Oligonucleotide set for detecting ESR1 mutation gene, detection method and application

Abstract

The present application relates to the field of biotechnology, and in particular to an oligonucleotide set for detecting a mutant ESR1 gene and its application. The present application provides a set of oligonucleotides for detecting mutations in the estrogen receptor gene α (ESR1). It is composed of an upstream primer shown in SEQ ID NO:1, a downstream primer shown in SEQ ID NO:2, and a probe shown in SEQ ID NO:3 and / or an upstream primer of a nucleotide sequence shown in SEQ ID NO:4, a downstream primer of a nucleotide sequence shown in SEQ ID NO:5, and a probe of a nucleotide sequence shown in SEQ ID NO:6. At a specific annealing temperature, the repressor probe is paired with a wild-type template to block the primer from binding to the wild-type template. At the Tm of the primer, the primer can specifically bind to the mutant template and extend, and amplify the gene mutation fragment in the sample DNA in large quantities through a PCR reaction. The present application also provides a detection kit for an oligonucleotide set and a method for detecting ESR1 gene mutations using a sequencing method, thereby achieving convenient and highly sensitive detection of multiple point mutations in the LBD region of ESR1.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and in particular refers to an oligonucleotide set for detecting ESR1 gene mutation and its application. Background Art

[0002] Breast cancer (HR) is a malignant tumor that occurs in the mammary epithelial tissue and is the most common malignant tumor in women. According to the latest data from the World Health Organization's International Agency for Research on Cancer (IARC) in 2020, the number of new cases of breast cancer reached 2.26 million, officially replacing lung cancer as the world's number one cancer. Breast cancer is highly heterogeneous at the molecular level, and hormone receptor-positive (HR) breast cancer accounts for about 65%-70% of all breast cancers. Endocrine therapy is the preferred treatment option for HR patients. However, the biggest clinical challenge currently faced is the phenomenon of resistance to endocrine therapy. Almost all HR breast cancer patients will eventually develop resistance to endocrine therapy and disease progression. Studies have shown that mutations in the α gene (ESR1) of the estrogen receptor (ER) are an important driving factor for resistance to anti-tumor therapy. ESR1 gene mutations include amplification, rearrangement, and point mutations. ESR1 mutations cause increased ER activity and upregulated expression, and can produce spontaneous activity that is independent of estrogen. Studies have shown that the ESR1 gene encodes ERα protein. The ligand domain (LBD) of ERα protein is the site where the protein binds to estrogen and plays an important role in the process of ER promoting cell proliferation. ERα mutation hotspots are mainly located near tyrosine 537, aspartic acid 538, and glutamic acid 380 in the LBD domain. Mutations at these positions may cause changes in protein conformation, leading to activation of ligand-independent ER signaling pathways, which may ultimately lead to endocrine therapy resistance and promote distant tumor metastasis.

[0003] Due to the above mutations, breast cancer patients are prone to drug resistance. Therefore, it is necessary to detect ESR1 gene mutations from the beginning of the clear diagnosis of hormone receptor-positive breast cancer. Detection runs through the entire process of endocrine therapy, and a reasonable screening and monitoring system is built for hormone receptor-positive breast cancer patients to detect ESR1 gene mutations as early as possible so as to adjust to a better treatment plan in time. Currently, the methods reported for detecting ESR1 mutations are mainly second-generation sequencing technology and third-generation sequencing technology. Among them, the second-generation sequencing technology IlluminaNGS has high sensitivity, but it relies on expensive instruments and analysis software, which greatly increases the detection cost, and the cycle is long and the analysis process is cumbersome, making it difficult to be used as a universal technology. Targeted sequencing based on second-generation sequencing has a long sequencing cycle and high cost, and only a few models of sequencing instruments are approved for clinical diagnosis. It is difficult to dynamically and conveniently monitor the dynamic evolution of ESR1 mutations. Third-generation sequencing technologies such as ddPCR (droplet digital PCR) and ARMs+RT-PCR (mutation retardation amplification system) are also unable to detect multiple point mutation sites through a single PCR amplification. Corresponding probes need to be designed for each mutation. They cannot meet the needs of simultaneous detection of multiple point mutations of ESR1 and cannot adapt to the complex and changeable conditions of clinical samples.

[0004] Based on the demand for ESR1 gene mutation detection in clinical experiments and clinical diagnosis and treatment to simultaneously detect polymorphic mutations in multiple segments and multiple sites within LBD with high detection sensitivity. We excluded ddPCR, ARMs+RT-PCR, IlluminaNGS and targeted sequencing based on second-generation sequencing, and chose to make a technical breakthrough from the pyrosequencing detection technology currently widely used in hospital laboratories. Pyrosequencing technology is a new sequencing technology based on the cascade reaction of four enzymes for quantitative sequence analysis. It is suitable for sequencing and analysis of short sequences. It directly determines the DNA sequence of patient samples and compares it with the known DNA sequence to make a judgment on the condition of breast cancer patients. It can not only guide clinical medication, but also predict the future development of the disease. At present, pyrosequencing technology is widely used in drug resistance analysis, personalized tumor treatment and virus typing. Because pyrosequencing can directly obtain the target nucleotide fragment, it is called the "gold standard" in molecular diagnosis.

[0005] Although pyrophosphate sequencing alone has good real-time performance, it still has the problem of insufficient detection sensitivity. The methods recorded in the literature to improve its detection sensitivity often involve designing corresponding primers or probes for each point mutation, which complicates the amplification step and makes it difficult to achieve practical clinical application.

[0006] The main problems with pyrosequencing are insufficient sensitivity and an overly complex amplification system. We designed two sets of specific repressor probes and paired primers to perform targeted amplification of multiple ESR1 mutant genes, thereby improving the detection sensitivity of pyrosequencing and achieving the purpose of simultaneously detecting multiple gene mutations. Summary of the invention

[0007] In view of the unique variation characteristics of the ligand binding domain (LBD) in the functional mutation region of ESR1, the inventors designed a multi-point mutation targeted amplification system with a repressor probe as the core, combined with pyrophosphate sequencing, which can inhibit the amplification of wild-type sequences and effectively amplify mutant sequences in large quantities.

[0008] To achieve the above objectives, the present application first provides an oligonucleotide set for detecting estrogen receptor gene α (ESR1) mutations, which is composed of an upstream primer of the nucleotide sequence shown in SEQ ID NO:1, a downstream primer of the nucleotide sequence shown in SEQ ID NO:2, and a probe of the nucleotide sequence shown in SEQ ID NO:3; and / or is composed of an upstream primer of the nucleotide sequence shown in SEQ ID NO:4, a downstream primer of the nucleotide sequence shown in SEQ ID NO:5, and a probe of the nucleotide sequence shown in SEQ ID NO:6.

[0009] In the PCR system, the upstream primer, the downstream primer and the probe are used in a ratio of Fprimer:Rprimer≤1 and Switch:Rprimer≥1, preferably 1:2-10:5-10.

[0010] Furthermore, the 3' end of the probe sequence has a modification group that can prevent the 3' end exonuclease and 3' end polymerase from functioning, thereby preventing the probe from extending directly. Optionally, the group is C3 spacer.

[0011] Furthermore, the 5' end of the probe is spaced 1-5 bp away from the 3' end of the upstream primer.

[0012] In some embodiments of the present application, the annealing (melting) temperature of the probe is 1-10° C. higher than the annealing temperature of the mutation detection primer. Alternatively, the melting temperature (Tm) value of the probe is 1-6° C. higher than the melting temperature (Tm) value of the mutation detection primer.

[0013] In some embodiments of the present application, the final concentration of each of the above primers in the PCR amplification system is 0.2-2.5 uM.

[0014] The present application effectively prevents the amplification of the absolutely dominant wild sequence in the wild and mutant mixed sequence containing trace mutant sequences through the combination of the above two groups of upstream and downstream primers and specific probes, and only targets the amplification of a large number of mutant gene sequences; and can effectively amplify multi-point mutation sequences to provide a DNA template for pyrophosphate sequencing.

[0015] Secondly, the present application also provides the use of the above nucleotide group in the preparation of a kit for detecting mutations in the α gene (ESR1) of the estrogen receptor (ER).

[0016] Then, the present application provides a kit for detecting multiple mutations of the ESR1 gene mediated by a repressor probe, the kit comprising an upstream primer of the nucleotide sequence shown in SEQ ID NO:1, a downstream primer of the nucleotide sequence shown in SEQ ID NO:2, and a probe of the nucleotide sequence shown in SEQ ID NO:3; and / or an upstream primer of the nucleotide sequence shown in SEQ ID NO:4, a downstream primer of the nucleotide sequence shown in SEQ ID NO:5, and a probe of the nucleotide sequence shown in SEQ ID NO:6; the primers are thiolated and the probes are modified in a variety of ways, for example, the 3' end has a group that can shield the direct extension of the primer, for example, a spacer-type modification C3Spacer. And a premixed system for PCR; specifically, the premixed system comprises TaqDNA polymerase, PCR buffer, and a mixture of dNTPs.

[0017] The use of primers, probe combinations, and enzymes in the kit can effectively prevent the amplification of the dominant wild-type sequence in the mixed sample, and only amplify a trace amount of mutant sequences. As a result, it is possible to achieve convenient, highly immediate, and highly sensitive detection of single-site polymorphisms, trace amounts, and multi-site mutations in the highly heterogeneous LBD mutation region of the ESR1 small segment, and monitor the condition changes of breast cancer patients at a lower cost.

[0018] Finally, the present application provides a method for detecting ESR1 mutant genes using repressor probes and primers. The method for detecting gene mutations comprises the following steps:

[0019] An upstream primer of the nucleotide sequence shown in SEQ ID NO:1, a downstream primer of the nucleotide sequence shown in SEQ ID NO:2, and a probe of the nucleotide sequence shown in SEQ ID NO:3 and / or an upstream primer of the nucleotide sequence shown in SEQ ID NO:4, a downstream primer of the nucleotide sequence shown in SEQ ID NO:5, and a probe of the nucleotide sequence shown in SEQ ID NO:6 are synthesized and diluted, and then mixed in a desired ratio; the upstream primer, the downstream primer, and the probe can be used in a ratio of Fprimer:Rprimer≤1 and Switch:Rprimer≥1, preferably upstream primer:downstream primer:probe=1:2-10:5-10; then the sample DNA to be tested is added to form a mixture; the mixture is mixed with a premixed system including TaqDNA polymerase, PCR buffer and dNTPs to form a PCR amplification system;

[0020] The mixed PCR amplification system is placed in a PCR instrument to carry out PCR reaction;

[0021] In the PCR program, the annealing / extension temperature is 50-55°C for 1 minute, for a total of 35-40 cycles, to obtain the PCR product; finally, the PCR product is purified and sequenced.

[0022] Furthermore, the final concentration of the upstream and downstream primers and the probe in the PCR amplification system is 0.2uM to 2.5uM. Furthermore, the PCR system is 25-100ul.

[0023] The technical progress achieved by the present invention is that a specific repressor probe that can be fully complementary to the wild-type sequence is provided, and the probe has a higher melting temperature (TM value) than the primer, and still maintains a double-stranded state of complementary pairing with the wild-type sequence at the set PCR extension temperature, so chain extension cannot be carried out; and the mutant sequence has a relatively low melting temperature because it cannot be fully complementary to the probe, and presents a melting state at the set PCR extension temperature, so chain extension can be carried out smoothly. Thereby achieving the purpose of targeted amplification of mutant sequences. Through the monitoring of the fluorescent signal during the PCR process, the probe designed in the present application has a large difference in the melting temperature on the wild-type sequence and the mutant sequence, and the melting peak can be well separated, and the mutant sequence can be effectively amplified in large quantities, providing more DNA templates for subsequent sequencing. Therefore, without increasing the overall process of pyrophosphate sequencing, the sensitivity of pyrophosphate sequencing is greatly improved, and multiple mutation sites and specific mutation types can be accurately distinguished at the same time relative to methods such as digital PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The amplification melting curve diagram according to the present application is shown, wherein Part A is the amplification melting curve diagram when the upstream and downstream primer ratio is 1:1, and when the upstream and downstream primer ratio is 1:1, the probe peak signal is not displayed; Part B is the amplification melting curve diagram when the upstream and downstream primer ratio is 1:10 (asymmetric amplification of R>F), and when the upstream and downstream primer ratio is 1:10, the probe peak signal is obvious; wherein, WT is the wild-type sequence, 1610 is the 1607TA mutant sequence, 1138 is the 1138GC mutant sequence, WT1610 is a 1:1 mixture of the wild-type sequence and the 1607TA mutant sequence, and WT1138 is a 1:1 mixture of the wild-type sequence and the 1138GC mutant sequence.

[0025] Figure 2 The melting curves of the repressor probe according to the present application on the wild-type sequence and the mutant sequence are shown; A is the melting peak of the 1610 probe on the wild-type sequence and the 1607TA mutant sequence respectively; B is the melting peak of the 1610 probe in a 1:1 mixed template system of the wild-type and 1607TA mutant sequences; C is the melting peak of the 1138 probe 1 on the wild-type sequence and the 1138GC mutant sequence respectively; D is the melting peak of the 1138 probe 1 in a 1:1 mixed template system of the wild-type and 1138GC mutant sequences, wherein switch1610 represents the probe bound to the 1610 region, and switch1138 represents the probe bound to the 1138 region.

[0026] Figure 3 The amplification effect of the 1610 probe on the wild-type sequence and mutant sequence templates according to the present application is shown; A is the amplification effect of the 1610 probe on the 1607TA mutant sequence; B is the amplification effect of the 1610 probe on the wild-type sequence; C is the amplification effect of the 1610 probe in the wild-type and 1607TA mutant 1:1 mixed template system (green peak) and the mutant sequence template system (orange peak).

[0027] Figure 4 The figure shows the signal peak of the first generation sequencing of the product obtained after PCR amplification using the 1610 probe according to the present application.

[0028] Figure 5 The melting peak and amplification effect diagram using the 1138switch2 probe according to the present application are shown.

[0029] Figure 6 The melting peak and amplification effect diagram using the 1138switch3 probe according to the present application are shown.

[0030] Figure 7The diagram shows the amplification effect when the ratio of upstream and downstream primers to probes is 1:X:1 according to the present application; A is the amplification effect when Fprimer:Rprimer:Switch=1:1:1; B is the amplification effect when Fprimer:Rprimer:Switch=1:2:1; C is the amplification effect when Fprimer:Rprimer:Switch=1:5:1; D is the amplification effect when Fprimer:Rprimer:Switch=1:10:1; wherein F (Fprimer) is the upstream primer, R (Rprimer) is the downstream primer, and S (Switch) is the repression probe.

[0031] Figure 8 The amplification effect diagram when the upstream and downstream primers and probes are matched in a ratio of 1:2:X according to the present application is shown; A is the amplification effect diagram when Fprimer:Rprimer:Switch=1:2:1; B is the amplification effect diagram when Fprimer:Rprimer:Switch=1:2:2; C is the amplification effect diagram when Fprimer:Rprimer:Switch=1:2:5; D is the amplification effect diagram when Fprimer:Rprimer:Switch=1:2:10.

[0032] Fig. 9 The diagram shows the amplification effect when the upstream and downstream primers and probe ratio is 1:5:X according to the present application; A is the amplification effect diagram when Fprimer:Rprimer:Switch=1:5:1; B is the amplification effect diagram when the primer ratio is Fprimer:Rprimer:Switch=1:5:2; C is the amplification effect diagram when the primer ratio is Fprimer:Rprimer:Switch=1:5:5; D is the amplification effect diagram when the primer ratio is Fprimer:Rprimer:Switch=1:5:10.

[0033] Fig.10 The diagrams of amplification effects when the ratio of upstream and downstream primers to probes according to the present application is 1:10:X are shown; A is the diagram of amplification effects when Fprimer:Rprimer:Switch=1:10:1; B is the diagram of amplification effects when Fprimer:Rprimer:Switch=1:10:2; C is the diagram of amplification effects when Fprimer:Rprimer:Switch=1:10:5; and D is the diagram of amplification effects when Fprimer:Rprimer:Switch=1:10:10.

[0034] Fig.11 A diagram showing the PCR amplification process in a sequencing instrument according to the present application is shown.

[0035] Fig.12 The working principle diagram of amplifying mutant sequences using a repressor probe according to the present application is shown, wherein Forward primer represents the upstream primer, Switch represents the repressor probe, Long hybridizing region represents the long hybridizing region, Bridge represents the bridge, and C3 spacer represents the spacer. DETAILED DESCRIPTION

[0036] In view of the fact that most of the current PCR (polymerase chain reaction) diagnostic methods for ESR1 mutations are still limited to detecting single-point mutations. The applicant has made improvements based on the existing technology to achieve simultaneous detection of multiple mutations, so as to better realize the judgment and monitoring of breast cancer disease changes. The present invention is based on Blocker technology to detect multiple mutation sites, and designs an oligonucleotide that is completely matched and complementary to the mutation site region in the wild-type sequence and modified at the 3' end to prevent its extension as a repressor probe. The Tm value (melting temperature) of the repressor probe is higher than the Tm value of the primer. Therefore, after the denatured double-stranded DNA template is opened, at a specific annealing temperature (Tm), the repressor probe preferentially binds to the wild-type template to block the mutation detection primer from binding to the wild-type template, and then at the annealing temperature (Tm) of the mutation detection primer, the detection primer binds to the mutant template, and the fragment of the gene mutation to be detected in the sample DNA is enriched and amplified by PCR reaction.

[0037] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] If the specific conditions are not specified in the following experiments, they are all carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be obtained through commercial purchase. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the contents recorded can be applied to this application.

[0039] In order to better explain the present invention, the professional terms involved in the present invention are explained as follows. In the context of the present invention, the term "primer" used is two artificially synthesized oligonucleotide sequences, and the primers are divided into two types: forward primer and reverse primer. The forward primer is complementary to the 5' end of the target DNA sequence, and the reverse primer is complementary to the 3' end of another DNA template chain, serving as the starting point of DNA replication. Under the catalysis of TaqDNA polymerase, with dNTP as raw material, the primer extends from 5' to 3' direction to form a new DNA fragment. The term "repressor probe" used is an artificially synthesized oligonucleotide sequence that can fully match and complement the wild-type mutation site region sequence and is modified at the 3' end to prevent its extension. The annealing temperature (Tm value) of the repressor probe is higher than the annealing temperature (Tm value) of the primer. Therefore, after the DNA template is denatured and the double-stranded DNA is opened, at the annealing temperature (Tm) of the repressor probe, the repressor probe preferentially binds to the wild-type template, thereby blocking the binding of the primer to the wild-type template. Then, at the annealing temperature (Tm) of the primer, the primer binds to the mutant template, and the PCR reaction is used to enrich and amplify the gene mutation fragments in the sample DNA, so that the mutant sequence template can be amplified in large quantities.

[0040] Example 1 Investigating the ratio of primers and probes

[0041] 1. Design primers and suppressor probes for mutation sites

[0042] The human ESR1 (estrogen receptor gene) gene sequence (NCBI Reference Sequence: NG_008493.2) published in the NCBI database was used as a wild-type sequence template. By designing a specific repressor probe for the wild-type sequence and optimizing the PCR reaction system, high-sensitivity detection of multiple mutation sites was achieved.

[0043] First, according to the LBD domain of ESR1, three upstream primers named 1610F1-F3 (upstream-Fprimer) and one downstream primer 1610R (downstream-Rprimer), 1610 repressor probe 1610S (switch-probe) were designed for the gene mutation near sites 536, 537 and 538. Three upstream primers named 1138F1-F3 (upstream-Fprimer) and one downstream primer 1138R (downstream-Rprimer), 1138 repressor probe 1138S1 (switch-probe) were also designed for the gene mutation at site 380. The length of the above primers and probes is 15-22bp, and the distance between the 5' end of the probe and the 3' binding position of the upstream primer on the sequence is at least 0-5bp. The primer sequences, modifications and TM values ​​are shown in Tables 1 and 2.

[0044] Table 1: Upstream and downstream primers and probes were synthesized by Sangon Biotech Co., Ltd.

[0045]

[0046]

[0047] Note: The order marked in the table is the sequence order from the 5-end to the 3-end. The number of groups marked with X in the sequence list means that X modification groups of this type are used. The order in which the modification groups are located in the sequence order is the sequential position of the group in the synthetic primer.

[0048] Table 2 shows the modifications made to primers and probes

[0049]

[0050] 2. Screen out suitable primer pairs by examining the TM value

[0051] PRIMER5 calculation revealed that the GC contents of 1610F3 and 1610R as well as 1138F1 and 1138R were similar, and the TM values ​​were closer (see Table 1). Therefore, the upstream and downstream primers 1610F3 and 1610R were selected to amplify the mutant sequence near the 536-538 position, and 1138F1 and 1138R were selected to amplify the mutant sequence near the 380 position.

[0052] 3. Through the significant specificity of the melting peak, explore the proportion of upstream primers, downstream primers and probes

[0053] Since the concentration of primers affects specificity, the optimal final concentration of primers in the system (excluding probes) is generally between 0.1-0.5 μM. Higher primer concentrations will lead to non-specific product amplification. Too many primers will produce misleading or primer dimers, while too low a concentration will reduce PCR amplification efficiency. Under certain conditions, setting different upstream and downstream primer concentrations can improve amplification efficiency.

[0054] As a wild-type closed system, the repressor probe can specifically bind to the wild-type template, thereby blocking the non-specific binding of the primer to the wild-type template. In addition, the modified group at the 3' end of the repressor probe can block the amplification of the probe sequence. Therefore, the concentration of the repressor probe as a blocker can directly affect the efficiency and specificity of PCR amplification. If the blocker concentration is too low, it cannot effectively prevent wild-type amplification, and if the concentration is too high, the PCR amplification efficiency will be reduced. The blocker concentration is generally higher than the primer concentration, and it is common to be 1-2 times the primer concentration. The specific concentration needs to be determined through optimization testing. Based on this, the present application sets the upstream and downstream primer ratio to Fprimer:Rprimer=1:1 or 1:10 and adds probes according to Fprimer:Switch=1:1 or does not add switch1 probes. The specific ratios are shown in Table 3

[0055] Table 3, Primer mix ratios are as follows:

[0056] F-primer(5uM) R-primer(5uM) Switch-probe(5uM) 1 1 1 1 1 1 10 1 10 1

[0057] Then, all mutant sequences including the wild-type sequence and all the point mutation sites involved in this application were synthesized. Each sequence was connected to the pUC-GW-Kan vector (Azenta) to become a synthetic ESR1 point mutation expression vector, which was used as a PCR template in the test phase. The qPCR system is shown in Table 4. The dye method fluorescence quantitative PCR was used in the test to explore the experimental conditions.

[0058] Table 4 qPCR system is as follows

[0059]

[0060] The PCR program was as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing / extension 1) at 50°C for 1 min for 40 cycles; melting curve analysis 2) at 95°C for 15 s, 45°C for 1 min, and 95°C for 1 s.

[0061] Experimental Results

[0062] In the systems of 1610F3 (upstream primer) and 1610R (downstream primer), 1138F1 (upstream primer) and 1138R (downstream primer) ratio = 1:1, upstream primer: downstream primer: probe = 1:1:1, and upstream primer: downstream primer = 1:10, no significant and specific melting peak could be obtained ( Figure 1 A). In the PCR system with a ratio of upstream primer: downstream primer: probe = 1:10:1, the WT sequence, 1607TA base mutation gene sequence, 1138GC mutation gene sequence, 1:1 mixed sequence of WT and 1607TA mutation gene, and 1:1 mixed sequence of WT and 1138GC mutation gene were amplified respectively, and significant specific melting peaks were obtained ( Figure 1 B).

[0063] Therefore, it was determined that the PCR amplification method with an asymmetric ratio of primers and probes was used to obtain a significant and specific melting peak.

[0064] Example 2: Investigating the melting curve of the probe to obtain a suitable annealing temperature

[0065] The mutant sequence and wild-type sequence were amplified using the PCR system and reaction conditions of upstream primer: downstream primer: probe = 1:10:1 in Example 1. The melting peaks of the 1610switch and 1138switch probes in the wild-type and mutant sequence single template systems were as follows: Figure 2 A and 2C. The melting peaks of 1610switch and 1138switch probes in the wild-type and mutant mixed template system are shown in Figure 2 B and 2D. As shown in the figure, the starting temperature (TM) of the mutant sequence melting peak is 55°C, and the actual melting temperature (TM) of the 1610switch probe is 61°C, which is 6°C higher than the TM value of the primer. This ensures that the probe binds to the target fragment before the primer during annealing, thereby preventing the amplification of the wild-type sequence. As can be seen from the figure, there is a very significant difference between the probe Tm in the wild-type (WT) sequence system and the probe Tm in the mutant (MUT) sequence system. Therefore, a melting temperature of 61°C is more appropriate.

[0066] According to the melting temperature of the 1610switch probe, 1610F3-primer and 1610R-primer were determined as upstream and downstream primers. Amplification at a temperature 55°C lower than the melting peak starting temperature of the mutant sequence can maximize the inhibition of amplification of the wild-type sequence, so the primer annealing temperature is set to be no higher than 55°C, between 50-55°C.

[0067] However, the actual melting temperature of the 1138switch probe is 46°C, which is lower than the starting temperature of the mutant sequence melting peak of 55°C. The melting temperature is too low and cannot bind to the target fragment before the primer, so it needs to be adjusted again.

[0068] Example 3: Verification of amplification effect using 1610 switch probe in wild-type and mutant sequence template systems

[0069] 1) Sequence amplification was performed using 1610F3-primer and 1610R-primer as upstream and downstream primers and 1610switch probe at a ratio of 1:10:1 and an annealing temperature of 50-55°C. The results showed that normal amplification was possible in the mutant sequence template system ( Figure 3 A), in the system with wild-type sequence template, amplification inhibition occurs ( Figure 3 B). Probe melting peak of the amplified product of the wild-type and mutant 1:1 mixed system ( Figure 3 C green) overlaps with the probe melting peak of the amplified product of the mutant sequence template system ( Figure 3 C orange). The results showed that the amplification product of the wild-type and mutant 1:1 mixed system was similar to the amplification product of the mutant sequence template system, and the amplification product of the mutant sequence had a greater proportion.

[0070] 2) Amplify the sequence of the wild-type and mutant 1:1 mixed system without adding the 1610 switch probe system, and the amplified product is a mixed signal of the wild-type and mutant ( Figure 4A). When the wild-type and mutant mixed system sequences were amplified by adding the 1610switch probe system, the amplified product only had mutant sequence signals ( Figure 4 B). This indicates that the repressor probe can effectively inhibit the amplification of the target fragment in the wild-type sequence.

[0071] Example 4 Redesign of 1138 switch probe to increase melting temperature

[0072] Since the actual melting temperature of the 1138 switch probe in Example 2 is 46°C, which is lower than the starting temperature of the mutant sequence melting peak of 55°C, the melting temperature is too low to prevent the primer from binding to the wild sequence and extending, and further adjustment is required.

[0073] Here, the probe for position 1138 was redesigned (as shown in Table 5) to increase the melting temperature of the probe. The mutant melting temperature of the new probe "1138switch2" is 66°C ( Figure 5 A orange peak), because the annealing temperature needs to be lower than the melting temperature of the mutant sequence, the melting temperature of the probe "1138switch2" is too high, resulting in a large gap between the Tm value of the 1138switc2 probe and the Tm value of the 1610 site probe (61°C). The 66°C probe 1138switch2 and the 1610 site probe cannot be amplified in the same PCR system using the same amplification conditions. When the annealing temperature of the 1138 site probe used is 60°C, the probe and the mutant sequence can be fully complementary, thereby hindering the amplification of the mutant sequence. However, in the wild-type and mutant 1:1 mixed template system, the effect of targeted amplification of the mutant sequence still cannot be produced ( Figure 5 B orange peak), further design adjustments are required.

[0074] Table 5

[0075]

[0076] In view of the problem that the melting temperature of the 1138switch probe is too high or too low, the 1138switch3 probe (Table 6) was designed again, so that the mutant melting temperature of the new probe "1138switch3" is between 56°C ( Figure 6 Orange peak), the temperature is appropriate, and the melting peaks of the mutant sequence and the wild-type sequence can be well separated. The probe meets the use requirements. According to the melting temperature of the 1138switch3 probe, 1138F1-primer and 1138R-primer are determined as upstream and downstream primers.

[0077] Table 6

[0078]

[0079] Example 5 Optimizing the ratio between upstream and downstream primers and probes

[0080] In order to achieve better targeted amplification efficiency and provide more DNA templates for pyrophosphate sequencing, different ratios of downstream primers (R-primer) were set in the gradient (as shown in Table 7), and the effects of primer pairings with different ratios on the amplification effect were compared.

[0081] Table 7 Fprimer:Rprimer:Switch-probe=1:X:1

[0082] F-primer R-primer Switch-probe F-primer R-primer Switch-probe 1 1 1 1 2 5 1 2 1 1 2 10 1 5 1 1 5 2 1 10 1 1 5 5 1 1 2 1 5 10 1 1 5 1 10 2 1 1 10 1 10 5 1 2 2 1 10 10

[0083] The sequence amplification results are as follows

[0084] 1)Fprimer:Rprimer:Switch-probe=1:X:1

[0085] By changing the ratio of R-primer, it was found that the biggest impact of R-primer is to interfere with the intensity of the probe melting peak signal. At the same time, the increase in the R-primer ratio will also reduce the actual proportion of the probe (Switch-probe) in the system, thereby affecting the targeted amplification effect of the mutant sequence. F-primer and Switch-probe are both base complementary pairing sequences of antisense sequences. F-primer is located upstream of Switch-probe, at least 1-5bp apart. The inhibitory effect of the probe prevents the chain extension of F-primer. Therefore, the actual amplification inhibition target of F-primer for Switch-probe is not easy to be too high. At the same time, since the biotin-labeled R-primer amplification product is the actual sequencing template when it is finally put on the machine, the total ratio of Fprimer to Rprimer has a greater impact on the intensity of the final sequencing signal. When the Rprimer ratio is high (such as Fprimer:Rprimer=1:10), the product concentration is lower than when the Rprimer ratio is low, but the amount of mutant sequence template amplified can still be used to achieve the sequencing goal. The results are as follows Figure 7 As shown in AD.

[0086] 2)Fprimer:Rprimer:Switch-probe=1:2:X

[0087] When Fprimer:Rprimer=1:2 is fixed ( Figure 8 AD), when the ratio of Switch-probe is higher than that of Rprimer, a stronger mutant sequence targeted amplification effect is shown ( Figure 8 C and Figure 8 D).

[0088] 3)Fprimer:Rprimer:Switch-probe=1:5:X

[0089] When Fprimer:Rprimer=1:5 is fixed ( Fig. 9 AD), so that when the ratio of Switch-probe is greater than or equal to Rprimer, a stronger mutant sequence targeted amplification effect is exhibited ( Fig. 9 C and Fig. 9 D).

[0090] 4)Fprimer:Rprimer:Switch-probe=1:10:X

[0091] When Fprimer:Rprimer=1:10 ( Fig.10 AD), so that when the ratio of Switch-probe is greater than or equal to Rprimer, a stronger mutant sequence targeted amplification effect is exhibited ( Fig.10 D).

[0092] Example 6 Pyrosequencing Verification

[0093] Based on the melting peak heights of the above mutant sequences and wild-type sequences, it was preliminarily confirmed that when the ratio of Switch-probe was greater than or equal to Rprimer, a stronger mutant sequence targeted amplification effect was exhibited. Therefore, different PrimerMix configurations of upstream primer: downstream primer: probe = 1:2:5 / 1:5:5 / 1:5:10 / 1:10:10 were used for 1610switch and 1138switch3, respectively. After amplification of the templates containing the 1607TA mutant sequence and the 1138GC mutant sequence, pyrophosphate sequencing was performed according to the method disclosed in Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res. 2001.11(1):3-11.

[0094] The results show (Table 8) that when Fprimer:Rprimer:Switch-probe is 1:2:5 / 1:5:5 / 1:5:10 / 1:10:10, the amplification ratio of 1607TA mutant sequence is increased by more than 20%, and the ratio of 1138GC mutant sequence is increased by more than 10%. Especially when Fprimer:Rprimer:Switch-probe=1:5:10, after targeted amplification, the ratio of plasmid template 1607TA mutant sequence is increased from 50% to 82.83%. The ratio of plasmid template 1138GC mutant sequence is increased from 50% to 67.07%, and the ratio of ctDNA template 1138GC mutant sequence is increased from 2% to 21.67%, with the best mutant sequence targeted amplification effect. Therefore, the ratio of PrimerMix to probe was set to 1:2:5 / 1:5:5 / 1:5:10 / 1:10:10, Fprimer:Rprimer≤1 and Switch:Rprimer≥1.

[0095] Table 8

[0096]

[0097] Example 7 Different gradient ratios of wild-type sequence plasmid and mutant sequence plasmid to verify the sensitivity of 1610switch and 1138switch probe amplification systems

[0098] 1) Using the ESR1 wild-type plasmid, the 1607TA mutant plasmid was continuously diluted with a 2-fold gradient, and a total of 16 dilution gradients were set. After targeted amplification, pyrophosphate sequencing was performed. The results showed (Table 9) that the system could amplify the 50% mutant sequence to 93% at the lowest and the 0.0015% mutant sequence to 16% at the highest under the set multiple gradient ratios, indicating that the system has a good effect of targeted amplification of mutant sequences in the case of large and trace mutant sequence proportions.

[0099] Table 9

[0100]

[0101] 2) The 1138GC mutant plasmid and the ESR1 wild-type plasmid were used for gradient ratio testing of the 1138switch probe amplification system. The 1138GC mutant plasmid was diluted with the ESR1 wild-type plasmid in a 2-fold gradient, and 16 dilution gradients were set. After targeted amplification, pyrophosphate sequencing was performed. The results showed (Table 10) that the performance of the 1138switch probe amplification system was slightly lower than that of the 1610switch probe amplification system, but it could also meet the requirements for the use of amplified sequencing templates.

[0102] Table 10

[0103]

[0104]

[0105] According to the above investigation results

[0106] 1) The reaction conditions of the above experiments were: PCR pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, annealing / extension 1) at 50°C for 1 minute for 40 cycles. Melting curve analysis 2) at 95°C for 15 seconds, 45°C for 1 minute, and 95°C for 1 second.

[0107] 2) The qPCR system is as follows

[0108]

[0109] 3) Primer mix ratio:

[0110] F-primer(5uM) R-primer(5uM) Switch-probe(5uM) 1 1-10 2-10

[0111] Example 8: Verification of 1610switch and 1138switch probe amplification systems using blood samples from breast cancer patients

[0112] According to the sample size requirements of the Phase I clinical trial, 30 peripheral blood samples from ER-positive breast cancer patients from the Cancer Hospital of the Chinese Academy of Medical Sciences were collected, and ctDNA was extracted, and the next-generation sequencing and pyrophosphate sequencing based on this targeted amplification method were performed simultaneously. The results showed that the system in this application can amplify multiple mutations near 1610 and 1138, and obtain excellent sequencing results.

[0113] The specific results are shown in Table 11

[0114]

[0115]

[0116] According to the above sequencing results, the mutation types detected by this application include D538G-1613A>G, Y537S-1610A>C, Y537N-1609T>A, Y537C-1610A>G, L536H-1607T>A, L536P-1607T>C, L536R-1607T>G, and E380Q-1138G>C. It is proved that the use of this primer pair can effectively and accurately detect multiple point mutations near the 537th tyrosine, the 538th aspartic acid, and the 380th glutamic acid in the LBD domain, providing an auxiliary monitoring means for grasping the real-time changes of the disease.

[0117] In summary, this application designs a blocking probe that can specifically bind to the wild-type mutation region sequence and is used together with a detection primer with selective binding. Since the repressor probe has a higher TM value, it can preferentially bind to the wild-type target sequence and prevent its extension, and the detection primer can bind to the mutant target nucleic acid sequence, thereby enriching the mutant variants of the target nucleic acid sequence in the nucleic acid sample through the PCR reaction, and combining pyrophosphate sequencing to detect the specific mutant gene through the change of the detection signal of the detection probe, so as to achieve the purpose of specific selective detection. Compared with digital PCRARMs+RT-PCR, which cannot detect multiple site mutations through a single PCR amplification, its detection effect on multiple point mutations is more significant, and it has the advantages of high sensitivity, short cycle and lower cost, which provides a powerful auxiliary means for monitoring the changes in the condition of breast cancer patients and adjusting treatment measures in time.

[0118] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for detecting estrogen receptor α gene mutation, characterized in that: The primer set includes a first primer set and a second primer set; The upstream primer of the first primer set is GCATGAAGTGCAAGAACGT*G*G*T, the downstream primer is 5'BiotinAAGTGGCTTTGGTCCGTCTC, the probe is 5'BHQ1* / i2OMeC / * / i2OMeC / *CTCTATGACC / i5NitInd / / i5NitInd / / i5NitInd / / i5NitInd / G / i6FAMdT / CTGGAGATGCTGGA* / i2OMeC / * / i2OMeG / (C3 Spacer)-3', and the concentration ratio of the upstream primer, the downstream primer and the probe is 1:5:10; and The upstream primer of the second primer set is GACCCTCCATGATCAGG*T*C*C, the downstream primer is 5'BiotinGTCCAAGAGCAAGTTAGGAGCA, the probe is 5'BHQ1* / i2OMeC / * / i2OMeC / * / i2OMeU / *TCTAGAATGT / i5NitInd / / i5NitInd / / i5NitInd / / i5NitInd / G / i6FAMdT / GCTAGAGATCCTGAT* / i2OMeG / * / i2OMeA / * / i2OMeU / (C3 Spacer)-3, and the concentration ratio of the upstream primer, the downstream primer and the probe is 1:5:10; Wherein, the * is thio modification.

2. Use of the primer set according to claim 1 in preparing a kit for detecting estrogen receptor α gene mutation, characterized in that: The estrogen receptor α gene mutations include: D538G-1613A>G, Y537S-1610A>C, Y537N-1609T>A, Y537C-1610A>G, L536H-1607T>A, L536P-1607T>C, L536R-1607T>G, and E380Q-1138G>C.

3. A kit comprising the primer set according to claim 1, characterized in that: The kit is used to detect estrogen receptor α gene mutations, and the estrogen receptor α gene mutations include: D538G-1613A>G, Y537S-1610A>C, Y537N-1609T>A, Y537C-1610A>G, L536H-1607T>A, L536P-1607T>C, L536R-1607T>G, and E380Q-1138G>C. The kit also includes a PCR premix system containing Taq DNA polymerase, PCR buffer and dNTPs.

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