Primer probe combination for detecting ESR1 gene drug-resistant mutation site based on digital PCR single tube and application of primer probe combination

Abstract

The invention discloses a primer and probe combination for detecting ESR1 gene drug-resistant mutation sites based on a digital PCR single tube and application of the primer and probe combination, and belongs to the technical field of molecular detection, sequences of primers are shown as SEQ ID NO.1-2, sequences of probes are shown as SEQ ID NO.3-4, and 5'ends of the two probes are marked with different fluorophores. According to the present invention, with the primer probe combination, the high-sensitivity and rapid detection of the common nine drug-resistant related mutation sites such as L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D and D538G of the ESR1 gene in the breast cancer through the single tube can be achieved, such that the ESR1 gene detection efficiency is substantially improved, and the detection cost is reduced so as to guide the endocrine treatment of the breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology, and in particular relates to a primer-probe combination for detecting drug resistance mutation sites in the ESR1 gene based on digital PCR single tube and its application. Background Technology

[0002] According to the latest global cancer burden data for 2022, there were 2.3 million new cases of breast cancer worldwide, with 670,000 deaths, making it the leading cause of cancer death among women globally. Approximately 60-70% of these breast cancers are estrogen receptor (ER) positive. ER binding to estrogen promotes the growth, proliferation, and survival of breast tumor cells. Endocrine therapy (ET) is the primary treatment for ER+ breast cancer. Drug therapy targeting ER is collectively referred to as endocrine therapy, which forms the foundation of breast cancer treatment and is integrated throughout the entire clinical process of breast cancer patients, from neoadjuvant / adjuvant therapy in early diagnosis to salvage therapy for recurrence and metastasis.

[0003] The ESR1 gene encodes estrogen receptor α (ERα) protein, an important transcription factor that plays a crucial role in regulating estrogen receptor-related signaling pathways. ESR1 gene mutations are a significant driver of endocrine therapy resistance. ESR1 mutations may occur in patients after aromatase inhibitor (AI) therapy, with the incidence varying depending on the location of metastatic lesions. Common types of ESR1 mutations include point mutations, insertions / deletions, and rearrangements, often associated with poor overall prognosis. To date, over 60 ESR1 point mutations have been detected in clinical breast cancer samples. However, most pathogenic mutations that can lead to endocrine therapy resistance are located in the ligand-binding domain (LBD) / AF2 domain. Y537S and D538G account for over 70% of ESR1 mutations found in resistant cases. ESR1 mutation is one of the important mechanisms of secondary resistance in ER+ breast cancer and is also an indicator of poor prognosis. Analyzing ESR1 gene mutation status is of great significance for guiding precision clinical treatment of breast cancer.

[0004] Circulating tumor DNA (ctDNA) consists of cell-free DNA fragments released into the bloodstream after tumor cells undergo apoptosis and necrosis. Its detection offers advantages such as high sensitivity, good homogeneity, and real-time performance, and has various potential clinical applications. ctDNA testing, as a non-invasive method, has become an alternative for gene testing in advanced breast cancer where tissue samples are unavailable. Multiple studies have shown that specific ctDNA mutations can guide appropriate treatment matching for patients with advanced breast cancer, and ESR1 gene mutations are often detectable in the plasma ctDNA of patients who have progressed after AI therapy. The European Society for Medical Oncology (ESMO) recommends that ctDNA testing should be prioritized for patients with ESR1 mutations in advanced breast cancer. However, cell-free DNA (cfDNA) in plasma is short in length and low in concentration, with ctDNA accounting for a negligible proportion, posing significant challenges to the sensitivity of detection techniques and the selection of candidate sites. Therefore, developing a cfDNA-based method for detecting ESR1 gene mutations is of great importance.

[0005] Currently reported methods for detecting ESR1 include first-generation sequencing and second-generation sequencing. First-generation sequencing is simple and inexpensive, but it requires a series of subsequent operations after PCR, making it prone to contamination and false positives, and it cannot detect trace nucleic acid mutations in blood. Second-generation sequencing is time-consuming and expensive. Summary of the Invention

[0006] To achieve rapid and efficient detection of ESR1 gene mutations in breast cancer, this invention provides a primer and probe combination and detection kit for ESR1 mutation detection based on a dPCR platform, which has high sensitivity and specificity and high cfDNA utilization.

[0007] This invention designs corresponding MGB fluorescent probes for the L536, Y537, and D538 mutation sites on exon 8 of the ESR1 gene based on the wild-type sequences. The 5' end of the probe is modified with HEX fluorescence, and the 3' end with an NFQ group. A non-mutant MGB fluorescent probe is also designed based on the wild-type sequence of the non-mutant sites, with a FAM fluorescent group at the 5' end and an NFQ group at the 3' end. When the mutation is wild-type, both probes can bind; when the mutation is mutant, only the non-mutant probe can bind. The upstream and downstream primers are designed based on the wild-type sequences, thus enabling the simultaneous detection of multiple mutation sites with a single primer-probe setup. See the primer-probe design scheme and amplification diagram for details. Figure 1 After the primer and probe designs are completed and screened, the reaction system is optimized to determine the optimal amount of system components and establish a detection method, which includes the following steps: (1) Based on the NM_000125.4 sequence of the transcript corresponding to ESR1, the location of the mutation site was determined, and the sequences before and after the site were obtained from the reference genome for primer and probe design.

[0008] (2) The designed primers and probes were screened to determine the primer and probe sequences used in this invention, as shown in Table 1 below.

[0009] Table 1 Primer and probe sequences used in this invention

[0010] (3) Optimize the amounts of components in the PCR reaction system, determine the formulations for different reaction tubes and the PCR amplification reaction procedure. Simultaneously, prepare the corresponding negative and positive control samples to complete the kit development. The formulations are shown in Table 2. When using the kit, add 5 μL of cfDNA template to each reaction tube. The PCR amplification reaction procedure is shown in Table 3.

[0011] Table 2. Recipe for ESR1 gene mutation detection reaction tube 1

[0012] Table 3 PCR amplification reaction procedure

[0013] (4) Interpretation of results: The FAM channel represents the wild-type + mutant gene copy number, the HEX channel represents the wild-type gene copy number, and the mutant gene copy number = FAM channel copy number - HEX channel copy number. Mutation ratio = (FAM channel copy number - HEX channel copy number) / FAM channel copy number.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) Based on the digital PCR (dPCR) platform, this invention designs a primer and probe combination for ESR1 gene mutation detection, which enables sensitive and rapid detection of nine drug resistance-related mutation sites of the ESR1 gene commonly found in breast cancer, namely L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, and D538G, in a single tube. This greatly improves the efficiency of ESR1 gene detection and reduces the detection cost, so as to guide subsequent endocrine therapy.

[0015] (2) Using dPCR technology, the endpoint fluorescence signal values ​​generated by each mutation probe at different concentrations were determined by the digital PCR detection results of standards with different mutation frequencies, making the data statistics results more accurate. The ESR1 gene mutation detection system determined the background threshold of each mutation site in the detection system by the digital PCR detection results of wild-type templates. When detecting the mutation copy number of the sample, the mutation copy number is equal to the detection result minus the background threshold, which can make the results more accurate.

[0016] (3) The amplification length of the primers is designed to be within 100 bp, which is suitable for the detection of cfDNA extracted from peripheral blood and realizes liquid biopsy. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the primer and probe design scheme and amplification detection of the present invention.

[0018] Figures 2-1 to 2-6 This is a 2D dot matrix image for detecting different copy numbers of the D538G mutant plasmid in Example 1.

[0019] Figure 3 The results show the linearity of different mutation frequencies at the L536H mutation site in Example 1.

[0020] Figure 4 The results show the linearity of different mutation frequencies at the L536Q mutation site in Example 1.

[0021] Figure 5 The results show the linearity of different mutation frequencies at the L536R mutation site in Example 1.

[0022] Figure 6 The results show the linearity of different mutation frequencies at the L536P mutation site in Example 1.

[0023] Figure 7 The results show the linearity of different mutation frequencies at the Y537S mutation site in Example 1.

[0024] Figure 8 The results show the linearity of different mutation frequencies at the Y537C mutation site in Example 1.

[0025] Figure 9 The results show the linearity of different mutation frequencies at the Y537N mutation site in Example 1.

[0026] Figure 10 The results show the linearity of different mutation frequencies at the Y537D mutation site in Example 1.

[0027] Figure 11 The results show the linearity of different mutation frequencies at the D538G mutation site in Example 1. Detailed Implementation

[0028] Example 1 This embodiment tests the specificity of the reagents in this invention, whether they will react non-specifically with wild-type templates, and tests the detection limit of the reagents. The wild-type amplification template used in this embodiment is cell-free DNA (cfDNA) from healthy individuals without tumors, and the mutant amplification template is an ESR1 mutation site-positive plasmid artificially constructed in the laboratory, which has been digested with restriction endonucleases to form a linear structure.

[0029] I. Sample processing and template extraction quality control Peripheral blood was collected using cell-free DNA preservation tubes, with a collection volume of no less than 10 mL. Peripheral blood plasma DNA was extracted using nucleic acid extraction reagents (Plasma DNA (centrifugation column method)) from Xiamen Feishuo Biotechnology Co., Ltd. Specific procedures were followed according to the kit instructions. cfDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0), and the extraction quality and concentration were measured using a UV spectrophotometer. The cfDNA was diluted to 4 ng / μL with Tris-HCl (10 mmol / L, pH 8.0) as the DNA template for detection, and simultaneously diluted to 5 ng / μL as the background template.

[0030] Plasmids with different ESR1 gene mutation sites were linearized using HindIII-HF restriction endonuclease. The specific operating system and reaction procedure are shown in Tables 4-5. After the reaction, the digestion products were transferred to new 1.5 ml centrifuge tubes, and 50 µL of Tris-HCl (10 mmol / L, pH 8.0) was added and vortexed to obtain the plasmid stock solution. 2 µL of the plasmid stock solution was then serially diluted to 10⁻⁶. -5 Concentration is used for digital PCR detection to determine copy number. After determining the copy number, the plasmid is serially diluted to 10 copies / μL, 100 copies / μL, and 1000 copies / μL. For each of these three gradients, 1µL + 4µL of cfDNA (5 ng / μL) is used to prepare 5µL of template containing 10 copies, 100 copies, and 1000 copies of the plasmid, respectively. Simultaneously, 1µL (1000 copies / μL) of each plasmid is used + 4µL of Tris-HCl (10 mmol / L, pH 8.0) to prepare 5µL of pure plasmid template containing 1000 copies.

[0031] Table 4 Enzyme digestion reaction system

[0032] Table 5 Enzyme digestion reaction procedure

[0033] II. Preparation of the reaction solution The reaction solution was prepared according to Table 2. 38 reaction solutions were prepared. 5 μL of nuclease-free water, normal cfDNA (4 ng / μL), plasmid templates with 10 copies, 100 copies, and 1000 copies of each ESR1 mutation, and plasmid templates mixed with background were added to each reaction solution.

[0034] III. Digital PCR Detection Perform digital PCR detection according to the reaction procedure in Table 3.

[0035] IV. Statistical Analysis of Test Results The number of positive droplets in the FAM and HEX channels for each sample was counted, and the actual detected mutation copy number was calculated, as shown in Table 6. 2D dot matrix images of different copy number detections using the D538G mutant plasmid as an example are shown below. Figures 2-1 to 2-6 The linear results for different copy numbers of each mutation site (L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, D538G) under a wild-type background of 20 ng are as follows: Figure 3-11 As shown.

[0036] Table 6. Test results of plasmid systems with different copy numbers

[0037] In the above results, the number of positive droplets in the FAM channel and the number of positive droplets in the HEX channel of wild-type samples were equal, indicating no nonspecific reaction. Furthermore, the mutation sites L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, and D538G all showed linearity at different mutation frequencies above R-squared 0.99 with a wild-type background of 20 ng, and were detected normally at a mutation frequency of approximately 0.17% (calculated based on 1 ng of genome ~ 30 copies), indicating that the kit has high detection sensitivity and specificity.

[0038] Example 2 In this embodiment, a mixed plasmid template with different mutation sites and a single mutant plasmid template were used to verify that there was no difference in the detection of polyclonal and monoclonal clones in this invention. The specific steps are as follows: I. Sample processing and template extraction quality control Peripheral blood was collected using cell-free DNA preservation tubes, with a collection volume of no less than 10 mL. Peripheral blood plasma DNA was extracted using nucleic acid extraction reagents from Xiamen Feishuo Biotechnology Co., Ltd. [Plasma DNA (centrifugation column method)]. Specific procedures were followed according to the kit instructions. cfDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0), and the extraction quality and concentration were assessed using a UV spectrophotometer. The cfDNA was diluted to 5 ng / μL with Tris-HCl (10 mmol / L, pH 8.0) as a background template.

[0039] The plasmid was linearized using HindIII-HF restriction endonuclease. The specific operating system and reaction procedure are shown in Tables 4-5. After the reaction, the digestion product was transferred to a new 1.5 ml centrifuge tube, and 50 µL of Tris-HCl (10 mmol / L, pH 8.0) was added and vortexed to obtain the plasmid stock solution. 2 µL of the plasmid stock solution was then serially diluted to 10⁻⁶. -5 Concentration is used for digital PCR detection to determine the copy number. After determining the copy number, the plasmid is serially diluted to 100 copies / μL, and the template reaction solution is prepared according to Table 7.

[0040] Table 7. Reaction solutions for monoclonal and polyclonal mutant templates

[0041] II. Preparation of the reaction solution The reaction solution was prepared according to Table 2, and the amount of purified water in the system was adjusted according to the amount of template used. After preparation, the templates in Table 7 were added to the reaction solution.

[0042] III. Digital PCR Detection Perform digital PCR detection according to the PCR reaction procedure in Table 3.

[0043] IV. Statistical Analysis of Test Results The number of positive droplets in the FAM and HEX channels for each sample was counted, and the actual number of mutation copies detected was calculated, as shown in Table 8.

[0044] Table 8. Mutation detection results of monoclonal and polyclonal mutant templates.

[0045] The above results indicate that the detected copy number in both the monoclonal mutation system and the polyclonal system is within the normal range, demonstrating that the kit can detect ESR1 monoclonal and polyclonal mutations within the range.

[0046] Example 3 This embodiment uses next-generation sequencing methodology as a control method to verify the accuracy of the kit. Fifteen clinical samples diagnosed with breast cancer by pathology were collected. The specific steps are as follows: I. Sample processing and template extraction quality control Peripheral blood was collected using cell-free DNA preservation tubes, with a collection volume of no less than 10 mL. Peripheral blood plasma DNA was extracted using nucleic acid extraction reagents (Plasma DNA (centrifugation column method)) from Xiamen Feishuo Biotechnology Co., Ltd., following the kit instructions. cfDNA was eluted with Tris-HCl (10 mmol / L, pH 8.0), and the extraction quality and concentration were measured using a UV spectrophotometer. The cfDNA was diluted to 2 ng / μL with Tris-HCl (10 mmol / L, pH 8.0) as a template. One sample was used for next-generation sequencing, and the other sample was tested using the kit of this invention.

[0047] II. Preparation of the reaction solution Prepare the kit reaction solution according to Table 2, and add 5 μL of the above cfDNA template to each reaction tube.

[0048] III. Digital PCR Detection Perform digital PCR detection according to the PCR reaction procedure in Table 3.

[0049] IV. Statistical Analysis of Test Results The number of positive droplets in the FAM and HEX channels of 15 samples was counted, and the actual detected mutation copy number and mutation ratio were calculated. The consistency with the results of next-generation sequencing is shown in Table 9. As can be seen from the table, the mutation consistency rate of the results of this kit in detecting 15 clinical samples was 100% with the results of next-generation sequencing, and the mutation frequency deviation was within the normal range. This further demonstrates the accuracy of the primer-probe combination and kit of this invention in detecting ESR1 gene mutations L536H, L536Q, L536R, L536P, Y537S, Y537C, Y537N, Y537D, and D538G.

[0050] Table 9. Consistency analysis of the test results of this kit on 10 clinical samples and the results of next-generation sequencing.

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer-probe combination for detecting ESR1 gene drug-resistant mutation sites based on digital PCR single-tube detection, characterized in that, The sequence of the primer is shown as SEQ ID NO. 1-2, the sequence of the probe is shown as SEQ ID NO. 3-4, and the 5' end of the two probes is labeled with different fluorescent groups.

2. The primer probe combination of claim 1, wherein, The fluorescent group is selected from FAM, HEX, TET or Yakima Yellow.

3. The primer probe combination of claim 2, wherein, The 3' end of the two probes is labeled with any one of quenching groups NFQ, BHO-1, BHQ-2, BHQ-3, Dabcyl.

4. The primer probe combination of claim 3, wherein, The 3' end quenching group NFQ of the two probes is further connected with a minor groove binder MGB.

5. A product for detecting a drug-resistant mutation site of an ESR1 gene, characterized in that, The product contains the primer-probe combination according to any one of claims 1-4.

6. The product of claim 5, wherein, The product is a reagent or a kit.

7. Use of the primer-probe combination according to any one of claims 1-4 in the preparation of a product for detecting a drug-resistant mutation site of the ESR1 gene.

8. Use according to claim 7, characterized in that, The product is a kit.

9. Use according to claim 8, characterized in that, The reaction system when the primer-probe combination is used for dPCR is as follows: 。 10. Use according to claim 9, characterized in that, The reaction procedure of dPCR is as follows: 。

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