A method and kit for detecting ESR1 gene mutations based on cfDNA

Abstract

The embodiments of the present specification provide a method and kit for detecting ESR1 gene mutations based on cfDNA, the method comprising: obtaining a biological sample from a subject and extracting a cfDNA sample therefrom; (2) taking out two equal amounts of cfDNA samples from the cfDNA sample, adding them to a first container and a second container respectively for PCR amplification to detect whether the ESR1 gene in the biological sample has mutated; wherein the first container includes a primer pair specific to E380Q of the ESR1 gene, a primer pair specific to Y537C of the ESR1 gene, a primer pair specific to Y537N of the ESR1 gene, and a primer pair specific to Y537S of the ESR1 gene; and the second container includes a primer pair specific to L536H of the ESR1 gene, a primer pair specific to L536R of the ESR1 gene, a primer pair specific to L536P of the ESR1 gene, and a primer pair specific to D538G of the ESR1 gene.

Description

Technical Field

[0001] This specification relates to the field of biomedicine, and in particular to a method and kit for detecting ESR1 gene mutations based on cfDNA. Background Art

[0002] The ESR1 (Estrogen Receptor 1) gene encodes the human estrogen receptor alpha (ESRα) protein. Upon binding to estrogen, this protein activates a series of intracellular responses, 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 include L536H, Y537S, Y537C, Y537N, and D538G. These mutations alter the conformation of ESRα, enabling it to remain persistently activated even without estrogen binding.

[0003] 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. While new therapies for breast cancer are constantly being developed and improved, innate and acquired resistance to these drugs remains a major challenge. The tumor microenvironment is considered a major factor in conferring innate resistance to cancer therapy, and a significant proportion of patients develop resistance when treated with 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 demonstrated that activating mutations in the estrogen receptor (ESR1) gene play a significant role in driving resistance. These mutations confer endoplasmic reticulum transcriptional activity independent of estrogen ligands and are considered resistance mutations. Due to tumor heterogeneity, secondary resistance can occur. Cancer biomarkers vary across disease types and stages of progression, complicating early cancer detection and identification. “Liquid biopsies” targeting circulating tumor DNA (ctDNA) within cell-free DNA (cfDNA) are gaining prominence 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). In vitro and preclinical data suggest that ESR1 mutations confer complete resistance to AIs and partial resistance to ER agonists and antagonists. Detection of activating ESR1 mutations may be useful in guiding clinicians toward both endocrine and non-endocrine therapies. cfDNA fragments are relatively small, with a peak size of approximately 180 bp. The percentage of tumor-derived ctDNA in total cfDNA is individually variable and often too low to be detected.

[0004] Therefore, it is desirable to provide a cfDNA-based ESR1 mutation detection method with high sensitivity. Summary of the Invention

[0005] One or more embodiments of the present specification provide a method for detecting ESR1 gene mutation based on cfDNA, the method comprising: (1) obtaining a biological sample from a subject and extracting a cfDNA sample therefrom; (2) taking out two equal amounts of cfDNA samples from the cfDNA sample, adding the samples to a first container and a second container respectively for PCR amplification to detect whether the ESR1 gene in the biological sample has a mutation; wherein the first container comprises a primer pair specific for E380Q of the ESR1 gene, whose sequence is shown in SEQ ID NO:76 and SEQ ID NO:83, a primer pair specific for Y537C of the ESR1 gene, whose sequence is shown in SEQ ID NO:66 and SEQ ID NO:82, a primer pair specific for Y537N of the ESR1 gene, whose sequence is shown in SEQ ID NO:34 and SEQ ID NO:82, and a primer pair specific for Y537S of the ESR1 gene, whose sequence is shown in SEQ ID NO:23 and SEQ ID NO:82; the second container comprises a primer pair specific for L536H of the ESR1 gene, whose sequence is shown in SEQ ID NO: NO:45 and SEQ ID NO:82, and the primer pair specific for ESR1 gene L536R, whose sequence is shown in SEQ ID NO:57 and SEQ ID NO:82; the primer pair specific for ESR1 gene L536P, whose sequence is shown in SEQ ID NO:19 and SEQ ID NO:82; and the primer pair specific for ESR1 gene D538G, whose sequence is shown in SEQ ID NO:10 and SEQ ID NO:82.

[0006] In some embodiments, the first container and the second container further include a specific probe that can specifically bind to different sequences of the ESR1 gene, an internal reference probe, and an internal reference primer, the sequences of the specific probes are shown in SEQ ID NO:85 and SEQ ID NO:86, the ends of the specific probes are respectively provided with a fluorescent group and a fluorescence quenching group, the sequence of the internal reference probe is shown in SEQ ID NO:87, and the sequences of the internal reference primers are shown in SEQ ID NO:81 and SEQ ID NO:84.

[0007] In some embodiments, the first container and the second container further include a blocking probe having a sequence as shown in SEQ ID NO: 88, and the blocking probe is used to specifically bind to the non-mutated template in the sample to reduce the probability of the primer binding to the non-mutated template.

[0008] In some embodiments, the first container and the second container further include a probe of the wild sequence of ESR1 as shown in SEQ ID NO:91 and primers of the wild sequence of ESR1 as shown in SEQ ID NO:92 and SEQ ID NO:93, and the wild sequence probe and primers are used to estimate the mutation frequency range of ESR1 in the sample.

[0009] In some embodiments, the loading amount of the cfDNA sample is 20 ng.

[0010] In some embodiments, the optimal concentration of the primer pair is 600 nM.

[0011] In some embodiments, the optimal concentration of the specific probe is 300 nM, and the optimal concentration of the blocking probe is 100 nM.

[0012] One or more embodiments of the present specification further provide a kit for detecting ESR1 gene mutations based on cfDNA, the kit comprising a first container and a second container; the first container comprising a primer pair specific for ESR1 gene E380Q, whose sequences are shown in SEQ ID NO:76 and SEQ ID NO:83, a primer pair specific for ESR1 gene Y537C, whose sequences are shown in SEQ ID NO:66 and SEQ ID NO:82, a primer pair specific for ESR1 gene Y537N, whose sequences are shown in SEQ ID NO:34 and SEQ ID NO:82, and a primer pair specific for ESR1 gene Y537S, whose sequences are shown in SEQ ID NO:23 and SEQ ID NO:82; the second container comprising a primer pair specific for ESR1 gene L536H, whose sequences are shown in SEQ ID NO:45 and SEQ ID NO:82, and a primer pair specific for ESR1 gene L536R, whose sequences are shown in SEQ ID NO:57 and SEQ ID NO:83. NO:82, a primer pair specific for ESR1 gene L536P, whose sequences are shown in SEQ ID NO:19 and SEQ ID NO:82, and a primer pair specific for ESR1 gene D538G, whose sequences are shown in SEQ ID NO:10 and SEQ ID NO:82; the first container and the second container also include a specific probe that can specifically bind to different sequences of the ESR1 gene, an internal reference probe and an internal reference primer, the sequences of the specific probes are shown in SEQ ID NO:85 and SEQ ID NO:86, and the two ends of the specific probes respectively have a fluorescent group and a fluorescence quenching group, the sequence of the internal reference probe is shown in SEQ ID NO:87, and the sequence of the internal reference primers are shown in SEQ ID NO:81 and SEQ ID NO:84.

[0013] In some embodiments, the first container and the second container further include a blocking probe having a sequence as shown in SEQ ID NO: 88, and the blocking probe is used to specifically bind to the non-mutated template in the sample to reduce the probability of the primer binding to the non-mutated template.

[0014] In some embodiments, the first container and the second container further include a probe of the wild sequence of ESR1 as shown in SEQ ID NO:91 and primers of the wild sequence of ESR1 as shown in SEQ ID NO:92 and SEQ ID NO:93, and the wild sequence probe and primers are used to estimate the mutation frequency range of ESR1 in the sample.

[0015] In some embodiments, the starting amount of cfDNA sample detected using the kit is 20 ng.

[0016] In some embodiments, the concentration of the primer pair is 600 nM, the concentration of the specific probe is 300 nM, and the concentration of the blocking probe is 100 nM. DETAILED DESCRIPTION

[0017] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0018] the term

[0019] The terms "about" and "around" may describe a range of values ​​within a certain value, such as plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value, etc. For example, the term "about 10 mL" may include 9 mL to 11 mL.

[0020] As used herein, the terms "estrogen receptor," "ER," and "ESR1" are used interchangeably herein unless otherwise indicated. ESR1 may also be used to refer to the gene encoding the ER protein.

[0021] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides), and include naturally occurring (adenosine, guanidine, cytosine, uracil, and thymidine), non-naturally occurring, and modified nucleic acids. The term is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded and typically contain 5'-3' phosphodiester bonds, although in some cases, nucleotide analogs may have other bonds. The monomers are generally referred to as nucleotides.

[0022] The term "sample" or "biological sample" refers to any composition containing or assumed to contain nucleic acid, including tissue samples, liquid samples such as isolated cells, tissues or blood, etc. In some embodiments, the sample can be a plasma sample.

[0023] As used herein, the term "primer" refers to a short nucleic acid (oligonucleotide) that serves as a starting point for the synthesis of a polynucleotide chain by a nucleic acid polymerase under suitable conditions. A primer typically includes a region of at least one target hybridization that is at least substantially complementary to the target sequence (e.g., having 0, 1, 2, or 3 mismatches). 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 is directed in opposite directions relative to the target sequence and produces an amplified product under amplification conditions.

[0024] As used herein, the term "probe" refers to any molecule that is capable of selectively binding to a particular 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 is terminated with a fluorescent group and a fluorescence quenching group, respectively.

[0025] As used herein, the term "container" refers to a container that can hold reagents or assays. If the container is in a kit and holds reagents or is used for an amplification reaction, it can be closed or sealed to prevent contamination or evaporation. If the container is used for an assay, it can be open or accessible.

[0026] One aspect of the present disclosure provides a method for detecting ESR1 gene mutations based on cfDNA. The method comprises: (1) obtaining a biological sample from a subject and extracting a cfDNA sample from the sample.

[0027] In some embodiments, the subject can be a mammal. In some embodiments, the subject is a human.

[0028] In some embodiments, the biological sample is obtained in a non-invasive manner, for example, urine, skin, swab, saliva, blood, etc. In some embodiments, the biological sample is preferably a blood sample.

[0029] Methods for isolating DNA from biological samples are known, for example, using Roche DNA sample preparation kit. For specific operation steps, please refer to the kit's instructions.

[0030] (2) Two equal amounts of cfDNA samples are taken from the cfDNA sample, and are added to a first container and a second container respectively for PCR amplification to detect whether the ESR1 gene in the biological sample is mutated. The first container includes a primer pair specific for ESR1 gene E380Q, whose sequences are shown in SEQ ID NO: 76 and SEQ ID NO: 83, a primer pair specific for ESR1 gene Y537C, whose sequences are shown in SEQ ID NO: 66 and SEQ ID NO: 82, a primer pair specific for ESR1 gene Y537N, whose sequences are shown in SEQ ID NO: 34 and SEQ ID NO: 82, and a primer pair specific for ESR1 gene Y537S, whose sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 82; the second container includes a primer pair specific for ESR1 gene L536H, whose sequences are shown in SEQ ID NO: 45 and SEQ ID NO: 82, a primer pair specific for ESR1 gene L536R, whose sequences are shown in SEQ ID NO: 57 and SEQ ID NO: 82, and a primer pair specific for ESR1 gene L536P, whose sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 82. The primer pair shown in NO:82 and specific to ESR1 gene D538G has sequences shown in SEQ ID NO:10 and SEQ ID NO:82.

[0031] In some embodiments, the primer pair specific for ESR1 gene E380Q may further include: a downstream primer as shown in SEQ ID NO: 83 and any one or more upstream primers as shown in SEQ ID NO: 71-80.

[0032] In some embodiments, the primer pair specific for ESR1 gene Y537C may further include: a downstream primer as shown in SEQ ID NO: 82 and an upstream primer as shown in any one or more of SEQ ID NOs: 61-70.

[0033] In some embodiments, the primer pair specific for ESR1 gene Y537N may further include: a downstream primer as shown in SEQ ID NO: 82 and an upstream primer as shown in any one or more of SEQ ID NOs: 31-40.

[0034] In some embodiments, the primer pair specific for ESR1 gene Y537S may further include: a downstream primer as shown in SEQ ID NO: 82 and an upstream primer as shown in any one or more of SEQ ID NOs: 21-30.

[0035] In some embodiments, the primer pair specific for ESR1 gene L536H may further include: a downstream primer as shown in SEQ ID NO: 82 and any one or more upstream primers as shown in SEQ ID NO: 41-50.

[0036] In some embodiments, the primer pair specific for ESR1 gene L536R may further include: a downstream primer as shown in SEQ ID NO: 82 and any one or more upstream primers as shown in SEQ ID NO: 51-60.

[0037] In some embodiments, the primer pair specific for ESR1 gene L536P may further include: a downstream primer as shown in SEQ ID NO: 82 and an upstream primer as shown in any one or more of SEQ ID NOs: 11-20.

[0038] In some embodiments, the primer pair specific for ESR1 gene D538G may further include: a downstream primer as shown in SEQ ID NO: 82 and an upstream primer as shown in any one or more of SEQ ID NOs: 1-10.

[0039] In some embodiments, a 25 μl PCR reaction system is as follows:

[0040]

[0041] The above PCR system is merely illustrative, and in actual applications, the volume of the mixture and the content of each component therein can be proportionally increased or decreased.

[0042] In some embodiments, the PCR amplification program is 25° C., 10 min; 95° C., 5 min; (95° C., 30 s; 60° C., 60 s) 40 cycles; end.

[0043] In one embodiment, the loading amount of the cfDNA sample is about 20 ng.

[0044] In some embodiments, ESR1 gene E380Q, Y537C, Y537N, and Y537S mutation sites can be detected in the first container; and ESR1 gene L536H, L536R, L536P, and D538G mutation sites can be detected in the second container.

[0045] In some embodiments, the concentration of the primer pairs can be 200 nM, 250 nM, 300 nM, 400 nM, 600 nM, 800 nM.

[0046] In some embodiments, the optimal concentration of the primer pairs is 600 nM.

[0047] In some embodiments, the first container and the second container further include a specific probe that can specifically bind to different sequences of the ESR1 gene, an internal reference probe, and an internal reference primer, the sequences of the specific probes are shown in SEQ ID NO:85 and SEQ ID NO:86, the ends of the specific probes are respectively provided with a fluorescent group and a fluorescence quenching group, the sequence of the internal reference probe is shown in SEQ ID NO:87, and the sequences of the internal reference primers are shown in SEQ ID NO:81 and SEQ ID NO:84.

[0048] In some embodiments, the specific probe is an MGB-specific probe, which is linked to an MGB modifying group.

[0049] In some embodiments, the specific probe has a fluorescent reporter group and a fluorescent quencher group at both ends, respectively. 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.

[0050] In some embodiments, the fluorescent reporter group is preferably FAM; the fluorescent quencher group is preferably BHQ2.

[0051] In some embodiments, the first container and the second container further include a blocking probe having a sequence as shown in SEQ ID NO: 88, and the blocking probe is used to specifically bind to the non-mutated template in the sample to reduce the probability of the primer binding to the non-mutated template.

[0052] Because plasma cfDNA is extremely low, and the total amount of cfDNA in many samples is also low, it may contain fewer mutant sequences. To prevent nonspecific binding of primers to wild-type templates and increase the detection of target fragments, a blocking probe is added to the original primer-probe system. In the unannealed state, the ends of the blocking probe complement each other to form a double strand. At the appropriate annealing temperature, the double strands open and can specifically bind to the wild-type template, thus reducing the possibility of primer binding to the wild-type template at the annealing temperature and improving the accuracy of the primer.

[0053] In some embodiments, the concentration of the specific probe can be 200 nM, 250 nM, and 300 nM.

[0054] In some embodiments, the concentration of the blocking probe can be 100 nM, 200 nM, or 300 nM.

[0055] In some embodiments, the optimal concentration of the specific probe is 300 nM and the optimal concentration of the blocking probe is 100 nM.

[0056] In some embodiments, the first container and the second container further include a probe of the wild sequence of ESR1 with a sequence such as SEQ ID NO:91 and a primer of the wild sequence of ESR1 with a sequence such as SEQ ID NO:92 and SEQ ID NO:93. The probe and primer of the wild sequence are used to estimate the mutation frequency range of ESR1 in the sample.

[0057] The mutation frequency can be determined by dividing the number of copies of the mutant template by the total number of copies of the mutant template and the wild-type template. In some embodiments, the probe of the ESR1 wild-type sequence is capable of binding to all mutant templates and the wild-type template. The copy number of each mutant template and all templates (each mutant template and the wild-type template) can be determined by the CT value. The mutation frequency range of the mutation model is obtained by dividing the copy number of mutant templates with different mutation frequencies by the total copy number of the mutant template and the wild-type template.

[0058] The method for detecting ESR1 gene mutations provided in the embodiments of this specification can detect eight mutation sites (E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G) of the ESR1 gene based on cfDNA using fluorescent quantitative PCR (qPCR) technology. The method for detecting ESR1 gene mutations based on cfDNA effectively improves its detection sensitivity by using rationally screened upstream primers, self-designed specific probes, and wild-type sequence probes, and can accurately detect low-copy mutations, meeting the needs of routine clinical tissue sample testing.

[0059] In some embodiments, the methods are performed using a kit as described below.

[0060] Another aspect of this specification provides a kit for detecting ESR1 gene mutations based on cfDNA. In some embodiments, the kit is a kit for qPCR technology. The ESR1 gene mutations may include E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, D538G, and other mutation sites in the ESR1 gene.

[0061] In some embodiments, the kit comprises: a first container and a second container; the first container comprises a primer pair specific for ESR1 gene E380Q, whose sequences are shown in SEQ ID NO:76 and SEQ ID NO:83, a primer pair specific for ESR1 gene Y537C, whose sequences are shown in SEQ ID NO:66 and SEQ ID NO:82, a primer pair specific for ESR1 gene Y537N, whose sequences are shown in SEQ ID NO:34 and SEQ ID NO:82, and a primer pair specific for ESR1 gene Y537S, whose sequences are shown in SEQ ID NO:23 and SEQ ID NO:82; the second container comprises a primer pair specific for ESR1 gene L536H, whose sequences are shown in SEQ ID NO:45 and SEQ ID NO:82, a primer pair specific for ESR1 gene L536R, whose sequences are shown in SEQ ID NO:57 and SEQ ID NO:82, and a primer pair specific for ESR1 gene L536P, whose sequences are shown in SEQ ID NO: NO:19 and SEQ ID NO:82, and a primer pair specific for ESR1 gene D538G, whose sequences are shown in SEQ ID NO:10 and SEQ ID NO:82; the first container and the second container also include a specific probe that can specifically bind to different sequences of the ESR1 gene, an internal reference probe, and an internal reference primer, the sequences of the specific probes are shown in SEQ ID NO:85 and SEQ ID NO:86, and the two ends of the specific probes respectively have a fluorescent group and a fluorescence quenching group, the sequence of the internal reference probe is shown in SEQ ID NO:87, and the sequences of the internal reference primers are shown in SEQ ID NO:81 and SEQ ID NO:84.

[0062] In some embodiments, the first container and the second container further include a blocking probe having a sequence as shown in SEQ ID NO: 88, and the blocking probe is used to specifically bind to the non-mutated template in the sample to reduce the probability of the primer binding to the non-mutated template.

[0063] In some embodiments, the first container and the second container further include a probe of the wild sequence of ESR1 as shown in SEQ ID NO:91 and primers of the wild sequence of ESR1 as shown in SEQ ID NO:92 and SEQ ID NO:93, and the wild sequence probe and primers are used to estimate the mutation frequency range of ESR1 in the sample.

[0064] In some embodiments, the starting amount of cfDNA sample detected by the kit is 20 ng.

[0065] In some embodiments, the concentration of the primer pair is 600 nM, the concentration of the specific probe is 300 nM, and the concentration of the blocking probe is 100 nM.

[0066] In some embodiments, the kit includes a positive control solution and / or a blank control solution, wherein the positive control solution includes a mutant plasmid containing any one of the ESR1 gene mutation fragments E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G.

[0067] In some embodiments, the positive control solution is a mixture containing eight mutant plasmids, each containing ESR1 gene mutations E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G, respectively. These plasmids can be any plasmid known to those skilled in the art, and the eight plasmids can be at the same concentration.

[0068] In some embodiments, the mutant plasmid concentration of the positive control solution can be 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 4 copies / μl or 2000 copies / μl.

[0069] In some embodiments, the blank control solution comprises Tris-HCl buffer.

[0070] In some embodiments, the kit further comprises a PCR buffer, an enzyme mix, and / or other elements suitable for reverse transcription and amplification, such as cofactors or aptamers.

[0071] In some embodiments, the kit further includes consumables, for example, plates or tubes for nucleic acid preparation, tubes for sample collection, etc.

[0072] Another aspect of the present specification provides a primer set for detecting ESR1 gene mutation, which includes any one or more of the following primer combinations: primer combination 1, primer combination 2;

[0073] Wherein, primer combination 1 includes: a downstream primer shown in SEQ ID NO: 83, a specific probe shown in SEQ ID NO: 86, and an upstream primer selected from any one or more of SEQ ID NOs: 21-30, SEQ ID NOs: 31-40, SEQ ID NOs: 61-70, and SEQ ID NOs: 71-80;

[0074] Primer combination 2 includes the downstream primer shown in SEQ ID NO: 82, the specific probe shown in SEQ ID NO: 85, and any one or more upstream primers selected from SEQ ID NOs: 1-10, SEQ ID NOs: 11-20, SEQ ID NOs: 41-50, and SEQ ID NOs: 51-60;

[0075] In some embodiments, primer combination 1 can detect E380Q, Y537C, Y537N, and Y537S mutation sites.

[0076] In some embodiments, primer combination 1-a of sequences SEQ ID NO:76, SEQ ID NO:83, and SEQ ID NO:86 can detect E380Q.

[0077] In some embodiments, primer combination 1-b of sequences SEQ ID NO: 66, SEQ ID NO: 82, and SEQ ID NO: 85 can detect Y537C.

[0078] In some embodiments, primer combination 1-c of sequences SEQ ID NO: 34, SEQ ID NO: 82, and SEQ ID NO: 85 can detect Y537N.

[0079] In some embodiments, primer combination 1-d of sequences SEQ ID NO: 23, SEQ ID NO: 82, and SEQ ID NO: 85 can detect Y537S.

[0080] In some embodiments, primer combination 1 can detect L536H, L536R, L536P, and D538G mutation sites.

[0081] In some embodiments, primer combination 2-a of sequences SEQ ID NO:45, SEQ ID NO:82, and SEQ ID NO:85 can detect L536H.

[0082] In some embodiments, primer combination 2-b of sequences SEQ ID NO: 57, SEQ ID NO: 82, and SEQ ID NO: 85 can detect L536R.

[0083] In some embodiments, primer combination 2-c of sequences SEQ ID NO: 19, SEQ ID NO: 82, and SEQ ID NO: 85 can detect L536P.

[0084] In some embodiments, primer combination 2-d of sequences SEQ ID NO: 10, SEQ ID NO: 82, and SEQ ID NO: 85 can detect D538G.

[0085] In some embodiments, the specific probes shown in SEQ ID NO: 85 and SEQ ID NO: 86 have a fluorescent group and a fluorescence quenching group at both ends, respectively.

[0086] The above primer set can accurately detect 8 mutations of the ESR1 gene with high sensitivity and good specificity. In a 20ng DNA sample, 0.1% of mutations are accurately detected, except for the L536H mutant template.

[0087] In some embodiments, the primer set further includes an internal reference probe and an internal reference primer, the sequence of the internal reference probe is shown in SEQ ID NO: 87, the sequence of the internal reference primer is shown in SEQ ID NO: 81 and SEQ ID NO: 84, and the internal reference probe and internal reference primer are used for sample quality assessment.

[0088] In some embodiments, the primer set further includes a blocking probe having a sequence as shown in SEQ ID NO: 88, and the blocking probe is used to specifically bind to the non-mutated template in the sample to reduce the probability of the primer binding to the non-mutated template.

[0089] In some embodiments, the primer set further includes a probe of the wild sequence of ESR1 with a sequence such as SEQ ID NO:91 and a primer of the wild sequence of ESR1 with a sequence such as SEQ ID NO:92 and SEQ ID NO:93, and the probe and primer of the wild sequence are used to estimate the mutation frequency range of ESR1 in the sample.

[0090] Another aspect of the present specification provides a reagent for detecting ESR1 gene mutation, which includes the primer set described above.

[0091] The embodiments of this specification provide a method, primer set, reagent and kit for detecting ESR1 gene mutations based on cfDNA, which may bring about beneficial effects including but not limited to: (1) the standards used are prepared by using negative normal cfDNA and enzyme-digested mutant plasmids inserted with ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G mutation fragments at different copy number ratios, which can restore the characteristics of the test sample to the greatest extent, lay the foundation for the optimization of the reaction system, and play a decisive role in the system optimization process; (2) by detecting the mutation frequency standard in digital PCR, the CT value generated by each primer binding to the template at different concentrations can be accurately detected, thereby obtaining accurate and reliable data statistics; by detecting the wild-type template by digital PCR, the background concentration of different wild-type templates in the detection system can be clearly determined. In this way, when testing samples, the interference of non-specific amplification peaks of wild sequences on the test results can be effectively avoided, making the results more accurate; by detecting low mutation frequency standards through digital PCR, the sensitivity of the detection system can be accurately determined; (3) by detecting high mutation frequency standards through fluorescent PCR, the concentration of each probe is optimized, and the appropriate primer concentration is selected according to the difference in fluorescence intensity after the reaction of primers with different concentrations of each mutation. This method has accurate results and low cost; (4) The specific probe provided in the embodiment of this specification is a self-designed MGB specific probe with a short probe sequence and good specificity. The specific probes are selected through multiple combination optimization and have high amplification efficiency and high sensitivity; (5) The probe of the ESR1 wild sequence provided in the embodiment of this specification can be combined with the mutant template and the wild-type template, and the mutation frequency range of the mutation model is obtained by dividing the copy number of the mutant template with different mutation 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 in the embodiment of this specification can provide a reference for estimating the mutation frequency of the sample; (6) The method for detecting ESR1 gene mutation based on cfDNA provided in the embodiment of this specification is fast, efficient, and low-cost, and can detect 8 mutation sites (E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G) of the ESR1 gene based on trace cfDNA in biological samples through fluorescent quantitative PCR (qPCR) technology, and can quickly and accurately monitor whether the ESR1 gene of tumor patients has mutated, and timely monitor the patient's new gene mutation, thereby providing a strong basis for the formulation and adjustment of clinical treatment plans.

[0092] Example

[0093] The experimental methods in Examples 1-10 below, unless otherwise specified, are conventional methods. The experimental materials used in Examples 1-10 below, unless otherwise specified, were purchased from conventional biochemical reagent companies. The quantitative experiments in Examples 1-10 below, unless otherwise specified, were performed in triplicate, and the results were averaged.

[0094] The instruments used in Examples 1-10 mainly include: a vortex shaker, a high-speed centrifuge, a digital PCR (Xinyi Biotechnology Co., Ltd.), an A300 PCR, a SLAN-96s real-time PCR (Shanghai Hongshi Medical Technology Co., Ltd.), a droplet preparation instrument (drop maker M1), a droplet sample detector (chip reader R1) (Xinyi Manufacturing Technology (Beijing) Co., Ltd.), a biological safety cabinet, a water bath, a pipette, RainDropSense (RainDance Technologies), etc.

[0095] Example 1 Primer screening

[0096] Digital PCR was used to quantitatively detect the wild-type template (wild plasmid) and mutant template (mutant plasmid) and to screen the primers.

[0097] 1. Prepare the plasmids required for the experiment

[0098] (1) Theoretically, the number of copies of a single gene per 1ng of human DNA is approximately 300 copies. Dilute the concentration of wild-type plasmid DNA to 2.0×10 5 About copies / μl.

[0099] (2) After enzyme digestion, a mutant plasmid was obtained in which ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutant fragments were inserted. The calculation formula for the mutant plasmid copy number was 9.1×10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800bp); the concentration of the obtained mutant plasmid was diluted step by step to 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 4 copies / μl and 2000copies / μl.

[0100] 2. Primer screening

[0101] The sequences of primers and probes for ESR1 gene mutation detection are shown in Table 1. The specific binding of primers and probes to mutant templates and wild-type templates was tested respectively to screen out the optimal primers and probes for each mutation site.

[0102] Table 1 Primer and probe sequences

[0103]

[0104]

[0105]

[0106] The concentrations of the primer probes were 600 nM and 300 nM, respectively, and the concentration of the blocking probe was 100 nM. Except for the internal reference probe, all the above-mentioned specific probes were MGB probes, and the 5' end of the probe was modified with a FAM fluorescent group.

[0107] (1) Prepare the reaction system according to the following reaction system component table, as shown in Table 2. The copy number of the mutant template is 1000 copies, and the copy number of the wild-type template is 8000 copies.

[0108] Table 2 Reaction system components

[0109]

[0110] (2) The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0111] (3) After the above PCR reaction, the test 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 primers using mutant templates; Table 5 shows the test results of primers using wild-type templates.

[0112] Table 3 Results of digital PCR quantification of different mutant plasmids

[0113]

[0114] Table 4 Test results of primers in mutant templates

[0115]

[0116]

[0117] Table 5 Test results of primers in wild-type template

[0118] Sample name Ct Sample name Ct Sample name Ct Sample name Ct A8F1 W 31.87 HF1 W 36.79 R1 W NoCt P1 W 33.96 A8F2 W 38.12 HF2 W 37.81 R2 W NoCt P2 W 32.76 A8F3 W 34.03 HF3 W NoCt R3 W NoCt P3 W NoCt A8F4 W NoCt HF4 W NoCt R4 W NoCt P4 W 25.43 A8F5 W 36.41 HF5 W NoCt R5W NoCt P5 W NoCt A8F6 W NoCt HF6 W 38.40 R6W NoCt P6 W 38.30 A8F7 W NoCt HF7 W NoCt R7W NoCt P7 W NoCt A8F8 W 38.39 HF8 W 37.33 8W 39.34 P8 W 35.65 A8F9 W 37.22 HF9 W 37.36 R9W NoCt P9 W NoCt A8F10W 38.32 HF10 W 35.35 R10 W 37.86 P10 W 34.49 CF1 W 32.18 NF1 W 37.15 S1 W NoCt 3801W 34.29 CF2 W 38.88 NF2 W 38.56 S2 W NoCt 3802W 33.41 CF3 W 35.05 NF3 W NoCt S3 W NoCt 3803W NoCt CF4 W NoCt NF4 W NoCt S4 W NoCt 3804W 26.44 CF5 W 38.23 NF5 W NoCt S5W NoCt 3805W NoCt CF6 W NoCt NF6 W NoCt S6 W NoCt 3806W NoCt CF7 W NoCt NF7 W NoCt S7 W NoCt 3807W NoCt CF8 W NoCt NF8 W NoCt S8W NoCt 3808W 38.50 CF9 W NoCt NF9 W NoCt S9W NoCt 3809W NoCt CF10W NoCt NF10 W 38.88 S10 W NoCt 38010W 37.93

[0119] (4) Result analysis: The optimal primers were selected based on the CT values. The primers were screened based on the criteria of producing the minimum CT value under the mutant template and no CT value under the wild-type template. The optimal primers ultimately determined were F536R-7, F536P-9, F536H-5, F538-10, F537C-6, F537N-4, F537S-3, and F380-6.

[0120] Example 2 Primer concentration and probe concentration screening

[0121] Digital PCR was used to quantitatively detect mutant templates and screen primer concentrations.

[0122] 1. Prepare the plasmids required for the experiment

[0123] After enzyme digestion of the plasmid, a mutant plasmid was obtained in which ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutation fragments were inserted. The copy number of the mutant plasmid was calculated as 9.1×10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800bp); dilute the obtained plasmid concentration stepwise to 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 4 copies / μl, 2000copies / μl.

[0124] 2. Primer and probe concentration screening

[0125] Based on the mutant template, the specific binding degree of the primers and specific probes to the mutant template was tested respectively, and the optimal primer concentration and probe concentration for each mutation site were screened out.

[0126] (1) Prepare the reaction system according to the following reaction system component table, as shown in Table 6. The copy number of the mutant template is 1000 copies.

[0127] Table 6 Reaction system components

[0128]

[0129] (2) The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0130] (3) When the probe concentration was set at 300 nm, the primer concentrations were tested at 800 nm, 600 nm, and 400 nm, respectively. The results are shown in Table 7. When the primer concentration was 600 nm, the probe concentrations were tested at 300 nm, 250 nm, and 200 nm, respectively. The detection results are shown in Table 8.

[0131] Table 7 Detection results of different primer concentrations

[0132] Sample name Average Ct Sample name Average Ct Sample name Average Ct Sample name Average Ct R800nM 29.21 P800nM 29.11 538800nM 31.57 H800nM 29.88 R600nM 28.82 P600nM 28.25 538600nM 30.17 H600nM 29.01 R400nM 28.91 P400nM 28.36 538400nM 30.56 H400nM 30.23 C800nM 29.40 N800nM 30.66 S800nM 31.11 380800nM 31.78 C600nM 28.03 N600nM 30.29 S600nM 30.26 380600nM 30.44 C400nM 28.33 N400nM 31.54 S400nM 30.77 380400nM 30.673

[0133] Table 8 Detection results of different probe concentrations

[0134] Sample name Ct Sample name Ct Sample name Ct Sample name Ct R300nM 28.76 P300nM 28.34 538300nM 30.23 H300nM 29.12 R250nM 28.87 P250nM 28.75 538250nM 30.45 H250nM 29.61 R200nM 28.98 P200nM 29.16 538200nM 30.76 H200nM 30.63 C300nM 28.06 N300nM 30.49 S300nM 30.56 380300nM 30.24 C600nM 28.45 N600nM 30.76 S600nM 30.86 380600nM 30.54 C400nM 28.33 N400nM 31.34 S400nM 30.67 380400nM 30.97

[0135] (4) Result analysis: The optimal primer and probe concentrations were selected based on the CT values, with the primers producing the minimum CT value under the mutant template as the screening criterion. The optimal primer concentration was ultimately 600 nM, and the optimal probe concentration was 300 nM.

[0136] Example 3 Blocker probe concentration screening

[0137] Digital PCR was used to quantitatively detect wild-type and mutant templates and screen the optimal concentration of the blocker probe.

[0138] 1. Prepare the plasmids required for the experiment

[0139] (1) Dilute the concentration of wild plasmid DNA to about 2.0×10 5 copies / μl.

[0140] (2) After enzyme digestion of the plasmid, a mutant plasmid was obtained in which ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutation fragments were inserted. The formula for calculating the copy number of the mutant plasmid was 9.1×10 8 × plasmid concentration (ng / μl) ÷ plasmid length (2800bp); dilute the obtained plasmid concentration stepwise to 2.0×10 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 4 copies / μl, 2000copies / μl.

[0141] 2. Screening of optimal blocker probe concentration

[0142] Based on the mutant template and the wild-type template, the specific binding degree of the blocker probe to the mutant and wild-type templates was tested to screen the optimal blocker probe concentration.

[0143] (1) Prepare the reaction system according to the following reaction system component table, as shown in Table 9. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G. The copy number of the mutant template is 1000 copies, and the copy number of the wild-type template is 18,000 copies.

[0144] Table 9 Reaction system components

[0145]

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

[0147] (3) When the copy number of the wild-type template was 18,000 copies, the blocker probe concentrations were tested at 100 nm, 200 nm, and 300 nm, respectively. The detection results are shown in Table 10. At a blocker probe concentration of 100 nm, the detection results of each mutant template (copy number of 1,000 copies) were tested, and the results are shown in Table 11.

[0148] Table 10 Detection results of different concentrations of blocker probes

[0149]

[0150] Table 11 Detection results of 100nm blocker probe

[0151]

[0152] (4) Result analysis: When the concentration of the blocker probe was 100 nM, the primers could block the amplification of 18,000 copies of the wild-type template, while the mutant templates could be detected normally.

[0153] Example 4 Simulation sample template

[0154] Each mutant template was mixed with a wild-type template at a mutation frequency of approximately 13%, simulating template extraction from a 20ng sample. Digital PCR was used to quantitatively detect the wild-type and mutant templates.

[0155] 1. Prepare the reaction system according to the following reaction system component table, as shown in Table 12. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0156] Table 12 Reaction system components

[0157]

[0158] 2. The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0159] 3. After the above PCR reaction, the test results are shown in Table 13.

[0160] Table 13 Test results

[0161]

[0162]

[0163] 4. Result Analysis: When simulating a 20ng sample input, all mutant templates were detected normally and did not react nonspecifically with the wild-type template. The primer and probe in the reaction system were highly specific.

[0164] Example 5 Simulating sample templates with different mutation frequencies

[0165] Each mutant template was mixed with a wild-type template at mutation frequencies of approximately 10%, 1%, and 0.1%, simulating the presence of different mutation frequencies in templates extracted from a 20ng sample. Digital PCR was used to quantitatively detect the wild-type and mutant templates.

[0166] 1. Prepare the reaction system according to the following reaction system component table, as shown in Table 14. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0167] Table 14 Reaction system components

[0168]

[0169] 2. The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0170] 3. After the above PCR reaction, the test results are shown in Table 15.

[0171] Table 15 Test results

[0172]

[0173]

[0174] 4. Result analysis: When simulating a 20 ng sample input, except for the L536H mutant template, all other mutant templates can be detected at a mutation frequency of 0.1%.

[0175] Example 6 Estimating the mutation frequency of a sample

[0176] To estimate the mutation frequency range of the sample, a probe was designed to detect the wild-type ESR1 sequence. Digital PCR was used to quantitatively detect mutant and wild-type templates, with copies of 1000, 100, and 10 copies of each mutant template being tested.

[0177] 1. Prepare the reaction system according to the following reaction system component table, as shown in Table 16. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0178] Table 16 Reaction system components

[0179]

[0180]

[0181] 2. The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0182] 3. After the above PCR reaction, the test results are shown in Table 17.

[0183] Table 17 Test results

[0184] Sample name copies Ct Sample name copies Ct Sample name copies Ct E380Q-1000 ～1000 29.32 E380Q-100 ～100 32.32 E380Q-10 ～10 35.57 Y537S-1000 ～1000 32.16 Y537S-100 ～100 35.46 Y537S-10 ～10 38.86 Y537N-1000 ～1000 31.36 Y537N-100 ～100 34.06 Y537N-10 ～10 37.56 Y537C-1000 ～1000 33.61 Y537C-100 ～100 37.81 Y537C-10 ～10 39.81 D538G-1000 ～1000 34.12 D538G-100 ～100 37.02 D538G-10 ～10 39.82 L536H-1000 ～1000 34.32 L536H-100 ～100 37.42 L536H-10 ～10 39.72 L536R-1000 ～1000 29.54 L536R-100 ～100 32.26 L536R-10 ～10 35.76 L536P-1000 ～1000 31.43 L536P-100 ～100 34.39 L536P-10 ～10 37.68 ESR1 Wild ～1000 26.13 ESR1 Wild ～10000 22.83 ESR1 Wild ～5000 24.58

[0185] 4. Result analysis: The approximate number of mutation copies was selected based on the CT value. The approximate copy number was reflected by the CT value. Dividing it by the approximate copy number corresponding to the wild-type CT value of ESR1, the mutation frequency range of the mutation model can be obtained.

[0186] Example 7 Simulation sample

[0187] DNA was extracted from gene-edited cell lines containing ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S and D538G mutations. At the same time, cfDNA was extracted from negative plasma. After digital PCR quantification, they were mixed according to the mutation frequency of 1% to completely simulate the template extracted from the 20ng sample, and then tested using digital PCR.

[0188] 1. Prepare the reaction system according to the reaction system component table shown below, as shown in Table 18. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0189] Table 18 Reaction system components

[0190]

[0191]

[0192] 2. The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0193] 3. After the above PCR reaction, the test results are shown in Table 19.

[0194] Table 19 Test results

[0195] Sample name Mutation frequency or copies Ct E380Q-1% 1％ 33.86 Y537S-1% 1％ 36.66 Y537N-1% 1％ 35.73 Y537C-1% 1％ 36.81 gDNA-20ng 20ng(~6600) 26.43 D538G-1% 1％ 36.42 L536H-1% 1％ 36.87 L536R-1% 1％ 33.23 L536P-1% 1％ 35.11 gDNA-20ng 20ng(~6600) 26.62 ESR1 24.67

[0196] 4. Analysis of Results: Samples with a 1% mutation frequency simulated using both cell lines and negative plasma were detected normally. Based on the results of Example 6, it can be inferred that the simulated mutation frequency is in the 1% to 2% range, confirming the ability of the primer-probe formulation system provided in this specification to estimate mutation frequency.

[0197] Example 8 Comparison of primer and probe preparation systems

[0198] To demonstrate the effectiveness of the primer-probe system provided in this specification, a comparative experiment was conducted with the primer-probe system described in the Roche patent. A negative plasma template (wild-type template, 2 ml) and a mutant plasmid (mutant template) were mixed at a 1:100 ratio to simulate template extraction from a 20 ng plasma sample (digital PCR quantification). The specific steps were as follows:

[0199] 1. cfDNA and mutant plasmid extraction

[0200] (1) Extraction of plasma free DNA (cfDNA) using Roche DNA sample preparation kit. For specific operation steps, please refer to the kit's instructions.

[0201] (2) Plasmid extraction was performed using a plasmid miniprep kit (D1100, Beijing Solebow Technology Co., Ltd.). Specific steps can be found in the kit's instructions.

[0202] 2. Determination of cfDNA and mutant plasmid concentrations

[0203] The extracted cfDNA and plasmid were quantified using the Qubit™ dsDNA HS Assay Kit (Q32854). The specific experimental steps were referred to the instructions of the kit.

[0204] 3. Prepare the standard products required for the experiment

[0205] (1) Plasma cfDNA was diluted step by step to about 2.0×10 5 copies / μl.

[0206] (2) After enzyme digestion of the plasmid, a mutant plasmid was obtained in which ESR1 gene E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G mutation fragments were inserted. The formula for calculating the copy number of the mutant plasmid was 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 and 20copies / μl.

[0207] 4. Comparison of primer and probe preparation systems

[0208] (1) Prepare the PCR reaction system described in this specification and the Roche PCR reaction system according to the following reaction system component table, as shown in Tables 20-21. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0209] Table 20 Reaction system components

[0210]

[0211] Table 21 Roche PCR reaction system components

[0212]

[0213]

[0214] (2) The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0215] (3) After the above PCR reaction, the detection results are shown in Table 22.

[0216] Table 22QPCR test results

[0217]

[0218] (4) Analysis of Results: When detecting common ESR1 gene mutations, the CT values ​​detected using the primer-probe formulation provided in this specification were all lower than those detected using the Roche primer-probe formulation. Therefore, the primer-probe formulation provided in this specification is slightly superior to the Roche primer-probe formulation. Furthermore, this specification can be combined with the data in Example 6 to roughly estimate the mutation frequency of the sample.

[0219] Comparison of Example 9 with and without blocker probe

[0220] This example compares the detection results when the primer-probe system is prepared with and without a blocker probe. A negative plasma template (2 ml) and a mutant plasmid were mixed at a 1:100 ratio to simulate template extraction from a 20 ng plasma sample. A negative plasma template (4 ml) and a mutant plasmid were mixed at a 1:100 ratio to simulate template extraction from a 40 ng plasma sample.

[0221] 1. Prepare the reaction system according to the following reaction system component table, as shown in Table 23. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0222] Table 23 Reaction system components

[0223]

[0224] 2. The PCR amplification program was 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0225] 3. After the above PCR reaction, the test results are shown in Table 24.

[0226] Table 24 Test results

[0227]

[0228] 4. Result analysis: After adding the blocker probe, the blocker probe in the system binds to the wild-type template, so that the primer does not bind to the wild-type template to produce a CT value. In the system with the blocker probe added, the binding of the primer to the wild-type template is reduced, resulting in a smaller CT value. Therefore, adding a blocker probe to the system of primer probe preparation can improve the specificity of the primer, and then the mutation frequency range of the sample can be more accurately calculated based on the data of Example 6.

[0229] Example 10 Upgrading blocker probe verification

[0230] This embodiment upgrades the blocker probe.

[0231] 1. First, digital PCR was used to quantify each mutant template and wild-type template at 1000 copies / μl. Then, digital PCR was used to test the effect of adding different blocker probes (including upgraded blocker probes and ordinary blocker probes) on the CT value detection of the sample.

[0232] 2. Prepare the reaction system according to the following reaction system component table, as shown in Table 25. System 1 detects the ESR1 gene mutation sites Y537C, Y537N, Y537S, and E380Q, and System 2 detects the ESR1 gene mutation sites L536H, L536P, L536R, and D538G.

[0233] Table 25 Reaction system components

[0234]

[0235] 3. The PCR amplification program is 25°C, 10 min; 95°C, 5 min; (95°C, 30 s; 60°C, 60 s) 40 cycles; end.

[0236] 4. After the above PCR reaction, the test results are shown in Table 26.

[0237] Table 26 Test results

[0238]

[0239]

[0240] 5. Result analysis: When the template input amount is the same, the CT value detected by the system with the upgraded blocker probe is slightly lower than that detected by the system with the ordinary blocker probe, indicating that the upgraded blocker probe does not affect the binding of primers to templates.

[0241] While the basic concepts have been described above, it will be apparent to those skilled in the art that the detailed disclosure is merely illustrative and does not limit this specification. Although not explicitly stated herein, various modifications, improvements, and revisions to this specification may be made by those skilled in the art. Such modifications, improvements, and revisions are suggested in this specification and remain within the spirit and scope of the exemplary embodiments of this specification.

[0242] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[0243] Similarly, it should be noted that, in order to simplify the description of this specification and facilitate understanding of one or more embodiments of the invention, the foregoing descriptions of the embodiments of this specification sometimes combine multiple features into a single embodiment or its description. However, this disclosure method does not mean that the subject matter of this specification requires more features than those recited in the claims. In fact, an embodiment may have fewer features than all of the features of a single embodiment disclosed above.

[0244] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values ​​are as accurate as possible within the feasible range.

[0245] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, and documents, cited in this specification is hereby incorporated by reference in its entirety. This includes application history documents that are inconsistent with or conflict with the content of this specification, as well as documents (currently or subsequently attached to this specification) that limit the broadest scope of the claims of this specification. It should be noted that if the descriptions, definitions, and / or terminology used in the accompanying materials are inconsistent or conflicting with the content of this specification, the descriptions, definitions, and / or terminology used in this specification will control.

[0246] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. A primer probe set for detecting ESR1 gene mutations based on cfDNA, in, The primer probe set includes 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 includes a primer pair for detecting an ESR1 gene E380Q mutation, a primer pair for detecting an ESR1 gene Y537C mutation, a primer pair for detecting an ESR1 gene Y537N mutation, and a primer pair for detecting an ESR1 gene Y537S mutation, the sequences of the primer pair for detecting an ESR1 gene E380Q mutation are shown in SEQ ID NO:76 and SEQ ID NO:83, the sequences of the primer pair for detecting an ESR1 gene Y537C mutation are shown in SEQ ID NO:66 and SEQ ID NO:82, the sequences of the primer pair for detecting an ESR1 gene Y537N mutation are shown in SEQ ID NO:34 and SEQ ID NO:82, and the sequences of the primer pair for detecting an ESR1 gene Y537S mutation are shown in SEQ ID NO:23 and SEQ ID NO:82; The primer probe set in the second container includes a primer pair for detecting the ESR1 gene L536H mutation, a primer pair for detecting the ESR1 gene L536R mutation, a primer pair for detecting the ESR1 gene L536P mutation, and a primer pair for detecting the ESR1 gene D538G mutation. The sequences of the primer pair for detecting the ESR1 gene L536H mutation are shown in SEQ ID NO:45 and SEQ ID NO:82, the sequences of the primer pair for detecting the ESR1 gene L536R mutation are shown in SEQ ID NO:57 and SEQ ID NO:82, the sequences of the primer pair for detecting the ESR1 gene L536P mutation are shown in SEQ ID NO:19 and SEQ ID NO:82, and the sequences of the primer pair for detecting the ESR1 gene D538G mutation are shown in SEQ ID NO:10 and SEQ ID NO:82, wherein: The primer probe set in the first container and the primer probe set in the second container also include specific probes shown as SEQ ID NO: 85 and SEQ ID NO: 86 and a blocking probe shown as SEQ ID NO: 88, and the two ends of the specific probes respectively have a fluorescent group and a fluorescence quenching group.

2. The primer probe set according to claim 1, wherein The primer probe set in the first container and the primer probe set in the second container also include 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.

3. The primer probe set according to claim 1, wherein The blocking probe is used to specifically bind to the non-mutation template in the sample, thereby reducing the probability of the primer binding to the non-mutation template.

4. The primer probe set according to claim 1, wherein The primer probe set in the first container and the primer probe set in the second container also include a probe of the wild sequence of ESR1 as shown in SEQ ID NO:91 and primers of the wild sequence of ESR1 as shown in SEQ ID NO:92 and SEQ ID NO:

93. The wild sequence probe and primers are used to estimate the mutation frequency range of ESR1 in the sample.

5. The primer probe set according to claim 1, wherein The sample loading amount of the cfDNA was 20 ng.

6. 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 for detecting the ESR1 gene E380Q mutation, a primer pair for detecting the ESR1 gene Y537C mutation, a primer pair for detecting the ESR1 gene Y537N mutation, and a primer pair for detecting the ESR1 gene Y537S mutation, wherein the sequences of the primer pair for detecting the ESR1 gene E380Q mutation are shown in SEQ ID NO:76 and SEQ ID NO:83, the sequences of the primer pair for detecting the ESR1 gene Y537C mutation are shown in SEQ ID NO:66 and SEQ ID NO:82, the sequences of the primer pair for detecting the ESR1 gene Y537N mutation are shown in SEQ ID NO:34 and SEQ ID NO:82, and the sequences of the primer pair for detecting the ESR1 gene Y537S mutation are shown in SEQ ID NO:23 and SEQ ID NO:82; The second container comprises a primer pair for detecting the ESR1 gene L536H mutation, a primer pair for detecting the ESR1 gene L536R mutation, a primer pair for detecting the ESR1 gene L536P mutation, and a primer pair for detecting the ESR1 gene D538G mutation, wherein the sequences of the primer pair for detecting the ESR1 gene L536H mutation are shown in SEQ ID NO:45 and SEQ ID NO:82, the sequences of the primer pair for detecting the ESR1 gene L536R mutation are shown in SEQ ID NO:57 and SEQ ID NO:82, the sequences of the primer pair for detecting the ESR1 gene L536P mutation are shown in SEQ ID NO:19 and SEQ ID NO:82, and the sequences of the primer pair for detecting the ESR1 gene D538G mutation are shown in SEQ ID NO:10 and SEQ ID NO:82; The first container and the second container further include a specific probe, an internal reference probe, and an internal reference primer. The sequences of the specific probes are shown in SEQ ID NO: 85 and SEQ ID NO:

86. The two ends of the specific probes are respectively provided with a fluorescent group and a fluorescence quenching group. The sequence of the internal reference probe is shown in SEQ ID NO:

87. The sequences of the internal reference primers are shown in SEQ ID NO: 81 and SEQ ID NO:

84. The first container and the second container also include a blocking probe as shown in SEQ ID NO:

88.

7. The kit according to claim 6, wherein The blocking probe is used to specifically bind to the non-mutation template in the sample, thereby reducing the probability of the primer binding to the non-mutation template.

8. The kit according to claim 6, wherein The first container and the second container also include a probe of the wild sequence of ESR1 as shown in SEQ ID NO: 91 and primers of the wild sequence of ESR1 as shown in SEQ ID NO: 92 and SEQ ID NO:

93. The wild sequence probe and primers are used to estimate the mutation frequency range of ESR1 in the sample.

9. The kit according to claim 6, wherein The starting amount of cfDNA sample detected using the kit was 20 ng.

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