO: 85 and SEQ ID NO: 86 has a fluorescent group and a fluorescent quenching group at both ends, respectively.

[0061] The above primer set can accurately detect 8 mutations of ESR1 gene, with high sensitivity and good specificity. In a 20 ng DNA sample, except for the L536H mutant template, 0.1% of the mutation can be accurately detected.

[0062] In some embodiments, the primer set further comprises an internal reference probe and an internal reference primer, the sequence of the internal reference probe is shown in SEQ ID NO: 87, and the sequences of the internal reference primer are shown in SEQ ID NO: 81 and SEQ ID NO: 84, the internal reference probe and the internal reference primer are used for sample quality evaluation.

[0063] In some embodiments, the primer set further comprises a blocking probe with a sequence as shown in SEQ ID NO: 88, the blocking probe is used to specifically bind to the non-mutant template in the sample, thereby reducing the probability of primer binding to the non-mutant template.

[0064] In some embodiments, the primer set further comprises a probe of wild sequence of ESR1 with a sequence as shown in SEQ ID NO: 91 and a primer pair of wild sequence of ESR1 with sequences as shown in SEQ ID NO: 92 and SEQ ID NO: 93, the wild sequence probe and the primer pair are used to estimate the mutation frequency range of ESR1 in the sample.

[0065] An aspect of the present specification provides a reagent for detecting ESR1 gene mutation based on cfDNA, which comprises the above-mentioned primer set.

[0066] An aspect of the present specification provides an application of detecting ESR1 gene mutation based on cfDNA for estimating the mutation frequency of ESR1 gene in a sample, which comprises the above-mentioned primer set.

[0067] An aspect of the present specification provides a method for detecting ESR1 gene mutation based on cfDNA, comprising: (1) obtaining a biological sample of an individual and extracting a cfDNA sample therefrom.

[0068] In some embodiments, the individual can be a mammal. In some embodiments, the individual is a human.

[0069] In some embodiments, the biological sample includes, but is not limited to, cells, tissues, or blood, etc., such as, for example, purified or isolated components of cells, tissues, or blood. For another example, frozen or fresh tissues, or from liquid samples. In some embodiments, the biological sample is obtained in a non-invasive manner, such as, for example, urine, skin, swab, saliva, blood, etc. In some embodiments, the biological sample is preferably a blood sample.

[0070] Methods for isolating DNA from a biological sample are known, such as, for example, using Roche cobas® TaqMan® DNA Test. The DNA sample preparation kit, the specific operation steps can refer to the instructions of the kit.

[0071] (2) Take two equal amounts of cfDNA samples from the cfDNA sample, and add them into a first container and a second container respectively for PCR amplification, to detect whether the ESR1 gene in the biological sample is mutated. Among them, the first container includes a primer pair specific to ESR1 gene E380Q, the sequence of which is shown in SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequence of which is shown in SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequence of which is shown in SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene Y537S, the sequence of which is shown in SEQ ID NO: 23 and SEQ ID NO: 82; the second container includes a primer pair specific to ESR1 gene L536H, the sequence of which is shown in SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which is shown in SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which is shown in SEQ ID NO: 19 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene D538G, the sequence of which is shown in SEQ ID NO: 10 and SEQ ID NO: 82.

[0072] In some embodiments, the sequence of the primer pair specific for the ESR1 gene E380Q has at least 90% similarity to SEQ ID NO: 76 and SEQ ID NO: 83. In some embodiments, the sequence of the primer pair specific for the ESR1 gene E380Q has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 76 and SEQ ID NO: 83.

[0073] In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537C has at least 90% similarity to SEQ ID NO: 66 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537C has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 66 and SEQ ID NO: 82.

[0074] In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537N has at least 90% similarity to SEQ ID NO: 34 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537N has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 34 and SEQ ID NO: 82.

[0075] In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537S has at least 90% similarity to SEQ ID NO: 23 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific for the ESR1 gene Y537S has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 23 and SEQ ID NO: 82.

[0076] In some embodiments, the sequence of the primer pair specific for the ESR1 gene L536H has at least 90% similarity to SEQ ID NO: 45 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific for the ESR1 gene L536H has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 45 and SEQ ID NO: 82.

[0077] In some embodiments, the sequence of the primer pair specific to the ESR1 gene L536R has at least 90% similarity to SEQ ID NO: 57 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific to the ESR1 gene L536R has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 57 and SEQ ID NO: 82.

[0078] In some embodiments, the sequence of the primer pair specific to the ESR1 gene L536P has at least 90% similarity to SEQ ID NO: 19 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific to the ESR1 gene L536P has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 19 and SEQ ID NO: 82.

[0079] In some embodiments, the sequence of the primer pair specific to the ESR1 gene D538G has at least 90% similarity to SEQ ID NO: 10 and SEQ ID NO: 82. In some embodiments, the sequence of the primer pair specific to the ESR1 gene D538G has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 10 and SEQ ID NO: 82.

[0080] For example only, a PCR amplification reaction system (25 μΐ) is as follows:

[0081] The above PCR system is only illustrative, and in practical applications, the volume of the mixture and the content of each component therein can be proportionally expanded or reduced. Among them, ESR-F is the upstream primer of the ESR1 gene, and ESR-R is the downstream primer of the ESR1 gene.

[0082] In some embodiments, the PCR amplification procedure is 25 °C, 10 min; 95 °C, 5 min; (95 °C, 30 s; 60 °C, 60 s) 40 cycles; end.

[0083] In an embodiment, the loading amount of the cfDNA sample is about 20 ng.

[0084] In some embodiments, the ESR1 gene E380Q, Y537C, Y537N, Y537S mutation site can be detected in the first container, and the ESR1 gene L536H, L536R, L536P, D538G mutation site can be detected in the second container.

[0085] In some embodiments, the concentration of the primer pair can be 400-800 ng. In some embodiments, the concentration of the primer pair is 400 nM, 600 nM, 800 nM. In some embodiments, the optimal concentration of the primer pair is 600 nM.

[0086] In some embodiments, the first container further comprises a first specific probe and a second specific probe, and the second container further comprises a third specific probe.

[0087] In some embodiments, the first specific probe has a fluorescent group and a fluorescent quenching group at two ends respectively, and can specifically bind to the sequence of the ESR1 gene E380Q, and the sequence of the first specific probe is shown as SEQ ID NO: 86. In some embodiments, the sequence of the first specific probe has at least 90% similarity with SEQ ID NO: 86. In some embodiments, the sequence of the first specific probe has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similarity with SEQ ID NO: 86.

[0088] In some embodiments, the second specific probe and the third specific probe have the same sequence shown as SEQ ID NO: 85, and the second specific probe and the third specific probe have a fluorescent group and a fluorescent quenching group at two ends respectively, and the second specific probe and the third specific probe can specifically bind to the sequences of the ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G respectively. In some embodiments, the second specific probe and the third specific probe have at least 90% similarity with SEQ ID NO: 85. In some embodiments, the second specific probe and the third specific probe have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similarity with SEQ ID NO: 85.

[0089] In some embodiments, the first container and the second container further comprise a reference probe and a reference primer pair respectively, and the sequence of the reference probe is shown as SEQ ID NO: 87, and the sequences of the reference primer pair are shown as SEQ ID NO: 81 and SEQ ID NO: 84.

[0090] In some embodiments, the sequence of the internal control probe has at least 90% similarity to SEQ ID NO: 87. In some embodiments, the sequence of the internal control probe has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 87.

[0091] In some embodiments, the sequence of the internal control primer pair has at least 90% similarity to SEQ ID NO: 81 and SEQ ID NO: 84. In some embodiments, the sequence of the internal control primer pair has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 81 and SEQ ID NO: 84.

[0092] In some embodiments, the specific probe is a MGB specific probe, which is linked with a MGB modification group.

[0093] In some embodiments, the specific probe contains a fluorescent group. Illustratively, the specific probe has a fluorescent reporter group and a fluorescent quencher group at its two ends, respectively. The fluorescent reporter group includes, but is not limited to, FAM, JOE, TET, Cal Fluor Gold 540, HEX, VIC, Cal Fluor Orang 560, TAMRA, Cyanine 3 (CY3), Quasar 570, Cal Fluor Red 590, ROX, Texas Red, Cyanine 5 (CY5), Quasar 670, and Cyanine 5.5 (CY5.5); and the fluorescent quencher group includes, but is not limited to, TAMRA, DABCYL, NFQ, and BHQ1-3.

[0094] In some embodiments, the fluorescent reporter group is selected from any one of FAM, HEX, ROX, or CY5; and the fluorescent quencher group is selected from any one of TAMRA, BHQ1, BHQ2, or NFQ.

[0095] In some embodiments, the fluorescent reporter group is preferably FAM; and the fluorescent quencher group is preferably BHQ2.

[0096] In some embodiments, the concentration of the specific probe can be 200-300 nM. In some embodiments, the concentration of the specific probe is 200 nM, 250 nM, and 300 nM. In some embodiments, the optimal concentration of the specific probe is 300 nM.

[0097] In some embodiments, the first container and the second container further comprise a blocking probe with a sequence as shown in SEQ ID NO: 88, which is used to specifically bind to the non-mutated template in the sample, so as to reduce the probability of the primer binding to the non-mutated template.

[0098] In some embodiments, the sequence of the blocking probe has at least 90% similarity to SEQ ID NO: 88. In some embodiments, the sequence of the blocking probe has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 88.

[0099] In some embodiments, the concentration of the blocking probe can be 100-300 nM. In some embodiments, the concentration of the blocking probe is 100 nM, 200 nM, 300 nM. In some embodiments, the optimal concentration of the blocking probe is 100 nM.

[0100] Because the content of cfDNA in plasma is extremely small, and the total amount of cfDNA in many samples is also not high, the mutant sequence contained therein can be even less. In order to prevent non-specific binding of primers to wild-type templates and increase the detection of target fragments, blocking probes are added to the original primer probe preparation system. In a non-annealing state, the beginning and end of the blocking probe will complement each other to form double-stranded, and at a suitable annealing temperature, the double-stranded will open and can specifically bind to the wild-type template, thereby reducing the possibility of primer binding to the wild-type template at the annealing temperature, and improving the accuracy of the primer.

[0101] In some embodiments, the first container and the second container further comprise a probe of wild sequence of ESR1 with sequence as shown in SEQ ID NO: 91 and a primer pair of wild sequence of ESR1 with sequence as shown in SEQ ID NO: 92 and SEQ ID NO: 93, and the probe and primer pair of wild sequence are used to estimate the mutation frequency range of ESR1 in the sample.

[0102] In some embodiments, the sequence of the probe of wild sequence of ESR1 has at least 90% similarity to SEQ ID NO: 91. In some embodiments, the sequence of the probe of wild sequence of ESR1 has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 91.

[0103] In some embodiments, the sequence of the primer pair of wild sequence of ESR1 has at least 90% similarity to SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, the sequence of the primer pair of wild sequence of ESR1 has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO: 92 and SEQ ID NO: 93.

[0104] The mutation frequency can be determined by dividing the number of mutant template copies by the total number of mutant and wild-type template copies. In some embodiments, the probe for the ESR1 wild sequence is capable of binding to all mutant and wild-type templates. The copy number of each mutant template and all templates (each mutant template and wild-type template) can be determined by the CT value. The mutation frequency range for the mutation model is obtained by dividing the number of mutant template copies for different mutation frequencies by the total number of mutant and wild-type template copies.

[0105] The method for detecting ESR1 gene mutations provided by the embodiments of the present specification can detect eight mutation sites (E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G) of the ESR1 gene based on cfDNA by using the fluorescence quantitative PCR (qPCR) technique. The method for detecting ESR1 gene mutations based on cfDNA effectively improves the detection sensitivity by using the reasonably screened upstream primers, the specifically designed specific probes, and the wild sequence probes, can accurately detect low copy mutations, and can meet the detection needs of clinical routine tissue samples.

[0106] An aspect of the present specification provides a method for determining whether two or more ESR1 mutations are present in a sample from an individual, the method comprising: (1) obtaining a cfDNA sample from the individual.

[0107] In some embodiments, the individual is a breast cancer patient or a breast cancer patient who has developed resistance to AI treatment.

[0108] In some embodiments, the amount of the cfDNA sample is about 20 ng.

[0109] (2) performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P, and D538G.

[0110] In some embodiments, performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample comprises: (1) determining whether the cfDNA sample comprises ESR1 gene E380Q, Y537C, Y537N, and Y537S in a first container; and (2) determining whether the cfDNA sample comprises ESR1 gene L536H, L536R, L536P, and D538G in a second container.

[0111] In some embodiments, the qPCR is performed in two vessels to determine the presence of four or more ESR1 mutations.

[0112] In some embodiments, the individual is provided a treatment upon determining the presence of the ESR1 mutation.

[0113] Another aspect of the present specification provides a method of treating an individual suffering from breast cancer or breast cancer that is resistant to AI treatment, the method comprising: (1) obtaining a cfDNA sample from the individual; (2) performing PCR in two vessels to determine the presence of two or more ESR1 mutations in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P, and D538G; and (3) providing the individual a treatment upon determining the presence of the ESR1 mutation.

[0114] Another aspect of the present specification provides a kit for detecting ESR1 gene mutations based on cfDNA. In some embodiments, the kit is a kit for qPCR technique. The ESR1 gene mutations can include ESR1 gene E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, D538G, etc. mutation sites.

[0115] In some embodiments, the kit comprises: a first container and a second container; the first container comprises a primer pair specific to ESR1 gene E380Q, the sequence of which is shown as SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequence of which is shown as SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequence of which is shown as SEQ ID NO: 34 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537S, the sequence of which is shown as SEQ ID NO: 23 and SEQ ID NO: 82; the second container comprises a primer pair specific to ESR1 gene L536H, the sequence of which is shown as SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which is shown as SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which is shown as SEQ ID NO: 19 and SEQ ID NO: 82, a primer pair specific to ESR1 gene D538G, the sequence of which is shown as SEQ ID NO: 10 and SEQ ID NO: 82; the first container and the second container further comprise a specific probe capable of specifically binding to different sequences of ESR1 gene, a reference probe and a reference primer pair, the sequence of the specific probe is shown as SEQ ID NO: 85 and SEQ ID NO: 86, the specific probe has a fluorescent group and a fluorescent quencher group at its two ends, the sequence of the reference probe is shown as SEQ ID NO: 87, the sequence of the reference primer pair is shown as SEQ ID NO: 81 and SEQ ID NO: 84.

[0116] In some embodiments, the first container and the second container further comprise a blocking probe with the sequence shown as SEQ ID NO: 88, which is used to specifically bind to non-mutated templates in the sample, thereby reducing the probability of primer binding to non-mutated templates.

[0117] In some embodiments, the first container and the second container further comprise a probe with the wild sequence of ESR1, the sequence of which is shown as SEQ ID NO: 91, and a primer pair with the wild sequence of ESR1, the sequence of which is shown as SEQ ID NO: 92 and SEQ ID NO: 93, which are used to estimate the mutation frequency range of ESR1 in the sample.

[0118] In some embodiments, the starting amount of the cfDNA sample detected by the kit ranges from 10 ng to 30 ng, preferably 20 ng.

[0119] In some embodiments, the concentration of the primer pair is in the range of 200-800 ng, preferably 600 nM, the concentration of the specific probe is in the range of 200-300 nM, preferably 300 nM, and the concentration of the blocking probe is in the range of 100-300 nM, preferably 100 nM.

[0120] In some embodiments, the kit comprises a positive control and / or a blank control. The positive control comprises a mutant plasmid containing any one of ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G mutant fragments.

[0121] In some embodiments, the positive control is a mixture containing eight mutant plasmids, each of which contains ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G mutant fragments. The plasmid can be a plasmid well known to those skilled in the art, and the concentrations of the eight plasmids can be the same.

[0122] In some embodiments, the concentration of the mutant plasmid of the positive control can be 2.0 x 10 6 copies / μl, 2.0 x 10 5 copies / μl, 2.0 x 10 4 copies / μl, or 2000 copies / μl.

[0123] In some embodiments, the blank control comprises a Tris-HCl buffer.

[0124] In some embodiments, the kit further comprises a PCR buffer, an enzyme mixture, and / or other elements suitable for reverse transcription and amplification, such as co-factors or aptamers.

[0125] In some embodiments, the kit further comprises consumables, such as plates or test tubes for nucleic acid preparation, test tubes for sample collection, and the like.

[0126] The primer group, method, reagent and kit for detecting ESR1 gene mutation based on cfDNA provided by the embodiments of the present specification can bring beneficial effects including but not limited to: (1) the standard sample is prepared by using negative normal cfDNA and mutant plasmid with ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G mutation fragments after enzyme digestion, and different copy number ratios, which can maximize the characteristics of the detection sample, lay the foundation for the optimization of the reaction system, and play a decisive role in the system optimization process; (2) the mutant frequency standard sample is detected by digital PCR, which can accurately detect the CT value of the primer binding to the template under different concentrations, and then obtain accurate and reliable data statistics; the wild type template is detected by digital PCR, which can accurately detect the background concentration of different wild type templates in the system. Therefore, when detecting the sample, the interference of the non-specific amplification peak of the wild sequence on the detection result can be effectively avoided, and the result is more accurate; the low mutant frequency standard sample is detected by digital PCR, which can accurately determine the sensitivity of the detection system; (3) the high mutant frequency standard sample is detected by fluorescence PCR, so as to optimize the concentration of each probe. According to the difference in fluorescence intensity after the reaction of each mutant with different concentrations of primers, the appropriate concentration of primers is selected. The method has accurate results and low cost; (4) the specific probe provided by the embodiments of the present specification is a self-designed MGB specific probe, which has a short probe sequence and good specificity. The specific probe is selected by multiple combinations, has high amplification efficiency and high sensitivity; (5) the probe of the ESR1 wild sequence provided by the embodiments of the present specification can be combined with the mutant template and the wild type template. The mutant frequency range of the mutant model is obtained by dividing the copy number of the mutant template with different mutant frequencies by the total copy number of the mutant template and the wild type template (determined according to the CT value). The method for detecting ESR1 gene mutation based on cfDNA provided by the embodiments of the present specification can provide a reference for estimating the mutant frequency of the sample; (6) the method for detecting ESR1 gene mutation based on cfDNA provided by the embodiments of the present specification is rapid, efficient, low in cost, and can detect 8 mutant sites (E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G) of ESR1 gene based on trace cfDNA in biological samples by fluorescence quantitative PCR (qPCR) technology. The method can quickly and accurately monitor whether the ESR1 gene of the tumor patient is mutated, timely monitor the new gene mutation of the patient, and thus provide a strong basis for the formulation and adjustment of the clinical treatment plan.

[0127] The experimental methods in the following Examples 1-10 are conventional methods unless otherwise specified. The experimental materials used in the following Examples 1-10 are commercially available from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following Examples 1-10 are set up in triplicate unless otherwise specified, and the results are averaged.

[0128] The instruments used in Examples 1-10 include mainly vortex shaker, high-speed centrifuge, digital PCR (Xinyi Biotechnology Co., Ltd.), A300 PCR, SLAN-96s real-time PCR (Shanghai Hongshi Medical Technology Co., Ltd.), drop maker (drop maker M1), chip reader (chip reader R1) (Xinyi Manufacturing Technology (Beijing) Co., Ltd.), biological safety cabinet, water bath, pipette, RainDrop Sense (RainDance Technologies), etc.

[0129] The wild-type template (wild-type plasmid) and the mutant template (mutant plasmid) are quantitatively detected by digital PCR, and the primers are screened.

[0130] 1. Preparation of plasmids required for experiments

[0131] (1) In theory, the number of single gene copies of human DNA per 1 ng is about 300 copies. The concentration of wild-type plasmid DNA is diluted to about 2.0 x 10 5 copies / μl.

[0132] (2) The mutant plasmid containing the inserted ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutant fragments is obtained by enzyme digestion. The calculation formula for the copy number of the mutant plasmid is 9.1 x 10 8 copies / μl, 2.0 x 10 6 copies / μl, 2.0 x 10 5 copies / μl, 2.0 x 10 4 copies / μl, and 2000 copies / μl.

[0133] 2. Primer screening

[0134] The ESR1 gene mutation detection primer and probe sequences are shown in Table 1. The specific binding degree of the primers and probes to the mutant template and the wild-type template is tested, respectively, to screen the optimal primer and probe for each mutation site. Table 1 Primer and probe sequences

[0135] The concentrations of the primer probe and the blocking probe were 600 nM and 100 nM, respectively. The specific probes described above were MGB probes, and the 5' end of the probe was modified with a FAM fluorescent group.

[0136] (1) The reaction system was prepared according to the reaction system component table shown in Table 2. The copy number of the mutant template was 1000 copies, and the copy number of the wild type template was 8000 copies. Table 2 Reaction system component table

[0137] (2) The PCR amplification procedure was 25°C for 10 min, 95°C for 5 min, 40 cycles of (95°C for 30 s, 60°C for 60 s), and end.

[0138] (3) After the above PCR reaction, the detection results are shown in Tables 3-5. Table 3 shows the results of digital PCR quantification of different mutant plasmids. Table 4 shows the test results of the primer using the mutant template; and Table 5 shows the test results of the primer using the wild type template. Table 3 Results of digital PCR quantification of different mutant plasmids Table 4 Test results of the primer in the mutant template Table 5 Test results of the primer in the wild type template

[0139] (4) Result analysis: The optimal primer was selected based on the CT value, and the primer was screened according to the standard that the primer produced the smallest CT value in the mutant template and did not produce CT value in the wild type template. The finally determined optimal primer was F536R-7, F536P-9, F536H-5, F538-10, F537C-6, F537N-4, F537S-3, and F380-6. Example 2 Screening of primer concentration and probe concentration

[0140] The mutant template was quantitatively detected by digital PCR, and the primer concentration was screened.

[0141] 1. Preparation of plasmids required for the experiment

[0142] The mutant plasmid obtained after enzyme digestion of the plasmid contained the inserted ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutant fragments. The copy number of the mutant plasmid was calculated according to the formula 9.1 x 10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800 bp); and the obtained plasmid concentration was diluted to 2.0 x 106 copies / μl, 2.0 x 10 5 copies / μl, 2.0 x 10 4 copies / μl, 2000 copies / μl.

[0143] 2. Screening of primer and probe concentrations

[0144] The specific binding of the primer and the specific probe to the mutant template was tested respectively based on the mutant template, and the optimal primer concentration and probe concentration for each mutation site were screened.

[0145] (1) The reaction system was prepared according to the following reaction system component table, as shown in Table 6. Among them, the copy number of the mutant template was 1000 copies. Table 6 Reaction system component table

[0146] (2) The PCR amplification procedure was 25°C for 10 min; 95°C for 5 min; (95°C for 30 s; 60°C for 60 s) for 40 cycles; and end.

[0147] (3) When the probe concentration was set to 300 nm, the primer concentration was tested, and the primer concentration was set to 800 nm, 600 nm and 400 nm respectively, and the results are shown in Table 7. When the primer concentration was 600 nm, the probe concentration was tested, and the probe concentration was set to 300 nm, 250 nm and 200 nm respectively, and the detection results are shown in Table 8. Table 7 Detection results of different primer concentrations Table 8 Detection results of different probe concentrations

[0148] (4) Result analysis: The optimal primer and probe concentrations were selected according to the CT value, and the smallest CT value of the primer under the mutant template was used as the screening standard. The optimal primer concentration was finally screened to be 600 nm, and the optimal probe concentration was 300 nm.

[0149] The wild type template and the mutant template were quantitatively detected by digital PCR, and the optimal concentration of the blocker probe was screened.

[0150] 1. Preparation of plasmids required for the experiment

[0151] (1) The concentration of the wild type plasmid DNA was diluted to about 2.0 x 10 5 copies / μl.

[0152] (2) The mutant plasmid obtained after plasmid digestion, which contains the inserted ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G mutant fragment. The copy number of the mutant plasmid is calculated by the formula: 9.1 x 10 8 copies / μl ÷ plasmid length (2800bp); the obtained plasmid concentration is diluted to 2.0 x 10 6 copies / μl, 2.0 x 10 5 copies / μl, 2.0 x 10 4 copies / μl, 2000 copies / μl.

[0153] 2. Optimal blocker probe concentration screening

[0154] Based on the mutant template and the wild type template, the specific binding degree of the blocker probe to the mutant and wild type template is tested, and the optimal blocker probe concentration is screened.

[0155] (1) The reaction system is prepared according to the following reaction system component table, as shown in Table 9. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, wherein the mixed upstream primer of ESR1 gene Y537C, Y537N, Y537S and E380Q is represented as CNS380-F, the mixed downstream primer is represented as CNS380-R, and the specific probe for detecting ESR1 gene E380Q is represented as specific probe 380. System 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G, wherein the mixed upstream primer of ESR1 gene L536H, L536P, L536R and D538G is represented as RPH538-F, the mixed downstream primer is represented as RPH538-R, and the specific probe for detecting ESR1 gene Y537C, Y537N, Y537S, L536H, L536P, L536R and D538G is represented as specific probe 536-538. The upstream primer of the internal reference is represented as RPPH1-F, and the downstream primer of the internal reference is represented as RPPH1-R. The copy number of the mutant template is 1000 copies, and the copy number of the wild type template is 18000 copies. Table 9 Reaction system component table

[0156] (2) The PCR amplification program is 25℃, 10min; 95℃, 5min; (95℃, 30s; 60℃, 60s) 40 cycles; end.

[0157] (3) When the copy number of the wild type template is 18000 copies, the blocker probe concentration is tested, and the blocker probe concentration is set to 100 nm, 200 nm and 300 nm, respectively, and the detection results are shown in Table 10. When the blocker probe concentration is 100 nm, the detection of each mutant template (1000 copies) is tested, and the results are shown in Table 11. Table 10 Detection results of different concentrations of blocker probe Table 11 Detection results of 100 nm blocker probe

[0158] (4) Result analysis: When the concentration of the blocker probe is 100 nm, the primer can prevent the amplification of the 18000 copies of the wild type template, and each mutant template can be normally detected. Example 4 Simulated sample template

[0159] Each mutant template and wild type template are mixed according to a mutation frequency of about 13%, simulating the template extracted from 20 ng of sample. Digital PCR is used to quantitatively detect the wild type template and the mutant template.

[0160] 1. According to the following reaction system component table, the reaction system is prepared as shown in Table 12. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Table 12 Reaction system component table

[0161] 2. The PCR amplification program is 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0162] 3. After the above PCR reaction, the detection results are shown in Table 13. Table 13 Detection results

[0163] 4. Result analysis: When simulating 20 ng of sample input, each mutant template can be normally detected, and there is no non-specific reaction with the wild type template. The primers and probes in the reaction system have good specificity. Example 5 Simulated sample template with different mutation frequencies

[0164] Each mutant template and wild type template are mixed at a mutation frequency of about 10%, 1% and 0.1%, simulating the presence of different mutation frequencies in the template extracted from 20 ng of sample. Digital PCR is used to quantitatively detect the wild type template and the mutant template.

[0165] 1、According to the following reaction system component table, the reaction system is prepared as shown in Table 14. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Table 14 Reaction system component table

[0166] 2、The PCR amplification program is 25℃, 10min; 95℃, 5min; (95℃, 30s; 60℃, 60s) 40 cycles; end.

[0167] 3、After the above PCR reaction, the detection results are shown in Table 15. Table 15 Detection results

[0168] 4、Result analysis: when simulating 20ng sample input, except for L536H mutant template, other mutant templates can be detected at a mutation frequency of 0.1%. Example 6 Estimation of mutation frequency of sample

[0169] In order to estimate the mutation frequency range of the sample, a probe for detecting the wild type sequence of ESR1 is designed. Digital PCR is used to quantitatively detect mutant and wild type templates, and the copy number of each mutant template is 1000 copies, 100 copies and 10 copies.

[0170] 1、According to the following reaction system component table, the reaction system is prepared as shown in Table 16. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Table 16 Reaction system component table

[0171] 2、The PCR amplification program is 25℃, 10min; 95℃, 5min; (95℃, 30s; 60℃, 60s) 40 cycles; end.

[0172] 3、After the above PCR reaction, the detection results are shown in Table 17. Table 17 Detection results

[0173] 4、Result analysis: according to the CT value to select the approximate number of mutant copies, the approximate copy number is reflected by the CT value, and the mutation frequency range of the mutant model can be obtained by dividing the approximate copy number corresponding to the CT value of ESR1 wild type. Example 7 Simulated sample

[0174] DNA from the gene edited cell lines containing ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S and D538G mutations were extracted, and cfDNA from negative plasma was also extracted. After quantification by digital PCR, they were mixed at a mutation frequency of 1%, completely simulating the template extracted from 20 ng of samples, and then tested by digital PCR.

[0175] 1. The reaction system was prepared as shown in Table 18 according to the reaction system component table shown below. System 1 detects mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Among them, the upstream primer of the ESR1 wild sequence is represented as ESR1 wild sequence-F, and the downstream primer of the ESR1 wild sequence is represented as ESR1 wild sequence-R. Table 18 Reaction system component table

[0176] 2. The PCR amplification program is 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0177] 3. After the above PCR reaction, the detection results are shown in Table 19. Table 19 Detection results

[0178] 4. Result analysis: The 1% mutation frequency sample simulated by cell lines and negative plasma can be normally detected. According to the results of Example 6, it can be inferred that the simulated mutation frequency is in the range of 1% to 2%, which verifies the ability of the primer probe preparation system provided in the specification to estimate the mutation frequency. Example 8 Comparison of primer probe preparation system

[0179] In order to prove the effectiveness of the primer probe preparation system provided in the specification, a comparative experiment was carried out with the primer probe preparation system in the Roche patent. The negative plasma template (wild type template, 2 ml) and mutant plasmid (mutant template) were mixed at a ratio of 1:100 to simulate the template extracted from 20 ng of plasma sample (quantified by digital PCR), and the specific steps are as follows:

[0180] 1. Extraction of cfDNA and mutant plasmid

[0181] (1) Extract the plasma free DNA (cfDNA) using Roche cobas DNA sample preparation kit, and the specific operation steps can be referred to the instructions of the kit.

[0182] (2) Extraction of plasmid The plasmid extraction kit (D1100, Beijing Solabio Science and Technology Co., Ltd.) was used for extracting the plasmid, and the specific operation steps can be referred to the instruction manual of the kit.

[0183] 2. Concentration determination of cfDNA and mutant plasmid

[0184] The extracted cfDNA and plasmid were quantified using Qubit™ dsDNA HS Assay Kit (Q32854), and the specific experimental operation steps were referred to the instruction manual of the kit.

[0185] 3. Preparation of standard samples required for the experiment

[0186] (1) The plasma cfDNA was diluted to about 2.0 x 10 5 copies / μl.

[0187] (2) The mutant plasmid containing the inserted ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutant fragments was obtained after digestion of the plasmid. The copy number calculation formula of the mutant plasmid was 9.1 x 10 8 copies / μl x plasmid concentration (ng / μl) ÷ plasmid length (2800 bp); the obtained plasmid was diluted to 2.0 x 10 6 copies / μl, 2.0 x 10 5 copies / μl, 2.0 x 10 2 copies / μl, and 20 copies / μl.

[0188] 4. Comparison of the system prepared by the primer probe

[0189] (1) According to the following reaction system component table, as shown in Tables 20-21, the PCR reaction system of the present specification and the Roche PCR reaction system were prepared. Among them, system 1 detects the mutant sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutant sites of ESR1 gene L536H, L536P, L536R and D538G. Table 20 Reaction system component table Table 21 Component table of Roche PCR reaction system

[0190] (2) The PCR amplification program was 25℃, 10min; 95℃, 5min; (95℃, 30s; 60℃, 60s) 40 cycles; end.

[0191] (3) After the above PCR reaction, the detection results are shown in Table 22. Table 22 QPCR detection results

[0192] (4) Result analysis: when detecting common ESR1 gene mutations, the CT value detected by the primer probe preparation system provided in the specification is mostly lower than the CT value detected by the Roche primer probe preparation system, so the primer probe preparation system provided in the specification is slightly better than the Roche primer probe preparation system. In addition, the mutation frequency of the sample can be roughly estimated according to the data in Example 6. Example 9 Comparison of primer probe preparation system with and without blocker probe

[0193] In this example, the detection results of the primer probe preparation system with and without blocker probe are compared. The negative plasma template (2 ml) and mutant plasmid are mixed at a ratio of 1:100 to simulate the template extracted from 20 ng of plasma sample; the negative plasma template (4 ml) and mutant plasmid are mixed at a ratio of 1:100 to simulate the template extracted from 40 ng of plasma sample.

[0194] 1. The reaction system was prepared according to the following reaction system component table, as shown in Table 23. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Table 23 Reaction system component table

[0195] 2. The PCR amplification program is 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0196] 3. After the above PCR reaction, the detection results are shown in Table 24. Table 24 Detection results

[0197] 4. Result analysis: after adding the blocker probe, the blocker probe in the system binds to the wild type template, so that the primer cannot bind to the wild type template to produce CT value. In the system with the addition of the blocker probe, the binding of the primer to the wild type template is reduced, resulting in a smaller CT value. Therefore, the addition of the blocker probe in the primer probe preparation system can improve the specificity of the primer, and thus more accurately estimate the mutation frequency range of the sample according to the data in Example 6. Example 10 Upgrade of blocker probe verification

[0198] In this example, the blocker probe is upgraded.

[0199] 1、Firstly, the mutant templates and wild type templates are quantified as 1000 copies / μl by digital PCR, and then the influence of adding different blocker probes (including upgraded blocker probes and ordinary blocker probes) on sample CT value detection is explored by digital PCR test.

[0200] 2、The reaction system is prepared according to the following reaction system component table, as shown in Table 25. Among them, system 1 detects the mutation sites of ESR1 gene Y537C, Y537N, Y537S and E380Q, and system 2 detects the mutation sites of ESR1 gene L536H, L536P, L536R and D538G. Table 25 Reaction system component table

[0201] 3、The PCR amplification procedure is 25℃, 10min; 95℃, 5min; (95℃, 30s; 60℃, 60s) 40 cycles; end.

[0202] 4、After the above-mentioned PCR reaction, the detection results are shown in Table 26. Table 26 Detection results

[0203] 5、Result analysis: under the condition of the same template input amount, the CT value detected by the system added with the upgraded blocker probe is slightly smaller than the CT value detected by the system added with the ordinary blocker probe, indicating that the upgraded blocker probe will not affect the combination of the primer and the template.

[0204] The foregoing description has been set forth to illustrate the basic concepts of the present specification. Obviously, the above detailed disclosure is only an example for those skilled in the art, and does not constitute a limitation on the present specification. Although the present specification does not explicitly state it, those skilled in the art can make various modifications, improvements and corrections to the present specification. Such modifications, improvements and corrections are suggested in the present specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of the present specification.

[0205] Meanwhile, specific words are used in the present specification to describe the embodiments of the present specification. As "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure or characteristic related to at least one embodiment of the present specification. Therefore, it should be emphasized and noted that the "an embodiment" or "one embodiment" or "one alternative embodiment" mentioned in different positions in the present specification does not necessarily mean the same embodiment. In addition, some features, structures or characteristics in one or more embodiments of the present specification can be properly combined.

[0206] For simplicity and to facilitate understanding of one or more embodiments, a description of an embodiment sometimes refers to multiple features coexisting in a single embodiment or description of an embodiment. Such a description, however, does not imply that the described features are necessarily coexisting in a single embodiment. Rather, such a description is used to facilitate understanding of one or more embodiments.

[0207] Some embodiments use numerical values to describe components, quantities of attributes. It should be understood that such numerical values used in the description of embodiments are in some examples modified by the adjectives "about," "approximately," or "substantially." Unless otherwise stated, "about," "approximately," or "substantially" indicate that the described value allows for a variation of ±20%. Accordingly, numerical parameters such as those outlined in the specification and claims are approximations, and can vary depending upon the desired properties sought to be obtained by the particular embodiments. In some embodiments, numerical parameters are determined by the limitations inherent in the various components used to practice the embodiments. While numerical ranges and parameters setting forth the broadest scope of embodiments herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, can contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0208] Each patent, patent application, patent publication, and other material, such as articles, books, specifications, publications, documents, and the like, referenced herein is hereby incorporated by reference in its entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented. Document(s) incorporated by reference to the extent possible are for the purpose of explaining and / or quantifying the embodiments disclosed herein and / or defining the terms used therein. To the extent there is a conflict between the description, definitions, and / or terms in the present document and that of the incorporated material, the present document controls. In addition, if a description, definition, and / or a term in the present document is more

[0209] Finally, it should be understood that the embodiments described herein are merely exemplary of the principles of the embodiments described herein. Other variations having essentially the same structure and function but different values for components and / or different arrangements of components can also be utilized. Accordingly, the embodiments described herein are not to be understood as limited to the embodiments described herein.

Claims

1. A primer probe set for detecting ESR1 gene mutation based on cfDNA, the primer probe set comprising a primer probe set in a first container and a primer probe set in a second container; wherein the primer probe set in the first container comprises a primer pair specific to E380Q of ESR1 gene; the primer probe set in the second container comprises a primer pair specific to L536H of ESR1 gene; the primer probe set in the first container further comprises a first specific probe, a second specific probe and a first blocking probe, and the primer probe set in the second container further comprises a third specific probe and a second blocking probe. 2.The primer probe set of claim 1, wherein sequences of the primer pair specific to E380Q of ESR1 gene are shown in SEQ ID NO: 76 and SEQ ID NO: 83 respectively. the primer probe set in the first container further comprises 3. The primer probe set of claim 1 or 2, wherein, a primer pair specific to Y537C of ESR1 gene; a primer pair specific to Y537N of ESR1 gene; and a primer pair specific to Y537S of ESR1 gene. 4.The primer probe set of claim 3, wherein, sequences of the primer pair specific to Y537C of ESR1 gene are shown in SEQ ID NO: 66 and SEQ ID NO: 82 respectively; sequences of the primer pair specific to Y537N of ESR1 gene are shown in SEQ ID NO: 34 and SEQ ID NO: 82 respectively; and sequences of the primer pair specific to Y537S of ESR1 gene are shown in SEQ ID NO: 23 and SEQ ID NO: 82 respectively. 5.The primer probe set of claim 1, wherein sequences of the primer pair specific to L536H of ESR1 gene are shown in SEQ ID NO: 45 and SEQ ID NO:

82. the primer probe set in the second container further comprises 6. The primer probe set of any one of claims 1-5, wherein, a primer pair specific to L536R of ESR1 gene; a primer pair specific to L536P of ESR1 gene; and a primer pair specific to D538G of ESR1 gene. 7.The primer probe set of claim 6, wherein, sequences of the primer pair specific to L536R of ESR1 gene are shown in SEQ ID NO: 57 and SEQ ID NO: 82 respectively; sequences of the primer pair specific to L536P of ESR1 gene are shown in SEQ ID NO: 19 and SEQ ID NO: 82 respectively; and sequences of the primer pair specific to D538G of ESR1 gene are shown in SEQ ID NO: 10 and SEQ ID NO: 82 respectively. the first specific probe has a fluorescent group and a fluorescent quencher group at two ends respectively, and the first specific probe can specifically bind to a sequence of E380Q of ESR1 gene.

8. The primer probe set of any one of claims 1-7, wherein, ​ 9. The primer probe set of claims 1-7, wherein, The second specific probe and the third specific probe are respectively provided with a fluorescent group and a fluorescent quenching group at two ends, and the second specific probe and the third specific probe can specifically bind to the sequences of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G.

10. The primer probe set of claim 1, wherein, The first specific probe is shown as SEQ ID NO: 86, and the second specific probe and the third specific probe have the same sequence shown as SEQ ID NO:

85.

11. The primer probe set of claim 1, wherein, The first blocking probe and the second blocking probe have the same sequence shown as SEQ ID NO:

88.

12. The primer probe set of claim 1, wherein, The blocking probe is used for specifically binding to a non-mutated template in a sample, so as to reduce the probability of primer binding to the non-mutated template.

13. The primer probe set of claim 1, wherein, The primer probe set in the first container and the primer probe set in the second container further comprise a reference probe and a reference primer pair, the sequence of the reference probe is shown as SEQ ID NO: 87, and the sequences of the reference primer pair are shown as SEQ ID NO: 81 and SEQ ID NO:

84.

14. The primer probe set of claim 1, wherein, The primer probe set in the first container and the primer probe set in the second container further comprise a probe of a wild sequence of ESR1 shown as SEQ ID NO: 91 and a primer pair of the wild sequence of ESR1 shown as SEQ ID NO: 92 and SEQ ID NO: 93, and the probe and the primer pair of the wild sequence are used for estimating the mutation frequency range of ESR1 in the sample.

15. The primer probe set of claim 1, wherein, The loading amount of the cfDNA sample is 20 ng.

16. The primer probe set of claim 1, wherein, The concentration range of the primer pair is 400-800 nM, and preferably 600 nM.

17. The primer probe set of claim 1, wherein, The concentration range of the specific probe is 200-300 nM, and preferably 300 nM, and the concentration range of the blocking probe is 100-300 nM, and preferably 100 nM.

18. A reagent for detecting an ESR1 gene mutation based on cfDNA, characterized by, The kit comprises the primer set according to any one of claims 1-17.

19. Use of detecting ESR1 gene mutation based on cfDNA for estimating the frequency of ESR1 gene mutation in a sample, characterized in that, The kit comprises the primer set according to any one of claims 1-17.

20. A kit for detecting ESR1 gene mutation based on cfDNA, characterized in that, The kit comprises a first container and a second container; The first container comprises a primer pair specific to ESR1 gene E380Q, the sequences of which are shown as SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequences of which are shown as SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequences of which are shown as SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene Y537S, the sequences of which are shown as SEQ ID NO: 23 and SEQ ID NO:

82. the second container comprises a primer pair specific to ESR1 gene L536H, the sequence of which is shown as SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which is shown as SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which is shown as SEQ ID NO: 19 and SEQ ID NO: 82, a primer pair specific to ESR1 gene D538G, the sequence of which is shown as SEQ ID NO: 10 and SEQ ID NO: 82; the first container further comprises a first specific probe capable of specifically binding to the sequence of ESR1 gene E380Q, a second specific probe capable of specifically binding to the sequence of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; the second container further comprises a third specific probe capable of specifically binding to the sequence of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; and the first container and the second container further respectively comprise an internal reference probe and an internal reference primer pair.

21. The kit of claim 20, wherein, the sequence of the first specific probe is shown as SEQ ID NO: 86; the second specific probe and the third specific probe have the same sequence, which is shown as SEQ ID NO: 85; and the sequence of the internal reference probe is shown as SEQ ID NO: 87, and the sequence of the internal reference primer pair is shown as SEQ ID NO: 81 and SEQ ID NO:

84.

22. The kit of claim 20, wherein the first container and the second container further respectively comprise a blocking probe with the sequence shown as SEQ ID NO: 88, which is used to specifically bind to non-mutated templates in the sample, thereby reducing the probability of primer binding to the non-mutated templates.

23. The kit of claim 20, wherein the first container and the second container further comprise a probe with the sequence of wild-type ESR1 shown as SEQ ID NO: 91 and a primer pair with the sequence of wild-type ESR1 shown as SEQ ID NO: 92 and SEQ ID NO: 93, which are used to estimate the mutation frequency range of ESR1 in the sample.

24. The kit of claim 20, wherein, The amount of cfDNA sample loaded for detection using the kit is 20 ng.

25. The kit of claim 20, wherein, The concentration of the primer pair is in the range of 400-800 nM, preferably 600 nM.

26. The kit of claim 20, wherein, The concentration of the specific probe is in the range of 200-300 nM, preferably 300 nM, and the concentration of the blocking probe is in the range of 100-300 nM, preferably 100 nM.

27. A method for detecting ESR1 gene mutation based on cfDNA, the method comprising: (1) obtaining a biological sample of an individual and extracting a cfDNA sample therefrom; and (2) taking two equal amounts of cfDNA sample from the cfDNA sample, respectively adding into a first container and a second container for PCR amplification, detecting whether the ESR1 gene in the biological sample is mutated; The first container comprises a primer pair specific to ESR1 gene E380Q, the sequence of which has at least 90% similarity with SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific to ESR1 gene Y537C, the sequence of which has at least 90% similarity with SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific to ESR1 gene Y537N, the sequence of which has at least 90% similarity with SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene Y537S, the sequence of which has at least 90% similarity with SEQ ID NO: 23 and SEQ ID NO:

82. The second container comprises a primer pair specific to ESR1 gene L536H, the sequence of which has at least 90% similarity with SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536R, the sequence of which has at least 90% similarity with SEQ ID NO: 57 and SEQ ID NO: 82, a primer pair specific to ESR1 gene L536P, the sequence of which has at least 90% similarity with SEQ ID NO: 19 and SEQ ID NO: 82, and a primer pair specific to ESR1 gene D538G, the sequence of which has at least 90% similarity with SEQ ID NO: 10 and SEQ ID NO:

82.

28. The method of claim 27, wherein, The first container further comprises a first specific probe capable of specifically binding to the sequence of ESR1 gene E380Q, and a second specific probe capable of specifically binding to the sequences of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; The second container further comprises a third specific probe capable of specifically binding to the sequences of ESR1 gene Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G; The sequence of the first specific probe has at least 90% similarity with SEQ ID NO: 86; and The second specific probe and the third specific probe have the same sequence, and the sequence of the second specific probe and the third specific probe has at least 90% similarity with SEQ ID NO:

85.

29. The kit of claim 27, wherein, The first container and the second container further comprise an internal reference probe and an internal reference primer pair, respectively, the sequence of the internal reference probe having at least 90% similarity with SEQ ID NO: 87, the sequence of the internal reference primer pair having at least 90% similarity with SEQ ID NO: 81 and SEQ ID NO:

84.

30. The method of claim 27, wherein, The first container and the second container further comprise a blocking probe having at least 90% similarity with SEQ ID NO: 88, respectively, the blocking probe being used to specifically bind to non-mutated templates in the sample, reducing the probability of primer binding to the non-mutated templates.

31. The method of claim 27, wherein, The first container and the second container further comprise a probe of wild sequence of ESR1 having at least 90% similarity with SEQ ID NO: 91 and a primer pair of wild sequence of ESR1 having at least 90% similarity with SEQ ID NO: 92 and SEQ ID NO: 93, respectively, the probe and the primer pair of wild sequence being used to estimate the mutation frequency range of ESR1 in the sample.

32. The method of claim 27, wherein, The amount of the cfDNA sample is 20 ng.

33. The method of claim 27, wherein, The concentration of the primer pair ranges from 400-800 nM, preferably 600 nM.

34. The method of claim 27, wherein, The concentration of the specific probe ranges from 200-300 nM, preferably 300 nM, and the concentration of the blocking probe ranges from 100-300 nM, preferably 100 nM.

35. A method for determining whether two or more ESR1 mutations are present in a sample from an individual, comprising, The method comprises: (1) obtaining a cfDNA sample from the individual; and (2) performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P and D538G.

36. The method of claim 35, wherein, The individual is a breast cancer patient or a breast cancer patient who is resistant to aromatase inhibitor treatment.

37. The method of claim 35, wherein, The method comprises performing quantitative PCR in two containers to determine whether four or more ESR1 mutations are present.

38. The method according to any one of claims 35-37, characterized by, The method further comprises providing treatment to the individual when the presence of ESR1 mutations is determined.

39. The method according to any one of claim 35, wherein, The performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample comprises: (1) determining whether the cfDNA sample comprises ESR1 gene E380Q, Y537C, Y537N and Y537S in a first container; and (2) determining whether the cfDNA sample comprises ESR1 gene L536H, L536R, L536P and D538G in a second container.

40. A method for treating individuals with breast cancer or those resistant to aromatase inhibitor therapy, characterized in that, The method comprises: (1) obtaining a cfDNA sample from the individual; and (2) performing quantitative PCR in two containers to determine whether four or more ESR1 mutations are present. (2) performing PCR in two containers to determine whether two or more ESR1 mutations are present in the cfDNA sample, the two or more ESR1 mutations comprising ESR1 gene E380Q, Y537C, Y537N, Y537S, L536H, L536R, L536P, and D538G; and (3) providing a treatment to the individual if the presence of an ESR1 mutation is determined.

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