Primer-probe set, kit and method for detecting ESR1 gene mutation

Abstract

Provided in the present invention are a primer-probe set, a kit, and a method for detecting ESR1 gene mutation. The primer-probe set comprises a primer set and a probe set for PCR amplification; the primer set comprises a wild-type primer set specific for wild-type ESR1, and an E380Q mutation primer set having the sequences shown in SEQ ID NO: 3-4.

Description

Primer and probe set, kit and method for detecting ESR1 gene mutations Cross-referencing

[0001] This application claims priority to Chinese patent application No. 202410954817.6, filed on July 16, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This manual relates to the field of ESR1 gene mutation detection technology, specifically to primer and probe sets, kits, and methods for detecting ESR1 gene mutations. Background Technology

[0003] The ESR1 (Estrogen Receptor 1) gene encodes the human estrogen receptor alpha (ESRα) protein. Upon binding to estrogen, ESRα activates a series of cell cycle responses, promoting cell growth and proliferation. Activating mutations in the ESR1 gene are closely related to the development and progression of breast cancer. The most common mutation types in the ESR1 gene are E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, and D538G. These mutations alter the conformation of ESRα, allowing it to remain persistently activated without binding to estrogen.

[0004] Breast cancer is one of the most common gynecological cancers. Over 70% of primary breast cancers are estrogen receptor (ER) positive, and many initially respond to endocrine therapy. Despite ongoing advancements in the development of new therapies for breast cancer, innate and acquired resistance to these drugs remains a challenge. The tumor microenvironment is considered a major factor conferring innate resistance to cancer treatment, while a significant proportion of patients develop resistance when receiving systemic anti-estrogens 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 identified as drivers of acquired resistance. Recent studies have highlighted the important role of activating mutations in the estrogen receptor (ESR1) gene in driving resistance. Key activating mutation hotspots identified include p.L536, p.Y537, and p.D538, which produce endoplasmic reticulum transcriptional activity independent of estrogen ligands and are considered resistance mutations. Secondary resistance also exists due to tumor heterogeneity. Cancer biomarkers vary across disease types and stages of disease progression, complicating early-stage cancer detection and identification. Circulating tumor DNA (ctDNA) is increasingly prominent as a “liquid biopsy” method for detecting and monitoring resistance to systemic therapies. Acquired resistance to hormone therapy may be based on activating mutations in the estrogen receptor gene (ESR1). Advantages of using cfDNA for tumor mutation detection include i) non-invasive collection, ii) availability at any time during disease course, and iii) real-time detection and dynamic monitoring with potentially fewer heterogeneity issues compared to tumor tissue testing. In vitro and preclinical data suggest that ESR1 mutations lead to complete resistance to AIs and partial resistance to ER agonists and antagonists. Detection of ESR1 activating mutations may guide clinicians in administering endocrine and non-endocrine therapies. cfDNA fragments are relatively small, with a peak size of approximately 180 bp. The percentage of tumor-derived DNA in total cfDNA is individually variable and often too low to be detected. Therefore, it is of great significance to develop a highly sensitive cfDNA-based ESR1 mutation detection method.

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

[0006] Microdroplet PCR (dPCR) is now revolutionizing the field of traditional PCR and redefining the detection limits for mutations. For many detection methods, dPCR offers significantly higher sensitivity than traditional PCR analysis and improves accuracy and precision by counting more molecules individually. The increased sensitivity of digital PCR is redefining our understanding of disease pathogenesis, progression, and recurrence. A particularly attractive application of digital PCR is the quantification of small numbers of mutant DNA molecules within a large pool of wild-type molecules, which is relevant to cancer research, especially the detection of minor alleles.

[0007] In view of this, there is an urgent need to provide a primer and probe set, kit and method for detecting ESR1 gene mutations, so as to achieve rapid and accurate monitoring of various mutations in the ESR1 gene of tumor patients, facilitate timely monitoring of the occurrence of new gene mutations in patients, and provide a basis for the formulation and adjustment of clinical treatment plans. Summary of the Invention

[0008] This specification provides one or more embodiments of a primer and probe set for detecting ESR1 gene mutations, the primer and probe set comprising a primer set and a probe set for PCR amplification; the primer set comprising a wild-type primer set specific to wild-type ESR1, and an E380Q mutation primer set with nucleotide sequences as shown in SEQ ID NO: 3-4.

[0009] This specification provides one or more embodiments of a kit for detecting ESR1 gene mutations, the kit comprising tube A and / or tube B, reaction reagents, positive control and negative control; tube A comprising the wild-type primer set from the primer-probe set described above; and tube B comprising the wild-type primer set from the primer-probe set described above and the E380Q mutation primer set.

[0010] This specification provides one or more embodiments of a method for detecting ESR1 gene mutations. The method includes: obtaining nucleic acid from a sample to be tested as a template; dividing the template into two equal portions and adding them to a first container and a second container in a kit, respectively, for digital PCR; determining, based on the amplification results of the digital PCR, whether the sample to be tested exhibits one or more ESR1 gene mutations; wherein the first container includes a wild-type primer set whose sequence has at least 90% similarity to SEQ ID NO: 1-2; and the second container includes a wild-type primer set whose sequence has at least 90% similarity to SEQ ID NO: 1-2, and a mutant primer set specific to ESR1 gene E380Q whose sequence has at least 90% similarity to SEQ ID NO: 3-4. Attached Figure Description

[0011] This specification will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting; in these embodiments, the same reference numerals denote the same structures, wherein:

[0012] Figure 1 is a graph of PCR amplification results of the E380Q mutation site according to some embodiments of this specification;

[0013] Figure 2 is a graph of PCR amplification results of the L536H mutation site according to some embodiments of this specification;

[0014] Figure 3 is a graph showing the PCR amplification results of the L536R mutation site according to some embodiments of this specification;

[0015] Figure 4 is a diagram of PCR amplification results of the L536P mutation site according to some embodiments of this specification;

[0016] Figure 5 is a graph showing the PCR amplification results of the Y537C mutation site according to some embodiments of this specification;

[0017] Figure 6 is a diagram of PCR amplification results of the Y537N mutation site according to some embodiments of this specification;

[0018] Figure 7 is a diagram of PCR amplification results of the Y537S mutation site according to some embodiments of this specification;

[0019] Figure 8 is a diagram of PCR amplification results of the D538G mutation site according to some embodiments of this specification;

[0020] Figure 9 shows the linear results of different mutation frequencies at the E380Q mutation site according to some embodiments of this specification;

[0021] Figure 10 shows the linear results of different mutation frequencies at the L536H mutation site according to some embodiments of this specification;

[0022] Figure 11 shows the linear results of different mutation frequencies at the L536R mutation site according to some embodiments of this specification;

[0023] Figure 12 shows the linear results of different mutation frequencies at the L536P mutation site according to some embodiments of this specification;

[0024] Figure 13 shows the linear results of different mutation frequencies at the Y537C mutation site according to some embodiments of this specification;

[0025] Figure 14 shows the linear results of different mutation frequencies at the Y537N mutation site according to some embodiments of this specification;

[0026] Figure 15 shows the linear results of different mutation frequencies at the Y537S mutation site according to some embodiments of this specification;

[0027] Figure 16 shows the linear results of different mutation frequencies at the D538G mutation site according to some embodiments of this specification. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] In the following embodiments, unless otherwise stated, all reagents used are assumed to be of analytical grade and are commercially available. Experimental methods not explicitly stated in this invention can be performed using conventional experimental methods, such as the basic biochemical molecular experimental methods published in *Molecular Cloning: A Laboratory Manual*, edited by J. Sambrook et al., published by Science Press in 2002, or according to the experimental methods explicitly stated by the reagent supplier. Except for a few definitions explicitly stated herein, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be used in this invention.

[0030] In some embodiments, the main reagents (a few common reagents are not listed) include digital PCR systems, E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, D538G and wild-type plasmid templates, extraction reagents, etc.

[0031] In some embodiments, the main instruments include a vortex mixer, a high-speed centrifuge, an A300PCR, a microdroplet preparation instrument, a microdroplet sample detector, a biosafety cabinet, a water bath, and a pipette, etc.

[0032] In some embodiments, the primer and probe set for detecting ESR1 gene mutations includes a primer set and a probe set for PCR amplification.

[0033] In some embodiments, the primer set includes a wild-type primer set specific to wild-type ESR1 and an E380Q mutant primer set with nucleotide sequences as shown in SEQ ID NO: 3-4. The nucleotide sequences of the wild-type primer set are shown in SEQ ID NO: 1-2.

[0034] Wild-type primer sets refer to primer and probe combinations specifically designed for the normal, unmutated version of a target gene during digital PCR amplification. Mutant primer sets refer to primer combinations specifically designed for the mutated version of a target gene during digital PCR amplification.

[0035] The target gene can be a gene fragment that requires specific amplification. Primer combinations can include upstream and downstream primers. The upstream and downstream primers are used to guide the digital PCR system to amplify the target gene from a specific location.

[0036] In some embodiments, the nucleotide sequence of the wild-type primer set has at least 90% similarity to SEQ ID NO:1-2. In some embodiments, the nucleotide sequence of the wild-type primer set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:1-2.

[0037] In some embodiments, the nucleotide sequence of the E380Q mutant primer set has at least 90% similarity to SEQ ID NO:3-4. In some embodiments, the nucleotide sequence of the E380Q mutant primer set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:3-4.

[0038] In some embodiments, the probe set includes at least one of the following: the Y537N mutant probe set with nucleotide sequences as shown in SEQ ID NO: 5-6; the Y537C mutant probe set with nucleotide sequences as shown in SEQ ID NO: 7-8; and the Y537S mutant probe set with nucleotide sequences as shown in SEQ ID NO: 9-10.

[0039] In some embodiments, the probe set further includes the D538G mutant probe set with nucleotide sequences as shown in SEQ ID NO: 11-12, the L536R mutant probe set with nucleotide sequences as shown in SEQ ID NO: 13-14, the L536P mutant probe set with nucleotide sequences as shown in SEQ ID NO: 15-16, and the L536H mutant probe set with nucleotide sequences as shown in SEQ ID NO: 17-18.

[0040] In some embodiments, the probe set further includes an E380Q mutant probe set with nucleotide sequences as shown in SEQ ID NO: 19-20, and a wild-type probe set with nucleotide sequences as shown in SEQ ID NO: 21-22.

[0041] A mutant probe set refers to a combination of probes that specifically detect mutated genes.

[0042] In some embodiments, the nucleotide sequence of the Y537N mutant probe set has at least 90% similarity to SEQ ID NO:5-6. In some embodiments, the nucleotide sequence of the Y537N mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:5-6.

[0043] In some embodiments, the nucleotide sequence of the Y537C mutant probe set has at least 90% similarity to SEQ ID NO:7-8. In some embodiments, the nucleotide sequence of the Y537C mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:7-8.

[0044] In some embodiments, the nucleotide sequence of the Y537S mutant probe set has at least 90% similarity to SEQ ID NO:9-10. In some embodiments, the nucleotide sequence of the Y537S mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:9-10.

[0045] In some embodiments, the nucleotide sequence of the D538G mutant probe set has at least 90% similarity to SEQ ID NO:11-12. In some embodiments, the nucleotide sequence of the D538G mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:11-12.

[0046] In some embodiments, the nucleotide sequence of the L536R mutant probe set has at least 90% similarity to SEQ ID NO:13-14. In some embodiments, the nucleotide sequence of the L536R mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:13-14.

[0047] In some embodiments, the nucleotide sequence of the L536P mutant probe set has at least 90% similarity to SEQ ID NO:15-16. In some embodiments, the nucleotide sequence of the L536P mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:15-16.

[0048] In some embodiments, the nucleotide sequence of the L536H mutant probe set has at least 90% similarity to SEQ ID NO:17-18. In some embodiments, the nucleotide sequence of the L536H mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:17-18.

[0049] In some embodiments, the nucleotide sequence of the E380Q mutant probe set has at least 90% similarity to SEQ ID NO:19-20. In some embodiments, the nucleotide sequence of the E380Q mutant probe set has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:19-20.

[0050] In some embodiments, the nucleotide sequence of the wild-type probe group has at least 90% similarity to SEQ ID NO:21-22. In some embodiments, the nucleotide sequence of the wild-type probe group has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to SEQ ID NO:21-22.

[0051] In some embodiments, a rapid and efficient detection system is provided, featuring a primer and probe set for ESR1 gene mutation genotyping detection (also known as a primer and probe set for detecting ESR1 gene mutations) with high specificity and sensitivity, high utilization of cell-free DNA, and high accuracy, as well as a kit using this primer and probe set. This primer and probe set can detect ESR1 gene mutations using a small number of samples and provide the mutation frequency of the samples. The primer and probe set for detecting ESR1 gene mutations includes multiple upstream primers, multiple downstream primers, and a mutation probe set for detecting ESR1 gene mutations E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G.

[0052] The nucleotide sequences of multiple upstream and downstream primers are shown in Table 1. Table 1: Primer-Probe Set Sequences

[0053] In some embodiments, in the Y537N mutant probe set, the Y537C mutant probe set, and the Y537S mutant probe set, at least one sequence in each mutant probe set has multiple locked nucleic acid modifications.

[0054] In some embodiments, in the D538G mutant probe set, L536R mutant probe set, L536P mutant probe set, L536H mutant probe set, and E380Q mutant probe set, at least one sequence in each mutant probe set has multiple locked nucleic acid modifications.

[0055] In some embodiments, the sequences of the wild-type probe set are modified with multiple locked nucleic acids.

[0056] In the sequences shown in Table 1, the bases following "+" indicate locked nucleic acid modifications, such as "+T" indicating locked nucleic acid modification of the base T. It should be noted that if there are discrepancies between the provided sequence list and the sequences shown in Table 1, the sequences in Table 1 shall prevail.

[0057] In some embodiments, the locked nucleic acid modifications in the Y537N mutant probe set, Y537C mutant probe set, and Y537S mutant probe set are as follows: As shown in SEQ ID NO: 5, the nucleotides at positions 7-10 and 12-13 from the 5' end are modified with locked nucleic acids. As shown in SEQ ID NO: 6, the nucleotides at positions 2, 5-7, and 10 from the 5' end are modified with locked nucleic acids. As shown in SEQ ID NO: 7, the nucleotides at positions 3-4, 6-8, and 10 from the 5' end are modified with locked nucleic acids. As shown in SEQ ID NO: 8, the nucleotides at positions 4-5, 7-9, and 11 from the 5' end are modified with locked nucleic acids. As shown in SEQ ID NO: 10, the nucleotides at positions 3, 5-7, and 9 from the 5' end are modified with locked nucleic acids.

[0058] In some embodiments, the locked nucleic acid modification in the D538G mutant probe set is as follows: The nucleotide sequence shown in SEQ ID NO: 11 is modified with locked nucleic acid at position 10 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 12 is modified with locked nucleic acid at positions 3-4 and 6-10 starting from the 5' end.

[0059] In some embodiments, the locked nucleic acid modifications in the L536R mutant probe set, L536P mutant probe set, and L536H mutant probe set are as follows: The nucleotide sequence shown in SEQ ID NO: 13 is modified with locked nucleic acid at positions 2-5 and 7-8 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 15 is modified with locked nucleic acid at positions 2-5 and 7-8 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 16 is modified with locked nucleic acid at positions 10 and 12 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 17 is modified with locked nucleic acid at positions 2-5 and 7-8 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 18 is modified with locked nucleic acid at positions 9-10 and 12 starting from the 5' end.

[0060] In some embodiments, the locked nucleic acid modification in the E380Q mutant probe set is as follows: The nucleotide sequence shown in SEQ ID NO: 19 is modified with locked nucleic acid at positions 9 and 12 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 20 is modified with locked nucleic acid at position 10 starting from the 5' end.

[0061] In some embodiments, the locked nucleic acid (LCA) modification in the wild-type probe set is as follows: The nucleotide sequence shown in SEQ ID NO: 21 is modified with a locked nucleic acid at positions 9-10 starting from the 5' end. The nucleotide sequence shown in SEQ ID NO: 22 is modified with a locked nucleic acid at positions 3-5 and 7-9 starting from the 5' end.

[0062] In some embodiments, the concentration ranges of the wild-type primer set and the mutant primer set can be preset. For example, the concentration range of the wild-type primer set and the mutant primer set is 500-700 nM, etc. Another example is the concentration range of the wild-type primer set and the mutant primer set being 550-650 nM, etc. Yet another example is the concentration range of the wild-type primer set and the mutant primer set being 580-620 nM, etc.

[0063] In some embodiments, the concentration range of the mutant probe set can be preset. For example, the concentration range of the mutant probe set is 200-400 nM, etc. Another example is 250-350 nM, etc. Yet another example is 280-320 nM, etc.

[0064] In some embodiments, the concentration range of the wild-type probe set can be preset. For example, the concentration range of the wild-type probe set is 100-300 nM, etc. Another example is a concentration range of 150-250 nM, etc. Yet another example is a concentration range of 180-220 nM, etc.

[0065] In some embodiments, the concentration of the primer set in the primer probe set is 600 nM, the concentration of the mutant probe set is 300 nM, and the concentration of the wild-type probe set is 200 nM.

[0066] In some embodiments, the mutant probe set other than the wild-type probe set can be MGB probes modified with locked nucleic acids, etc.

[0067] In some embodiments, the mutant probe set is labeled with a reporter group and a quencher group. For example, the mutant probe set with nucleotide sequences such as SEQ ID NO: 5-18 has a reporter group labeled at the 5' end and a quencher group labeled at the 3' end.

[0068] A reporter group can be a molecular group capable of generating a detectable signal (such as fluorescence, color change, electrochemical signal, etc.). A quenching group can be a molecular group capable of inhibiting the signal generated by the reporter group.

[0069] In some embodiments, the reporting group may include FAM, ROX, CY5, HEX, and CY5.5.

[0070] The primer and probe sets, kits, and methods for detecting ESR1 gene mutations are described below through several examples.

[0071] Example 1

[0072] To determine the optimal concentration of the primer-probe set, wild-type plasma template and mutant plasmid template were prepared at a predetermined template ratio (e.g., 1:1 ratio) to obtain a template with the theoretical mutation frequency. The preparation method is as follows:

[0073] S1: Sample extraction.

[0074] In some embodiments, plasma extraction can be performed using a cell-free DNA (cfdna) extraction kit, etc. For specific operating procedures, please refer to the product instructions of the cfdna extraction kit, etc.

[0075] In some embodiments, plasmid extraction is performed using a plasmid extraction kit, etc. For specific operating procedures, please refer to the instruction manual of the plasmid extraction kit, etc.

[0076] S2: Determination of the concentration of cfDNA and mutant plasmids.

[0077] In some embodiments, the extracted cfDNA and plasmids are quantified using a quantitative kit, etc. For specific experimental procedures, please refer to the instructions for use of the quantitative kit.

[0078] S3, prepare the standard products required for the experiment.

[0079] In some embodiments, based on the theoretical number of single gene copies per 1 ng of human DNA being approximately 300, cfDNA is serially diluted to a preset concentration (e.g., 2.0 × 10⁻⁶). 5 (copies / μl, etc.).

[0080] In some embodiments, the copy number of the plasmid obtained after enzyme digestion can be calculated using the following formula (1): S = (6.022 × 10⁻⁶) 23 ×10 -9 ×m1) / (m2×660) (1)

[0081] Where S represents the plasmid copy number (in copies / μL), m1 represents the plasmid concentration (in ng / μL), and m2 represents the plasmid length (e.g., 2800 bp). The plasmid copy number obtained after restriction enzyme digestion can be calculated by cutting the plasmid DNA with restriction endonucleases, and then estimating the plasmid copy number based on the concentration of the digestion product and the plasmid length.

[0082] In some embodiments, the obtained plasmid is serially diluted to 2.0 × 10⁻⁶. 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 2 copies / μl and 20 copies / μl, etc.

[0083] S4. Adjust the working concentrations of the primer set and probe set according to the development purpose, and determine the final experimental concentrations of the primer set and probe set.

[0084] In some embodiments, the concentrations of the primer sets are 800 nM, 600 nM, and 400 nM, and the concentrations of the probe sets are 200 nM, 300 nM, and 400 nM, respectively. Amplification templates (i.e., templates with theoretical mutation frequencies) are prepared using mutant plasmid templates and wild-type plasma templates at a copy number ratio of 1:1. The reaction system is prepared according to the reaction system composition table shown in Table 2. Table 2: Reaction system, primer concentration system / 30 μl

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

[0086] The results of screening for optimal primer-probe set concentrations are shown in Table 4. Table 4:

[0087] In some embodiments, when there is only one pair of primer-probe sets in each reaction system and the template amount of the amplification template is uniform, the optimal concentrations corresponding to multiple primer-probe sets are screened out.

[0088] Example 2

[0089] To demonstrate that there was no difference between the detection of the mixed system and the detection of the individual system, each plasmid was quantified using digital PCR and other methods with the common sequence as the probe set, and the mutant plasmid and wild plasmid were quantified to 1000 cop / ul.

[0090] Based on the development objective, the working concentrations of the primer and probe sets were adjusted to determine their final experimental concentrations. Amplification templates were prepared using mutant plasmid templates and wild-type plasma templates at a ratio of 1000 copies. The reaction system was prepared according to the reaction system component table shown in Table 5. Table 5

[0091] Perform the digital PCR amplification procedure according to Table 3.

[0092] The results for the multiple reaction systems in Table 5 are shown in Table 6. Table 6:

[0093] Conclusion: The difference in detected copy number between the mixed system and the single system is within the normal range, proving that there is no significant difference in detection between the mixed system and the single system. The normal range can be preset based on historical experience.

[0094] Example 3

[0095] To fully simulate real samples, cell line DNA and CtDNA were extracted from gene-edited cell lines containing one or more ESR1 gene mutations and from negative plasma, respectively. The one or more ESR1 gene mutations included E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, and D538G mutations.

[0096] After digital PCR quantification of the extracted cell line DNA and CtDNA, they were mixed according to a preset mixing ratio, and the detection results were tested. The preset mixing ratio can be pre-set based on historical experience. The reaction system was prepared according to the reaction system component table shown in Table 7. Table 7:

[0097] Perform amplification according to the digital PCR amplification procedure shown in Table 3.

[0098] The test results are shown in Table 8. Table 8:

[0099] Conclusion: The primer-probe set for detecting ESR1 gene mutations can distinguish different types of gene mutations using different fluorescence channels.

[0100] Example 4

[0101] To verify the specificity of the primer-probe set for detecting ESR1 gene mutations and whether it would react nonspecifically with the amplification template, templates extracted from wild-type (4 ml) plasma and mutant plasmid templates were prepared according to a preset template ratio to obtain a template with the theoretical mutation frequency (quantitatively analyzed using digital PCR). The preparation method is as follows:

[0102] 1. Sample extraction

[0103] In some embodiments, cell-free DNA in plasma is extracted from plasma samples using a sample preparation kit or similar device. For specific operating procedures, please refer to the product instructions for the sample preparation kit.

[0104] 2. Determination of cfDNA and mutant plasmid concentrations

[0105] In some embodiments, the extracted cfDNA and plasmids are quantified using a quantitative kit, etc. For specific experimental procedures, please refer to the instructions for use of the quantitative kit.

[0106] 3. Prepare the required standards for the experiment.

[0107] In some embodiments, based on the theoretical number of single gene copies per 1 ng of human DNA being approximately 300, cfDNA is diluted to a preset concentration (e.g., 1.0 × 10⁻⁶). 3 (copies / μl, etc.).

[0108] In some embodiments, the copy number of the plasmid obtained after enzyme digestion can be calculated using formula (1).

[0109] In some embodiments, the obtained plasmid is serially diluted to 2.0 × 10⁻⁶. 6 copies / μl, 2.0×10 5 copies / μl, 2.0×10 2 Copies / μl and 20 copies / μl, etc. Depending on the development objective, mutant plasmid templates and wild-type plasma templates were prepared into amplification templates according to the copy number ratio. The reaction system was prepared according to the reaction system component table shown in Table 9. Table 9:

[0110] Perform the digital PCR amplification procedure according to Table 3.

[0111] Tables 10-13 show the test results. Table 10: Table 11: Table 12: Table 13:

[0112] Figure 1 is a diagram of PCR amplification results of the E380Q mutation site according to some embodiments of this specification.

[0113] Figure 2 is a diagram of PCR amplification results of the L536H mutation site according to some embodiments of this specification.

[0114] Figure 3 is a graph showing the PCR amplification results of the L536R mutation site according to some embodiments of this specification.

[0115] Figure 4 is a diagram of PCR amplification results of the L536P mutation site according to some embodiments of this specification.

[0116] Figure 5 is a diagram of PCR amplification results of the Y537C mutation site according to some embodiments of this specification.

[0117] Figure 6 is a diagram of PCR amplification results of the Y537N mutation site according to some embodiments of this specification.

[0118] Figure 7 is a diagram of PCR amplification results of the Y537S mutation site according to some embodiments of this specification.

[0119] Figure 8 is a diagram of PCR amplification results of the D538G mutation site according to some embodiments of this specification.

[0120] In Figures 1 to 8, the VIC direction (i.e., the vertical axis in Figures 1, 3, 4, 7, and 8, and the horizontal axis in Figures 2, 5, and 6) represents the specific signal of the VIC fluorescent dye. The FAM direction, CY5 direction, ROX direction, and Q705 direction represent the specific signals of the FAM fluorescent dye, FCY5 fluorescent dye, ROX fluorescent dye, and Q705 fluorescent dye, respectively.

[0121] In Figure 1, data points with values ​​ranging from (5000, 8000) in the VIC direction and from (0, 1000) in the CY5 direction represent internal reference genes, while the remaining data points represent mutant genes. Internal reference genes are genes that are stably expressed within the cell.

[0122] In Figure 2, data points with values ​​ranging from (5000, 8000) in the VIC direction and from (0, 2000) in the FAM direction represent internal reference genes, while the remaining data points represent mutant genes.

[0123] In Figure 3, data points with values ​​ranging from (5000, 8000) in the VIC direction and from (0, 1000) in the Q705 direction represent internal reference genes, while the remaining data points represent mutant genes.

[0124] In Figure 4, data points with values ​​ranging from (5000, 8000) in the VIC direction and from (1, 3) in the ROX (10^3) ​​direction represent internal reference genes, while the remaining data points represent mutant genes.

[0125] In Figure 5, data points with values ​​ranging from (5000, 8000) in the VIC direction and from (0, 2000) in the FAM direction represent internal reference genes, while the remaining data points represent mutant genes.

[0126] In Figure 6, data points with values ​​ranging from (2000, 4000) in the VIC direction and from (0, 2000) in the FAM direction represent internal reference genes, while the remaining data points represent mutant genes.

[0127] In Figure 7, data points with values ​​ranging from (2000, 4000) in the VIC direction and from (2000, 4000) in the Q705 direction represent internal reference genes, while the remaining data points represent mutant genes.

[0128] In Figure 8, data points with values ​​ranging from (2000, 4000) in the VIC direction and from (0, 2) in the ROX (10^3) ​​direction represent internal reference genes, while the remaining data points represent mutant genes.

[0129] Conclusion: Based on Figures 1 to 8, Y537C, Y537N, Y537S, and D538G in System 1 do not specifically bind to L536H, L536R, L536P, E380Q, or the wild-type plasma template, and there is no non-specific binding between 537CNS and 538. In System 2, 536RPH and 380 do not specifically bind to 537CNS, 538, or the wild-type plasma template, and there is no non-specific binding between 536RPH and 380.

[0130] Example 5

[0131] Since the amount of cell-free DNA in plasma is very small, in order to test the detection limit performance of this reagent (primer and probe set for detecting ESR1 gene mutations), a preset number (such as 5000) of wild-type copy number was used as the background, and wild-type plasma template (negative sample) was mixed with mutant plasmid to simulate real sample.

[0132] In some embodiments, the reagent is tested according to a preset mutation ratio. The preset mutation ratio can be set in advance, such as 50%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc.

[0133] Prepare the reaction system according to the composition table shown in Table 14. Table 14:

[0134] Perform amplification according to the digital PCR amplification procedure shown in Table 3.

[0135] Figure 9 shows the linear results of different mutation frequencies at the E380Q mutation site according to some embodiments of this specification.

[0136] Figure 10 shows the linear results of different mutation frequencies at the L536H mutation site according to some embodiments of this specification.

[0137] Figure 11 shows the linear results of different mutation frequencies at the L536R mutation site according to some embodiments of this specification.

[0138] Figure 12 shows the linear results of different mutation frequencies at the L536P mutation site according to some embodiments of this specification.

[0139] Figure 13 shows the linear results of different mutation frequencies at the Y537C mutation site according to some embodiments of this specification.

[0140] Figure 14 shows the linear results of different mutation frequencies at the Y537N mutation site according to some embodiments of this specification.

[0141] Figure 15 shows the linear results of different mutation frequencies at the Y537S mutation site according to some embodiments of this specification.

[0142] Figure 16 shows the linear results of different mutation frequencies at the D538G mutation site according to some embodiments of this specification.

[0143] Conclusion: As shown in Figures 9 to 16, the mutation sites E380Q, L536H, L536R, L536P, Y537C, Y537N, Y537S, and D538G all showed linearity at different mutation frequencies above R-squared 0.999 at a wild-type copy number of 5000, and could be detected normally at a mutation frequency of 0.1%.

[0144] Example 6

[0145] In some embodiments, an accuracy comparison experiment was designed for this reagent. Ten samples that had already been confirmed positive by NGS were used to perform genotyping using a kit containing this reagent, to determine whether the genotyping results from the kit were consistent with the genotyping results from NGS. The reaction system was prepared according to the reaction system component table shown in Table 15. Table 15:

[0146] Perform the digital PCR amplification procedure shown in Table 3. The detection results are shown in Table 16. Table 16:

[0147] Conclusion: The genotyping results of the kit containing this reagent are consistent with those of NGS genotyping, but the time required is shorter and the cost is lower.

[0148] In some embodiments, the kit for detecting ESR1 gene mutations provided in this specification includes tubes A and / or tubes B, reaction reagents, positive control and negative control.

[0149] In some embodiments, the reaction reagents may contain the basic biochemical components required for amplification, such as DNA polymerase, buffer systems, and stabilizers. Positive controls may be standardized samples containing the mutant sequence. Negative controls may be control samples not containing the mutant sequence.

[0150] In some embodiments, tube A includes a wild-type primer set. Tube B includes a wild-type primer set and an E380Q mutant primer set.

[0151] In some embodiments, tube A further includes the Y537N mutant probe set, the Y537C mutant probe set, the Y537S mutant probe set, the D538G mutant probe set, and the wild-type probe set.

[0152] In some embodiments, tube B further includes the E380Q mutant probe set, the L536R mutant probe set, the L536P mutant probe set, the L536H mutant probe set, and the wild-type probe set.

[0153] In some embodiments, the concentration range of one or more primer sets in tubes A and B is 500-700 nM. The concentration range of multiple mutant probe sets in tubes A and B is 200-400 nM. The concentration range of wild-type probe sets in tubes A and B is 100-300 nM.

[0154] In some embodiments, the method for detecting ESR1 gene mutations provided in this specification includes: obtaining nucleic acid from a sample to be tested as a template, dividing the template into two equal portions, and adding them to a first container and a second container in the kit, respectively, for digital PCR. The first container includes a wild-type primer set (with sequences having at least 90% similarity to SEQ ID NO: 1-2). The second container includes a wild-type primer set (with sequences having at least 90% similarity to SEQ ID NO: 1-2) and a mutant primer set specific to E380Q of the ESR1 gene (with sequences having at least 90% similarity to SEQ ID NO: 3-4).

[0155] In some embodiments, the presence of one or more ESR1 gene mutations in the sample is determined based on the amplification results of digital PCR. One or more ESR1 gene mutations include E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, and D538G mutations.

[0156] In some embodiments, determining whether the sample to be tested has one or more ESR1 gene mutations includes determining whether Y537N mutation, Y537C mutation, Y537S mutation, D538G mutation is present in the first container, and determining whether E380Q mutation, L536H mutation, L536R mutation, L536P mutation, etc. are present in the second container.

[0157] In some embodiments, the sample to be tested includes at least one of cells, body fluids, or tissues. Body fluids include at least one of serum, plasma, or tissue fluid.

[0158] In some embodiments, when the presence of one or more ESR1 gene mutations is determined, the individual corresponding to the test sample is given an altered treatment. The individual may include individuals receiving endocrine therapy, individuals with breast cancer, etc.

[0159] The primer and probe sets for detecting ESR1 gene mutations provided in some examples of this specification have high specificity and amplification efficiency, and can be applied to digital PCR detection. Based on trace amounts of ESR1 gene mutations (E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G) in samples, various mutations in the ESR1 gene of tumor patients can be rapidly and accurately detected. This allows for timely monitoring of the occurrence of new gene mutations in patients, providing a basis for the formulation and adjustment of clinical treatment plans.

[0160] The kits for detecting ESR1 gene mutation genotyping (also known as ESR1 gene mutations) provided in some examples of this manual use standards prepared from the nucleic acid of ESR1-negative samples and enzyme-digested mutant plasmids containing E380Q, L536R, L536P, L536H, Y537C, Y537N, Y537S, and D538G mutant fragments of the ESR1 gene, in copy number ratios. Standards with different mutation frequencies play different roles. The use of cfDNA and plasmids in the standards can maximally restore the characteristics of the test samples, providing a solid foundation for optimizing the detection system and playing a decisive role in this optimization process.

[0161] The kits for detecting ESR1 gene mutations provided in some embodiments of this specification determine the endpoint fluorescence signal values ​​produced by each mutation probe at different concentrations using digital PCR detection results of standards with different mutation frequencies, making the data statistical results more accurate. The ESR1 gene mutation detection system provided in some embodiments of this specification determines the background threshold for each mutation site in the detection system using digital PCR detection results of wild-type templates. When detecting the mutation copy number of a sample, the mutation copy number is equal to the detection result minus the background threshold, which can make the detection results more accurate.

[0162] The mutation detection system of the kit for detecting ESR1 gene mutation typing provided in some embodiments of this specification can be determined by digital PCR detection results of low mutation frequency standards.

[0163] Some embodiments in this manual optimize the kit for multiplex detection of ESR1 gene mutation typing by optimizing the concentration of each probe based on the results of traditional real-time fluorescence PCR detection using high mutation frequency standards. The appropriate probe concentration is selected based on the difference in fluorescence intensity after the reaction of different concentrations of probes for each mutation. This method is accurate and low in cost.

[0164] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A primer probe set for detecting a mutation in the ESR1 gene, characterized by, The kit comprises a primer set and a probe set for PCR amplification. The primer set comprises a wild type primer set specific to wild type ESR1, and an E380Q mutant primer set with a nucleotide sequence as shown in SEQ ID NO: 3-4.

2. The primer probe set of claim 1, wherein, The probe set comprises a Y537N mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 5-6, a Y537C mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 7-8, and a Y537S mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 9-10.

3. The primer probe set of any one of claims 1-2, wherein, The probe set further comprises a D538G mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 11-12, a L536R mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 13-14, a L536P mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 15-16, and a L536H mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 17-18.

4. The primer probe set of any one of claims 1-3, wherein, The probe set further comprises an E380Q mutant probe set with a nucleotide sequence as shown in SEQ ID NO: 19-20, and a wild type probe set with a nucleotide sequence as shown in SEQ ID NO: 21-22.

5. The primer probe set of claim 2, wherein, In the Y537N mutant probe set, the Y537C mutant probe set, and the Y537S mutant probe set, at least one sequence in each mutant probe set is modified with a plurality of locked nucleic acids.

6. The primer probe set of any one of claims 3-4, wherein, In the D538G mutant probe set, the L536R mutant probe set, the L536P mutant probe set, the L536H mutant probe set, and the E380Q mutant probe set, at least one sequence in each mutant probe set is modified with a plurality of locked nucleic acids.

7. The primer probe set of claim 4, wherein, The sequences in the wild type probe set are all modified with a plurality of locked nucleic acids.

8. The primer probe set of claim 5, wherein, In the Y537N mutant probe set, the Y537C mutant probe set, and the Y537S mutant probe set, the locked nucleic acid modification is as follows: the nucleotide sequence as shown in SEQ ID NO: 5 is modified with a plurality of locked nucleic acids at the 7th-10th and 12th-13th nucleotides from the 5' end; the nucleotide sequence as shown in SEQ ID NO: 6 is modified with a plurality of locked nucleic acids at the 2nd and 5th-7th, 10th nucleotides from the 5' end; the nucleotide sequence as shown in SEQ ID NO: 7 is modified with a plurality of locked nucleic acids at the 3rd-4th and 6th-8th, 10th nucleotides from the 5' end; the nucleotide sequence as shown in SEQ ID NO: 8 is modified with a plurality of locked nucleic acids at the 4th-5th and 7th-9th, 11th nucleotides from the 5' end; and the nucleotide sequence as shown in SEQ ID NO: 10 is modified with a plurality of locked nucleic acids at the 3rd and 5th-7th, 9th nucleotides from the 5' end.

9. The primer probe set of claim 6, wherein, In the D538G mutant probe set, the locked nucleic acid modification is as follows: the nucleotide sequence as shown in SEQ ID NO: 11 is modified with a plurality of locked nucleic acids at the 10th nucleotide from the 5' end; and the nucleotide sequence as shown in SEQ ID NO: 12 is modified with a plurality of locked nucleic acids at the 10th nucleotide from the 5' end. the nucleotide sequence shown as SEQ ID NO: 12 is modified with the locked nucleic acid at the 3rd-4th and 6th-10th nucleotides from the 5' end.

10. The primer probe set of claim 6, wherein, the L536R mutant probe set, the L536P mutant probe set and the L536H mutant probe set are modified with the locked nucleic acid as follows: the nucleotide sequence shown as SEQ ID NO: 13 is modified with the locked nucleic acid at the 2nd-5th and 7th-8th nucleotides from the 5' end; the nucleotide sequence shown as SEQ ID NO: 15 is modified with the locked nucleic acid at the 2nd-5th and 7th-8th nucleotides from the 5' end; the nucleotide sequence shown as SEQ ID NO: 16 is modified with the locked nucleic acid at the 10th and 12th nucleotides from the 5' end; the nucleotide sequence shown as SEQ ID NO: 17 is modified with the locked nucleic acid at the 2nd-5th and 7th-8th nucleotides from the 5' end; and the nucleotide sequence shown as SEQ ID NO: 18 is modified with the locked nucleic acid at the 9th-10th and 12th nucleotides from the 5' end.

11. The primer probe set of claim 6, wherein, the E380Q mutant probe set is modified with the locked nucleic acid as follows: the nucleotide sequence shown as SEQ ID NO: 19 is modified with the locked nucleic acid at the 9th and 12th nucleotides from the 5' end; and the nucleotide sequence shown as SEQ ID NO: 20 is modified with the locked nucleic acid at the 10th nucleotide from the 5' end.

12. The primer probe set of claim 7, wherein, the wild type probe set is modified with the locked nucleic acid as follows: the nucleotide sequence shown as SEQ ID NO: 21 is modified with the locked nucleic acid at the 9th-10th nucleotides from the 5' end; and the nucleotide sequence shown as SEQ ID NO: 22 is modified with the locked nucleic acid at the 3rd-5th and 7th-9th nucleotides from the 5' end.

13. The primer probe set of any one of claims 2-4, wherein, the mutant probe set with the nucleotide sequence shown as SEQ ID NO: 5-18 is labeled with a reporter group at the 5' end and a quencher group at the 3' end.

14. The primer probe set of claim 13, wherein, the reporter group includes FAM, ROX, CY5, HEX and CY5.

5.

15. The primer probe set of claim 1, wherein, the nucleotide sequence of the wild type primer set is shown as SEQ ID NO: 1-2.

16. A kit for detecting a mutation in the ESRl gene, characterized in that, the kit includes an A tube and / or a B tube, reaction reagents, positive quality control and negative quality control; the A tube includes the wild type primer set in the primer probe set of claim 1; and the B tube includes the wild type primer set and the E380Q mutant primer set in the primer probe set of claim 1.

17. The kit of claim 16, wherein the A tube further includes a Y537N mutant probe set with the nucleotide sequence shown as SEQ ID NO: 5-6, a Y537C mutant probe set with the nucleotide sequence shown as SEQ ID NO: 7-8, a Y537S mutant probe set with the nucleotide sequence shown as SEQ ID NO: 9-10, a D538G mutant probe set with the nucleotide sequence shown as SEQ ID NO: 11-12 and a wild type probe set with the nucleotide sequence shown as SEQ ID NO: 21-22.

18. The kit of claim 16, wherein The B tube further comprises an E380Q mutant probe group with a nucleotide sequence as shown in SEQ ID NO: 19-20, an L536R mutant probe group with a nucleotide sequence as shown in SEQ ID NO: 13-14, an L536P mutant probe group with a nucleotide sequence as shown in SEQ ID NO: 15-16, an L536H mutant probe group with a nucleotide sequence as shown in SEQ ID NO: 17-18, and a wild type probe group with a nucleotide sequence as shown in SEQ ID NO: 21-22.

19. The kit of any one of claims 17-18, wherein, At least one sequence in each of the Y537N mutant probe group, the Y537C mutant probe group, the D538G mutant probe group, the L536R mutant probe group, the L536P mutant probe group, the L536H mutant probe group, and the E380Q mutant probe group is modified with a plurality of locked nucleic acids.

20. The kit of any one of claims 17-18, wherein, The sequences of the wild type probe group are all modified with a plurality of locked nucleic acids.

21. The kit of claim 16, wherein The concentration of one or more groups of primers in the A tube and the B tube ranges from 500-700nM; the concentration of the plurality of mutant probe groups in the A tube and the B tube ranges from 200-400nM; and the concentration of the wild type probe group in the A tube and the B tube ranges from 100-300nM.

22. A method of detecting a mutation in the ESRl gene, the method comprising, The method comprises: obtaining nucleic acid of a sample to be tested as a template; dividing the template into two equal parts and adding them into a first container and a second container in the kit for digital PCR; determining whether one or more ESR1 gene mutations occur in the sample to be tested according to the amplification results of the digital PCR; wherein the first container comprises a wild type primer group with a sequence having at least 90% similarity to SEQ ID NO: 1-2; and the second container comprises a wild type primer group with a sequence having at least 90% similarity to SEQ ID NO: 1-2, and a mutant primer group specific to ESR1 gene E380Q with a sequence having at least 90% similarity to SEQ ID NO: 3-4.

23. The method of claim 22, wherein, The kit comprises the kit of claim 16, the first container comprises the A tube of the kit of claim 16, and the second container comprises the B tube of the kit of claim 16.

24. The method of claim 22, wherein, The one or more ESR1 gene mutations comprise E380Q, L536H, L536P, L536R, Y537C, Y537N, Y537S, and D538G mutations.

25. The method of claim 22, wherein, The sample to be tested is a cell, a body fluid, or a tissue; and the body fluid comprises serum, plasma, or interstitial fluid.

26. The method according to any one of claims 22 to 25, characterized in that, The method further comprises: when the presence of the one or more ESR1 gene mutations is determined, providing a changed treatment to an individual corresponding to the sample to be tested.

27. The method according to any one of claims 22-25, characterized by, The determination of whether one or more ESR1 gene mutations occur in the sample to be tested according to the amplification results of the digital PCR comprises: determining whether Y537N mutation, Y537C mutation, Y537S mutation, and D538G mutation occur in the first container; and determining whether a E380Q mutation, a L536H mutation, a L536R mutation, a L536P mutation occurs in the second container.

28. A method of providing a modified treatment for an individual having breast cancer who is receiving endocrine therapy, the method comprising: obtaining a test sample from the individual, using nucleic acid of the test sample as a template; dividing the template into two equal parts, and adding the two equal parts into a first container and a second container of a kit, respectively, for digital PCR; determining whether one or more ESR1 gene mutations occur in the test sample according to amplification results of the digital PCR; and providing a modified treatment for the individual if the one or more ESR1 gene mutations are determined to occur.

Images