Digital PCR detection kit for detecting multiple mutation sites of ESR1 gene
By designing the ESR1 gene multiplex digital PCR detection kit based on Spacer structure and fluorescent blocking probe, the detection problem of multiple high-frequency mutation sites in breast cancer ctDNA samples was solved, and efficient and accurate multiple detection was achieved, suitable for clinical dynamic monitoring.
Patent Information
- Application Number
- CN202510785691.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-08-15
AI Technical Summary
The prior art is difficult to efficiently and easily detect multiple high-frequency mutation sites of the ESR1 gene in breast cancer ctDNA samples simultaneously, especially L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G and E380Q, and there is a problem of insufficient sensitivity and specificity.
Using a combination strategy of molecular beacon probes and fluorescent blocking probes based on Spacer structure, the ESR1 gene multiple digital PCR detection kit is designed to detect multiple mutation sites simultaneously through a single tube reaction, and the Spacer structure is used to improve probe stability and reduce background signals. The fluorescent blocking probe further inhibits non-specific fluorescent background.
High-throughput and high-sensitivity ESR1 gene mutation site detection is achieved, which can accurately identify multiple mutation sites in clinical samples. It is suitable for clinical detection scenarios with limited ctDNA sample size, improving detection efficiency and accuracy.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of digital PCR detection, and in particular to a digital PCR detection kit for detecting multiple mutation sites in the ESR1 gene. Background Art
[0002] The estrogen receptor 1 (ESR1) gene encodes estrogen receptor α (ERα), a key driver of breast cancer pathogenesis and progression. In hormone receptor-positive (HR+) breast cancer, in particular, the activation state of the ER signaling axis is closely associated with response to endocrine therapy. Activating mutations in the ESR1 gene often occur within the hormone-binding domain (LBD). These mutations lead to sustained activation of ERα, conferring resistance to endocrine drugs such as aromatase inhibitors (AIs) on tumor cells, and are a key mechanism of acquired drug resistance in advanced breast cancer.
[0003] Current research has revealed that hotspot mutations in ESR1 are primarily concentrated at the following amino acid residues: L536, Y537, D538, and E380. Common mutations include L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, and E380Q. These mutations are highly prevalent in patients who fail aromatase inhibitor therapy and are closely associated with shortened progression-free survival (PFS) and decreased overall efficacy. Therefore, they hold important guiding value in clinical decision-making.
[0004] Liquid biopsy, as a non-invasive, dynamic technology for monitoring patient conditions, is particularly suitable for clinical scenarios where tissue sample access is limited. As a core method of liquid biopsy, ctDNA testing is gradually replacing traditional tissue pathology testing in tumor molecular monitoring. Current methods for ESR1 mutation detection mainly include second-generation sequencing (NGS), fluorescence quantitative PCR (qPCR), and digital PCR. Although NGS has advantages such as high throughput and wide coverage, it has limitations such as complex operation, long cycle time, high cost, and high platform requirements, making it difficult to meet the needs of routine clinical rapid testing. While qPCR technology is simple to operate, it has poor sensitivity and specificity, and is particularly susceptible to background interference when detecting low-abundance mutations in ctDNA, resulting in insufficient accuracy.
[0005] Digital PCR (dPCR) technology, leveraging its high-throughput microzonation capabilities and the Poisson distribution mathematical model, enables absolute quantification of mutant molecules with detection sensitivities of 0.1% or even lower, making it particularly suitable for accurately identifying low-frequency variants in ctDNA. Furthermore, dPCR supports multiplexed detection, enabling simultaneous coverage of multiple mutation sites in a single-tube reaction, minimizing sample waste and improving overall sensitivity.
[0006] Currently, there is a lack of a digital PCR kit specifically designed to detect multiple high-frequency mutation sites in the LBD region of the ESR1 gene. This is particularly true for the combined detection of multiple mutations in breast cancer ctDNA samples. Therefore, developing a digital PCR kit with high sensitivity, strong specificity, and ease of use, covering the major clinical mutation sites of ESR1, would provide an important tool for the dynamic monitoring and personalized treatment of breast cancer. Summary of the Invention
[0007] To address the above problems, the present invention provides a digital PCR detection kit for detecting multiple mutation sites in the ESR1 gene. The kit detects the L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, and E380Q mutation sites of the ESR1 gene using a single digital PCR reaction tube. Each site is detected using its own spacer molecular beacon detection probe, which sequentially comprises a 5' stem sequence, a loop sequence, a flexible spacer structure, and a 3' stem sequence, forming a hairpin structure as a whole. The 5' end is labeled with a fluorescent group, the 3' end is labeled with a quencher group, and the 5' stem sequence and the 3' stem sequence complementary sequence form a stable stem structure. The 5' stem sequence and the loop sequence are completely complementary to the target sequence to be detected, and the loop sequence and the flexible spacer structure form the loop structure of the molecular beacon detection probe. The flexible spacer structure is a polyethylene glycol structure.
[0008] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is not less than three.
[0009] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 3-6.
[0010] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 6.
[0011] In one embodiment, the kit includes a spacer molecular beacon detection probe SEQ ID NO. 16 of L536Q, a spacer molecular beacon detection probe SEQ ID NO. 17 of L536H, a spacer molecular beacon detection probe SEQ ID NO. 18 of L536P, a spacer molecular beacon detection probe SEQ ID NO. 19 of L536R, a spacer molecular beacon detection probe SEQ ID NO. 20 of Y537N, a spacer molecular beacon detection probe SEQ ID NO. 21 of Y537C, a spacer molecular beacon detection probe SEQ ID NO. 22 of Y537D, a spacer molecular beacon detection probe SEQ ID NO. 23 of Y537S, a spacer molecular beacon detection probe SEQ ID NO. 24 of D538G, and a spacer molecular beacon detection probe SEQ ID NO. 25 of E380Q.
[0012] In one embodiment, the kit further comprises a wild-type spacer molecular beacon detection probe SEQ ID NO. 26 targeting the L536–D538 site.
[0013] In one embodiment, the kit further includes two fluorescent blocking probes SEQ ID NO. 27 and SEQ ID NO. 28; the sequences of the fluorescent blocking probes are complementary to the 6-base regions at the 5' ends of the corresponding multiple molecular beacon detection probes, and the 3' ends of the fluorescent blocking probes are labeled with a fluorescent quenching group. The fluorescent blocking probes bind to the 5' ends of the molecular beacon detection probes in a free state to form a stable secondary blocking structure.
[0014] In one embodiment, the kit further comprises two pairs of amplification primers, the first pair of primers covering the L536 to D538 region, and being used to amplify a fragment comprising the L536, Y537, and D538 sites, the upstream primer being SEQ ID NO. 12, and the downstream primer being SEQ ID NO. 13; the second pair of primers targeting the E380 site region, and being used to specifically amplify the E380Q mutation segment, the upstream primer being SEQ ID NO. 14, and the downstream primer being SEQ ID NO. 15.
[0015] The ESR1 digital PCR multiplex detection kit provided by this invention features 13 probes designed in a single-tube reaction system, covering 10 clinically significant ESR1 gene mutation hotspots in breast cancer: L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, E380Q, and their corresponding wild-type sites. All probes are dispensed into a single tube for multiplex reactions and labeled with five fluorophores, enabling accurate typing and quantification of mutant and wild-type signals.
[0016] Based on a combined optimization strategy of molecular beacon probes and fluorescent blocking probes, the inventors have developed a digital PCR multiplex detection kit for ESR1 gene mutations suitable for liquid biopsy (ctDNA). This innovative "one-tube assay" design simultaneously identifies 10 clinically frequent mutations within the hormone-binding domain (LBD) of the estrogen receptor α: L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, and E380Q. These mutations encompass key mutations recognized in current research and consensus as closely associated with aromatase inhibitor resistance and endocrine therapy failure. This design significantly improves detection throughput and sample utilization while maintaining high sensitivity and specificity, making it particularly suitable for clinical testing in settings with limited ctDNA sample quantities.
[0017] In order to solve the problems of specific interference and fluorescence background accumulation faced by the simultaneous detection of multiple mutation hotspot sites in the same reaction system, the present invention adopts the following optimization strategies: (1) Construction of molecular beacon probe based on Spacer structure: The present invention innovatively introduces a Spacer flexible connecting arm into the molecular beacon detection probe structure, which is located between the 3' stem sequence of the probe and the main target recognition region. The Spacer structure has the following key advantages: 1) Improved spatial flexibility: It enables short sequence probes to efficiently form a stable hairpin-shaped stem-loop structure, significantly improving the closure efficiency; 2) Effectively reduce background signals: The distance between the fluorescence and the quenching group is more stable in the closed loop state, and the fluorescence inhibition is more sufficient in the non-binding state; 3) Enhanced probe structure design compatibility: Spacer reduces the possibility of non-specific pairing between the 3' stem and the target, avoids interference with the probe Tm, and is conducive to the design of multi-probe co-detection; 4) Improved multiple mutation detection capabilities: When detecting multiple homologous mutations or multi-site mutations, the Spacer structure enables the probe to maintain more consistent structural stability and background suppression capabilities, thereby achieving high-throughput and high-sensitivity mutation site resolution detection. The introduction of the Spacer structure of the present invention reduces the risk of non-specific complementary pairing between the 3' stem and the target; enhances the structural stability of the probe in the free state; and improves the spatial conformational adaptability of the beacon probe after binding to the mutant sequence, thereby facilitating enzymatic cleavage and signal release. (2) Introduction of fluorescent blocking probe system: To further reduce the non-specific fluorescence background, this kit introduces a class of specially designed fluorescent blocking probes. The blocking probe is a short-chain oligonucleotide whose sequence is highly complementary to the 5' stem region of the main probe and is labeled with a fluorescent quenching group at the 3' end. When the target DNA is not bound to the target sequence, the blocking probe preferentially binds to the 5' end region of the molecular beacon to form a stable double-stranded structure, effectively blocking the fluorescence release channel, thereby significantly improving the signal-to-noise ratio of the system.
[0018] Clinical validation results showed that the results of 15 clinical samples tested using the kit were consistent with those from NGS sequencing, including 9 samples with positive results for different mutation types and 6 samples with negative results. This indicates that the composition of the present invention is highly effective in detecting ESR1 mutations in plasma cell-free DNA. Furthermore, the kit can accurately provide information on sample mutation rates, facilitating dynamic clinical monitoring of changes in mutation abundance. DETAILED DESCRIPTION
[0019] In order to enable those skilled in the art to better understand the technical solutions of this application, the present invention will be further described below with reference to the following embodiments. Obviously, the embodiments described are only part of the embodiments of this application, rather than all the embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of this application.
[0020] Example 1 Probe screening experiment To achieve single-tube detection of 10 hotspot mutation sites in the ESR1 gene (L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, and E380Q), the present invention selected two conserved sequence fragments located in the mutation-enriched region and designed amplification primers for each region: the first pair of primers covers the L536 to D538 region and is used to amplify the fragment containing the L536, Y537, and D538 sites; the second pair of primers targets the E380 site region and is used to specifically amplify the E380Q mutation segment.
[0021] A single-tube multiplex reaction strategy was used to detect the above two regions, and common sites were typed, including E380Q, D538G, and Y537S. Other sites L536Q, L536H, L536P, L536R, Y537N, Y537C, and Y537D were detected using probes of the same fluorescent channel. At the same time, corresponding fluorescent probes were designed for the wild-type sequences of sites 536-538 as internal standard genes to check whether the quality control detection system is amplified normally.
[0022] The primer and probe sequences are detailed in Table 1. Since the detection sites are concentrated, the mutant probe sequences of 536-538 are basically the same except for the mutation site. Therefore, the present invention uses molecular beacon probes for detection to reduce the background signal of the same labeled probe in the reaction system.
[0023] Tm and ΔTm were obtained using OligoAnalyzer from IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).
[0024] Table 1 Primer and probe sequences
[0025] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template and the wild-type template; lowercase letters represent the 3-terminal stem sequence that does not match the target; uppercase letters with underlines at the 3 ends represent the 3-terminal stem sequence that matches the target; the underlined bases in the middle represent the mutation sites.
[0026] The primer and probe combinations designed in Table 1 were used to construct a multiplex digital PCR reaction system. The specific operation process is as follows: (1) System preparation Prepare a basic PCR reaction system (total volume 30 µL) according to the table below.
[0027]
[0028] The final concentrations of primers and probes were 600 nM and 200 nM, respectively.
[0029] (2) Digital PCR workflow Microdroplet preparation: Using a droplet generation chip (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) and a sample preparation instrument (Xinyi Manufacturing Technology (Beijing) Co., Ltd.), 30 µL of PCR reaction system was added to the sample well of the droplet generation chip, and 180 µL of droplet generation oil was added to the oil well. The chip and 8-tube strips were placed in the preparation instrument and covered with a rubber gasket to prepare microdroplets.
[0030] PCR amplification: Place the 8-tube strip containing microdroplets on a PCR instrument for amplification. The amplification program is set as shown in the following table:
[0031] Droplet detection: After PCR, the 8-tube strip and droplet detection chip (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) were placed in a fixture. 430 µL and 500 µL of detection oil were added to the oil wells, respectively. The chip was covered with a rubber gasket and placed in a chip analyzer (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) for droplet detection.
[0032] Data Analysis: Each of the tens of thousands of microdroplets created using a droplet generation chip and sample preparation instrument functions as an independent PCR reactor. Most droplets contain no target gene or at least one. After PCR amplification, the chip analyzer detects fluorescence signals in each channel of each droplet, recording the peak height of the droplet signal. Droplets containing the target gene will be detected with a corresponding fluorescence signal. Fluorescence intensity within the droplet is digitized using a fluorescence classification threshold. Droplets with strong fluorescence are interpreted as "1" (positive), and those with weak fluorescence as "0" (negative). The number of "1" and "0" values is counted and corrected using a Poisson distribution model to calculate the total copy number of each fluorescently labeled target gene in the input template.
[0033] Plasmid-constructed mutant templates were mixed with fragmented human wild-type genomic DNA in appropriate proportions to create a simulated sample with a 10% mutation frequency (the mutant and wild-type template copies were 50 and 500 copies / μL, respectively, with 2 μL of the mixed template added to each reaction). Simultaneously, pure wild-type genomic DNA, unadulterated with mutations, was used as a control template to conduct a preliminary evaluation of the system's performance. The test results are shown in Table 2.
[0034] The detection results showed that none of the mutant probes produced nonspecific fluorescence signals in the wild-type template, demonstrating that the system has good ability to distinguish wild-type from mutants under the current conditions. Table 1 also shows that when the melting temperature (Tm) of each mutant probe reaches 65°C or above, the ΔTm value between it and the corresponding wild-type sequence is generally greater than 5°C, achieving good discrimination. Only the ΔTm for the Y537N site is slightly below 5°C. However, because the detection system also includes wild-type probes covering sites 536–538, the wild-type probes have a significant binding advantage when used with wild-type templates, specifically generating a fluorescent signal, while the mutant probes lack a significant signal, thus effectively distinguishing between the two.
[0035] However, because multiple mutant probes share the FAM fluorescence channel, the background signal in this channel is high, which affects the detection of positive signals. Specifically, no positive signals are observed when detecting mutant templates such as L536Q, L536H, and Y537D, even though these probes have been previously verified to have clear positive detection capabilities in single-plex assays. Therefore, further optimization of the reaction system is necessary to reduce background interference in the FAM channel and ensure that each mutant can also produce stable and recognizable positive signals in the complex system.
[0036] Table 2 Test results
[0037] The molecular beacon probe system was further optimized to reduce the background signal of the reaction system and improve the signal-to-noise ratio. By introducing a flexible Spacer18 group into the molecular beacon probe, the Spacer structure was placed in the probe loop region to enhance the stability of the hairpin structure, thereby further reducing the background signal. The probe sequences are shown in Table 3.
[0038] Table 3 Spacer molecular beacon probe sequences
[0039] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template and the wild-type template; lowercase letters are the 3-terminal stem sequences that do not match the target; Spacer 18 is 6 polyethylene glycol (PEG) units; the underlined bases in the middle represent the mutation site.
[0040] The reaction system preparation and digital PCR detection procedures are described above. Plasmid-constructed mutant templates were mixed with fragmented human wild-type genomic DNA in appropriate proportions to create a mock sample with a 10% mutation frequency (the copy numbers of the mutant and wild-type templates were 50 and 500 copies / μL, respectively, with 2 μL of mixed template added to each reaction). Pure, unadulterated wild-type genomic DNA was also used as a control template for evaluation. The test results are shown in Table 4. The use of the spacer probe further reduced the background signal in the system, improving the signal-to-noise ratio. Positive signals were detected for all mutant templates, with no cross-reactivity with the wild-type template. The copy numbers were consistent with the expected results. However, the background in the FAM channel remained high, resulting in a low signal-to-noise ratio. In particular, for the L536Q and Y537D templates, the positive signal and background were close.
[0041] Table 4. Test results
[0042] To further reduce background signals in the amplification reaction system, the present invention also designed a fluorescent blocking probe. This probe is a short, small molecule probe complementary to the 5' end sequence of the beacon probe. Its 3' end is labeled with a highly efficient quenching group (such as BHQ1) and does not carry a fluorescent group. Its mechanism of action is as follows: when the molecular beacon probe is in a free, unblocked state, the fluorescent blocking probe binds to the 5' end sequence, effectively quenching fluorescence and further blocking the nonspecific background signal generated by the free, unblocked probe in the system. The sequence is shown in Table 5.
[0043] Table 5. Fluorescence blocking probe sequences
[0044] The reaction system preparation and digital PCR detection procedures were as described above. A mock sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (mutant and wild-type concentrations of 50 and 500 copies / µL, respectively, with 2µL of each system used). This was then tested alongside the wild-type genome as a template. The results of the addition of a fluorescent blocking probe are shown in Table 6 below. The use of the fluorescent blocking probe further reduced the background signal in the FAM channel of the system, further improving the signal-to-noise ratio. Specific detection of each mutant and wild-type template was achieved, and the copy number results were consistent with the expected results.
[0045] Table 6 Detection results of adding fluorescent blocking probe
[0046] Example 2 Composition of the detection kit of the present invention This kit includes a reaction system containing L536Q, L536H, L536P, L536R, Y537N, Y537C, and Y537D (labeled with FAM fluorescence, not typing), E380Q (labeled with ROX fluorescence), D538G (labeled with CY5 fluorescence), and Y537S (labeled with CY5.5 fluorescence), and the wild-type 536-538 (labeled with HEX fluorescence, serving as an internal standard gene). Two nucleic acid reaction solutions (reagent A and reagent B), a negative control (DEPC-treated water), and a positive control (a mixture of each mutant plasmid) are packaged together and accompanied by instructions. This yields the kit for detecting 10 hotspot mutations in the ESR1 gene described herein. The compositions of the premix, test solution, negative control, and positive control are shown in Table 7.
[0047] Table 7 Components of the ESR1 gene mutation detection kit
[0048] Example 3 Clinical Sample Validation of the Kit (1) Reagent preparation: According to the number of samples to be tested, negative controls, and positive controls, take out the PCR reaction solution and calculate the number of each reaction solution to be packaged (n = number of samples + positive controls + negative controls). According to the required number of samples n, add 7.5 μL of PCR reagent A and 3 μL of PCR reagent B to the PCR reaction tubes respectively, mix thoroughly, centrifuge briefly, and set aside.
[0049] (2) Sample processing: The sample was 2 ml of anticoagulated plasma. Plasma-free DNA was extracted using a commercial extraction kit. After extraction, DNA was eluted using TE buffer. Nucleic acids were denatured at 85°C for 5 min before addition to the reaction tube.
[0050] 10.5 μl of PCR reaction solution B and 19.5 μl of free DNA to be tested were added to the reaction tube, and droplet generation, PCR amplification, and droplet detection were performed according to the procedures described in Example 1. After the PCR amplification reaction was completed, genotyping was performed based on the target copy number. The principles for determining the results are as follows: ① The copy number of each target and the wild-type copy number in the positive control are both greater than 50 copies; ② The copy number of each target in the negative control group is 0, and the wild-type copy number is no more than 5 copies; ③ If both of the above conditions are met, the experiment is considered successful, and typing is performed based on the copy number of each target in the sample to be tested. The interpretation criteria are as follows: when the copy number of a target is greater than 3 copies, and the copy number of the internal standard gene (wild-type + mutant copy number) is greater than 500 copies, the target is positive; if the copy number of all targets is no more than 3 copies, and the copy number of the internal standard gene (wild-type + mutant copy number) is greater than 500 copies, the sample is negative; if the copy number of the internal standard gene is less than 500 copies, the test result is invalid, and amplification inhibition may exist or the nucleic acid concentration is too low, and the nucleic acid needs to be re-extracted for testing.
[0051] The test results are shown in Table 8 below. A total of 15 clinical samples were tested, including 9 positive samples with different mutation types and 6 negative samples. The results detected using the kit of the present invention were consistent with the NGS sequencing results, demonstrating that the composition of the present invention is highly capable of detecting ESR1 mutations in plasma free DNA. Furthermore, the kit of the present invention can accurately provide information on the sample mutation rate, facilitating dynamic clinical monitoring of changes in mutation abundance.
[0052] Table 8 Clinical sample test results
[0053] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.
[0054] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.
Claims
1. A digital PCR detection kit for detecting multiple mutation sites in the ESR1 gene, characterized in that: The kit detects the mutation sites L536Q, L536H, L536P, L536R, Y537N, Y537S, Y537C, Y537D, D538G, and E380Q of the ESR1 gene using a single-tube digital PCR reaction tube; each site is detected using its own spacer molecular beacon detection probe, which sequentially includes a 5' stem sequence, a loop sequence, a flexible spacer structure, and a 3' stem sequence, forming a hairpin structure as a whole; the 5' end is labeled with a fluorescent group, the 3' end is labeled with a quenching group, and the 5' stem sequence and the 3' stem sequence complementary sequence form a stable stem structure; the 5' stem sequence and the loop sequence are completely complementary to the target sequence to be detected, and the loop sequence and the flexible spacer structure form the loop structure of the molecular beacon detection probe; and the flexible spacer structure is a polyethylene glycol structure.
2. The digital PCR detection kit according to claim 1, characterized in that The number of polyethylene glycol units in the flexible Spacer structure is not less than three.
3. The digital PCR detection kit according to claim 1, characterized in that The number of polyethylene glycol units in the flexible Spacer structure is 3-6.
4. The digital PCR detection kit according to claim 3, characterized in that The number of polyethylene glycol units in the flexible Spacer structure is 6.
5. The digital PCR detection kit according to claim 4, characterized in that The kit includes a spacer molecular beacon detection probe SEQ ID NO. 16 of L536Q, a spacer molecular beacon detection probe SEQ ID NO. 17 of L536H, a spacer molecular beacon detection probe SEQ ID NO. 18 of L536P, a spacer molecular beacon detection probe SEQ ID NO. 19 of L536R, a spacer molecular beacon detection probe SEQ ID NO. 20 of Y537N, a spacer molecular beacon detection probe SEQ ID NO. 21 of Y537C, a spacer molecular beacon detection probe SEQ ID NO. 22 of Y537D, a spacer molecular beacon detection probe SEQ ID NO. 23 of Y537S, a spacer molecular beacon detection probe SEQ ID NO. 24 of D538G, and a spacer molecular beacon detection probe SEQ ID NO. 25 of E380Q.
6. The digital PCR detection kit according to claim 5, characterized in that The kit also includes a wild-type spacer molecular beacon detection probe SEQ ID NO. 26 for the L536–D538 site.
7. The digital PCR detection kit according to claim 5, characterized in that The kit also includes two fluorescent blocking probes SEQ ID NO. 27 and SEQ ID NO. 28; the sequences of the fluorescent blocking probes are complementary to the 6-base regions at the 5' ends of the corresponding multiple molecular beacon detection probes mentioned above, and the 3' ends of the fluorescent blocking probes are labeled with a fluorescent quenching group. The fluorescent blocking probes bind to the 5' ends of the molecular beacon detection probes in a free state to form a stable secondary blocking structure.
8. The digital PCR detection kit according to claim 5, characterized in that The kit also includes two pairs of amplification primers. The first pair of primers covers the L536 to D538 region and is used to amplify a fragment containing the L536, Y537 and D538 sites. The upstream primer is SEQ ID NO. 12, and the downstream primer is SEQ ID NO.
13. The second pair of primers targets the E380 site region and is used to specifically amplify the E380Q mutation segment. The upstream primer is SEQ ID NO. 14, and the downstream primer is SEQ ID NO. 15.