Digital PCR detection kit for detecting multiple mutation sites of PIK3CA gene

Abstract

The invention provides a digital PCR (Polymerase Chain Reaction) detection kit for detecting multiple mutation sites of a PIK3CA gene, the detection of 11 mutation sites of the PIK3CA gene is realized through two digital PCR reaction tubes, namely a tube A and a tube B, the tube A is used for detecting E545K, E545A, E545G, E545D, Q546E and Q546R sites, and the tube B is used for detecting E542K, C420R, H1047R, H1047L and H1047Y sites; wherein each site is detected by using a respective spacer molecular beacon detection probe. According to the invention, a Spacer structure is introduced into a short-sequence high-specificity probe, so that the problems of background control and structure ring formation of a traditional molecular beacon in complex mutation detection are solved, and the detection accuracy and the application range are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of digital PCR detection, and particularly to a digital PCR detection kit for detecting multiple mutation sites of the PIK3CA gene. Background Art

[0002] The PIK3CA gene encodes the p110α subunit of phosphatidylinositol 3-kinase (PI3K), which is an important component of the pathway regulating cell proliferation, survival and metabolism. Mutations in this gene can lead to the continuous activation of the PI3K / AKT / mTOR signaling pathway, thereby inducing malignant transformation of cells. In breast cancer, PIK3CA is one of the most common driver genes. It is reported that activating mutations exist in about 30%-40% of patients, and the incidence rate is even higher in the Chinese population, reaching 44-50%. The common mutation hotspots are mainly distributed at sites such as exon 9 (E542K, E545K) and exon 20 (H1047R, H1047L).

[0003] PIK3CA mutations are not only important markers for molecular typing and prognosis judgment, but also closely related to the sensitivity of patients to treatment regimens such as CDK4 / 6 inhibitors and endocrine therapy. Therefore, accurately, rapidly and highly sensitively detecting PIK3CA mutations has important clinical value. With the development of the "liquid biopsy" technology, the detection of mutations based on circulating tumor DNA (ctDNA) in plasma has become an important means for non-invasive and dynamic monitoring of the disease state of patients. CtDNA detection has advantages such as strong repeatability and good real-time performance, and is particularly suitable for clinical scenarios where it is difficult to obtain tissue samples. Currently, the methods for detecting PIK3CA mutations include fluorescence quantitative PCR (qPCR), next-generation sequencing (NGS) and digital PCR (digital PCR), etc.

[0004] NGS (next-generation sequencing) has advantages such as high throughput and whole-genome coverage, but its popularization in clinical detection still faces many limitations: First, the NGS process is complex and involves multiple steps such as library construction, capture, on-machine sequencing and bioinformatics analysis; second, the operation cycle is long, usually taking 5-7 working days, making it difficult to meet the clinical demand for rapid result output; in addition, its high detection cost and high requirements for laboratory platforms and personnel operation levels also limit its application in grass-roots and routine scenarios to a certain extent.

[0005] Fluorescence quantitative PCR is commonly used in clinical practice due to its simple operation and low cost. Currently, it mainly detects gene mutation sites through the ARMS (Amplification Refractory Mutation System) technology. However, the ARMS-qPCR method has the following limitations: limited sensitivity, usually around 0.5% - 1%, making it difficult to meet the needs of detecting low-frequency mutations in ctDNA; low specificity, prone to false positives, especially affecting the interpretation when interfered by background heterozygous signals; limited detection sites, and currently, PIK3CA detection kits often only cover a few common mutation sites, unable to meet the increasingly rich clinical classification and companion diagnosis needs; to improve sensitivity, it is usually necessary to detect different sites / genotypes in separate tubes, and separating templates will further reduce the overall detection sensitivity under the condition of low ctDNA sample volume.

[0006] In contrast, digital PCR (dPCR) technology realizes the absolute quantification of target mutant molecules by performing high-throughput micropartitioning on samples, combined with the Poisson distribution statistical model, without relying on a standard curve, and can improve the detection sensitivity to 0.1% or even lower, and is more suitable for detecting a small amount of samples. Therefore, dPCR is particularly suitable for the accurate detection of PIK3CA mutations in the context of ctDNA liquid biopsy.

[0007] Currently, there is still a lack of a digital PCR detection kit with high coverage, high sensitivity, good repeatability, and specifically designed for multiple PIK3CA mutation sites. There is an obvious technical gap especially in the unified detection of multiple hot-spot mutations in ctDNA samples. Therefore, developing a multiplex detection kit that can simultaneously detect multiple PIK3CA gene mutation sites, with intuitive result interpretation and comprehensive evaluation, has important clinical application value. Summary of the Invention

[0008] To solve the above problems, the present invention provides a digital PCR detection kit for detecting multiple mutation sites of the PIK3CA gene. The kit detects 11 mutation sites of the PIK3CA gene through two digital PCR reaction tubes, namely tube A and tube B. Among them, tube A includes the detection of sites E545K, E545A, E545G, E545D, Q546E, and Q546R, and tube B includes the detection of sites E542K, C420R, H1047R, H1047L, and H1047Y. Each site is detected using its respective spacer molecular beacon detection probe. The molecular beacon detection probe sequentially includes a 5'-end stem sequence, a loop sequence, a flexible Spacer structure, and a 3'-end stem sequence, presenting an overall hairpin structure. The 5'-end is labeled with a fluorescent group, and the 3'-end is labeled with a quenching group. The complementary sequences of the 5'-end stem sequence and the 3'-end stem sequence form a stable stem structure. The 5'-end 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.

[0009] In one embodiment, the loop sequence is modified with locked nucleic acid.

[0010] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is not less than three.

[0011] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 3 - 6.

[0012] In one embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 6.

[0013] In one embodiment, tube A includes the spacer molecular beacon detection probe SEQ ID NO.27 for E545K, the spacer molecular beacon detection probe SEQ ID NO. 28 for E545A, the spacer molecular beacon detection probe SEQ ID NO. 29 for E545G, the spacer molecular beacon detection probe SEQ ID NO. 30 for E545D, the spacer molecular beacon detection probe SEQ ID NO. 31 for Q546E, and the spacer molecular beacon detection probe SEQ ID NO. 32 for Q546R.

[0014] In one embodiment, tube A further includes the wild-type detection probe SEQ ID NO. 33 for E545, the upstream primer SEQ ID NO. 13 for E542 - 546, and the downstream primer SEQ ID NO.14 for E542 - 546.

[0015] In one embodiment, the tube A further includes a fluorescence quenching probe B1 for blocking the spacer molecular beacon detection probes for E545K, E545A, and E545G, and its sequence is SEQ ID NO. 34; a fluorescence quenching probe B2 for blocking the spacer molecular beacon detection probes for Q546E, Q546R, and E545D, and its sequence is SEQ ID NO. 35; the sequence of the fluorescence quenching probe is complementary to the 6-base region at the 5'-end of each of the above molecular beacon detection probes, and the 3'-end of the fluorescence quenching probe is labeled with a fluorescence quenching group. The fluorescence quenching probe binds to the 5'-end in the free state of the molecular beacon detection probe to form a stable secondary blocking structure.

[0016] In one embodiment, the tube B further includes a spacer molecular beacon detection probe SEQ ID NO. 40 for H1047R, a spacer molecular beacon detection probe SEQ ID NO. 41 for H1047L, a spacer molecular beacon detection probe SEQ ID NO. 42 for H1047Y, a spacer molecular beacon detection probe SEQ ID NO. 44 for C420R, and a spacer molecular beacon detection probe SEQ ID NO. 45 for E542K.

[0017] In one embodiment, the tube B further includes a fluorescence quenching probe B3 for blocking the spacer molecular beacon detection probes for H1047R, H1047L, and H1047Y, and its sequence is SEQ ID NO. 46; the sequence of the fluorescence quenching probe is complementary to the 6-base region at the 5'-end of each of the above molecular beacon detection probes, and the 3'-end of the fluorescence quenching probe is labeled with a fluorescence quenching group. The fluorescence quenching probe binds to the 5'-end in the free state of the molecular beacon detection probe to form a stable secondary blocking structure.

[0018] In the present invention, the digital PCR detection kit for detecting multiple mutation sites of the PIK3CA gene can simultaneously detect 11 hotspot mutation sites, covering the key mutation regions common in breast cancer clinics, thus making the detection results more comprehensive and accurate. The kit has strong specificity and high sensitivity, can detect nucleic acid samples as low as 0.3 ng / μL, and is suitable for the precise detection of ctDNA in liquid biopsy.

[0019] The present invention is based on locked nucleic acid (LNA) probe technology. By shortening the probe length to enhance specificity and combining the design of Spacer molecular beacon probes and fluorescence quenching probes, the background signal of the system is effectively reduced. For sites without wild-type probes, the detection specificity is further improved by quenching probes. Common mutation sites are genotyped by different fluorescence labels, while relatively rare mutation sites are not genotyped, and a two-tube detection strategy is adopted, so as to efficiently and sensitively detect 11 mutation sites, reduce the template separation effect, and improve the overall sensitivity.

[0020] The present invention innovatively introduces a Spacer flexible linker into the molecular beacon detection probe structure, which is located between the stem sequence at the 3'-end of the probe and the main target recognition region to be detected. The Spacer structure has the following key advantages: 1) Improving spatial flexibility: enabling short-sequence probes to efficiently form a stable hairpin-like stem-loop structure, significantly enhancing the closing efficiency; 2) Effectively reducing the background signal: the distance between the fluorescence and quenching groups is more stable in the closed-loop state, and the fluorescence inhibition is more sufficient in the unbound state; 3) Enhancing the compatibility of the probe structure design: Spacer reduces the possibility of non-specific pairing between the stem at the 3'-end and the target, avoiding interference with the Tm of the probe, and facilitating the design of multi-probe co-detection; 4) Improving the ability to detect multiple mutations: when detecting multiple homologous mutations or multi-site mutations, the Spacer structure enables the probes to maintain more consistent structural stability and background suppression ability, thus achieving high-throughput and high-sensitivity detection and discrimination of mutation sites.

[0021] By introducing a Spacer structure into short-sequence highly specific probes, the present invention breaks through the problems of background control and structure looping in traditional molecular beacon detection for complex mutations, significantly improving the detection accuracy and applicable range. In addition, a short-chain auxiliary probe sequence (fluorescence quenching probe) is introduced into the system. Its 3'-end is labeled with a quenching group and is complementary to the 5-7 nt sequence at the 5'-end of the main probe to further quench the unclosed free probe structure. This probe system effectively improves the specific recognition ability of enzyme digestion-dependent molecular beacon probes in a high-homology background, making it possible to accurately detect multiple homologous mutation sites, especially suitable for end-point detection platforms with extremely high signal-to-noise ratio requirements such as digital PCR.

[0022] The present invention also introduces a fluorescence quenching probe. This probe is a short-chain oligonucleotide, whose sequence is specifically complementary to the 5-7 base region at the 5'-end of the main probe, and the 3'-end is labeled with a fluorescence quenching group. When the target is not bound, this short-chain probe can preferentially bind to the 5'-end region of the molecular beacon to form a stable secondary quenching complex structure, further enhancing the fluorescence inhibition ability of the beacon in the free state. This mechanism significantly reduces non-specific fluorescence release, improves the overall signal-to-noise ratio, and helps to enhance the accuracy and sensitivity of mutation detection.

[0023] Due to the application of the probe in the present invention, the detection sensitivity of the present invention reaches 0.1%, which is 2 to 10 times higher than that of the traditional real-time fluorescence PCR technology. The whole detection process is easy to operate. Through copy number quantitative analysis, the positive and negative of the sample can be clearly determined, and the mutation rate data can be provided, which is convenient for dynamically monitoring the change of mutation abundance and provides reliable support for clinical decision-making.

[0024] The clinical verification results show that the 20 plasma samples detected by the kit of the present invention are completely consistent with the NGS sequencing results, which proves that the kit has extremely high accuracy and is particularly suitable for the detection of PIK3CA mutations in plasma samples. The detection of this composition has strong specificity, comprehensive site detection, and intuitive and easy-to-interpret detection results, which can provide a more comprehensive and accurate basis for drug selection for clinicians, ensure the effectiveness and safety of treatment plans, and has important value for companion diagnosis. Especially in the precise diagnosis and treatment of breast cancer, it plays an irreplaceable role. Detailed implementation manners

[0025] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0026] Embodiment 1 Detection scheme of the PIK3CA mutation detection kit

[0027] The present invention provides a digital PCR (dPCR) detection kit and its detection method for 11 common mutation hotspots of the PIK3CA gene. The covered mutation sites include: E545K, E545A, E545G, E545D, Q546E, Q546R, E542K, C420R, H1047R, H1047L, and H1047Y. These mutations have a relatively high occurrence frequency in various clinical samples and have important clinical and biological significance.

[0028] When establishing the detection system, the single-tube multiplex reaction strategy was initially designed. (1) For the mutation sites of the E545 series (E545K / A / G / D) and Q546 series (Q546E / R), molecular beacon detection probes with the same fluorescence channel were used for detection; (2) For the mutation sites of E542K, C420R, and H1047X (H1047R / L / Y), molecular beacon detection probes with independent fluorescence channels were designed respectively; (3) At the same time, a corresponding fluorescence probe was also designed for the wild-type sequence of the H1047 site as an internal standard gene to quality control whether the detection system was amplified normally. The primer and probe sequences are shown in Table 1.

[0029] Tm and ΔTm were obtained by OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0030] Table 1. Primer and probe sequences used for one-tube detection

[0031] Note: ΔTm is the Tm difference between the mutant probe and the mutant template match and the wild-type template mismatch; lowercase letters are the 3'-end stem sequences that do not match the target; the capital letters with a horizontal line at the 3'-end represent the 3'-end stem sequences that match the target; the underlined bases in the middle represent the mutation sites.

[0032] Using the primer and probe combinations designed in Table 1, a multiplex digital PCR reaction system was constructed. The specific operation process is as follows: (1) System preparation Prepare the basic PCR reaction system (total volume 30 µL) according to the following table.

[0033]

[0034] Among them, the final concentration of each primer is 600 nM, and the final concentration of each probe is 200 nM.

[0035] (2) Digital PCR workflow Microdroplet preparation: Use a droplet generation chip (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) and a sample preparation instrument (Xinyi Manufacturing Technology (Beijing) Co., Ltd.). Add 30 µL of the PCR reaction system to the sample well of the droplet generation chip, add 180 µL of droplet generation oil to the oil well, place the chip and the 8-well strip tube into the preparation instrument, cover with a gasket, and perform microdroplet preparation.

[0036] PCR amplification: Place the 8-well strip tube containing microdroplets on the PCR instrument for amplification. The amplification program is set as shown in the following table:

[0037] Micro-droplet detection: After PCR is completed, place the 8-strip tube and the droplet detection chip (manufactured by NeoGenomics Technologies (Beijing) Co., Ltd.) into the fixture. Add 430 µL and 500 µL of detection oil to the oil holes respectively, cover with the rubber gasket, and put the chip into the chip analyzer (manufactured by NeoGenomics Technologies (Beijing) Co., Ltd.) for droplet detection.

[0038] Data analysis: Tens of thousands of micro-droplets prepared by the droplet generation chip and the sample preparation instrument are each an independent PCR reactor. Most micro-droplets do not contain the target gene to be detected or contain at least one target gene to be detected. After PCR amplification, the fluorescence signals of each channel of each micro-droplet are detected by the chip analyzer, and the peak height of the micro-droplet signal is recorded. Droplets containing the target gene to be detected will be detected with corresponding fluorescence signals. The fluorescence intensity in the micro-droplets is digitized through the fluorescence classification threshold. Micro-droplets with stronger fluorescence are judged as "1" (positive), and micro-droplets with weaker fluorescence are judged as "0" (negative). The numbers of "1" and "0" are counted and corrected through the Poisson distribution model, and the total copy number of the target genes with each fluorescence label in the input template can be calculated.

[0039] A mutant template constructed with plasmid and fragmented human wild-type genomic DNA are mixed in proportion to prepare a simulated sample with a 10% mutation frequency (the copy numbers of the mutant and wild-type templates are 50 copies / µL and 500 copies / µL respectively, and 2 µL of the mixed template is added to each reaction system). At the same time, pure wild-type genomic DNA without doped mutation is used as the control template to conduct a preliminary evaluation of the system performance. The detection results are shown in Table 2.

[0040] Table 2. Detection Results

[0041] Note: Non-specific detection signals are indicated by bold italic numbers.

[0042] During the preliminary detection process, it was found that there was a problem of relatively high background signal in some systems, especially under the condition of the wild-type template, obvious non-specific fluorescence signal interference occurred.

[0043] Specifically, among the E545X / Q546X series of mutation sites, except for E545G and Q546R, positive FAM signals corresponding to the mutant template can be detected for the rest, indicating that the binding efficiency of the probe to the mutant template is good. However, in the wild-type template, FAM positive signals still appear for the probes at these mutation sites, suggesting a certain degree of non-specific hybridization. Further analysis found that this problem is mainly due to the relatively low melting temperature difference (ΔTm) of some probes (<5℃), resulting in the probes still having a certain compatibility binding ability on the wild-type template and being difficult to effectively distinguish.

[0044] Similarly, in the H1047X series of mutation detections, although positive signals can be detected for all mutant templates, the background value is relatively high, and obvious non-specific signals are also observed in wild-type templates. Especially at the H1047Y locus, its ΔTm is relatively low, becoming the main source of non-specificity.

[0045] The C420R probe performs well, stably detecting signals in mutant templates and showing no obvious background in wild-type templates. The probe for the E542K locus can also accurately identify mutant templates, but there is also a certain degree of non-specific hybridization signal in wild-type templates.

[0046] To improve the overall detection specificity and reduce the background value of the system, the present invention proposes the following optimization strategies for the above-mentioned probes: Appropriately shorten the probe sequence length to increase the sensitivity to base mismatches; Introduce LNA (Locked Nucleic Acid) modified bases to enhance the overall binding stability of the probe and increase the Tm value (target Tm > 65 °C), thereby ensuring that the probe forms a stable binding only with the completely matched mutant template under strict hybridization conditions, effectively improving the detection specificity.

[0047] In addition, during the construction of the system, it was observed that the probes for the two loci of E545G and Q546R detected abnormally, and the expected FAM fluorescence signal was not detected. After in-depth analysis, it was found that this abnormal phenomenon was mainly due to a high sequence overlap between the E542K mutant probe and the E545X / Q546X series of mutant templates, resulting in competitive binding interference between the probes. The E542K probe was originally used to identify specific mutations at the 542nd site of the PIK3CA gene. However, since its recognition region highly coincides with the nucleotide sequences of the E545 / Q546 mutation sites, when the E542K probe hybridizes with these non-target mutant templates, the mismatched bases are located in the 5′-end region of the probe (this probe is a reverse probe). The PCR amplification system has a relatively high tolerance for mismatches at the 5′-end of the probe, enabling this probe to still form a partial stable binding with these non-target templates, thus "occupying" the binding sites of mutant templates in the reaction system. Due to the mismatch at the 5′-end of this binding structure, it affects the recognition and cleavage of the probe-template complex by Taq enzyme, resulting in the non-release of the fluorophore, manifested as no signal output in the detection channel, that is, the mutant template exists but is missed by the system, resulting in false negatives.

[0048] To effectively solve the above problem of interference between probes, the present invention proposes the following optimization strategies: The E542K probe is physically separated from the E545 / Q546 series of mutant probes in separate tubes. It is integrated with the C420R and H1047X series of probes and placed in the same reaction tube for detection. The E542K probe has a high sequence overlap with the E545 / Q546 template, which is prone to competitive binding interference. Independent detection can effectively avoid cross-interference and ensure the accuracy of signal output at the mutation site.

[0049] The E545 and Q546 series of mutant probes can be placed in one tube for multiplex detection. The mismatch sites of such probes are mostly located in the middle region of the probe, with good structural specificity and good recognition ability for the mutation site. As long as obvious non-specific signals do not appear in the wild-type template, stable multiplex detection can be achieved.

[0050] Example 2 Optimization Experiment of Probes for Separate Tube Detection According to Example 1, the present invention splits 11 sites into two reaction tubes for detection: Tube A: contains six sites, namely E545K, E545A, E545G, E545D, Q546E, and Q546R. These mutations are concentrated in exon 10 of the PIK3CA gene and have strong aggregation. The present invention distinguishes the main mutation site E545K by designing different fluorescent labels, and the remaining sites use the same fluorescent label for combined detection without further typing, thereby simplifying the system and reducing cross-interference.

[0051] Tube B: contains five sites, namely E542K, C420R, H1047R, H1047L, and H1047Y. To avoid signal interference, E542K is independent of the E545 / Q546 sites and is separately placed in Tube B for detection in the present invention. At the same time, since the C420R and H1047 series of mutation sites are distributed in different exons and there is no amplification interference between them, they can be detected together with E542K, thereby improving the detection efficiency.

[0052] (I) Selection of Reaction Conditions for Tube A The primers and probes used in Tube A (covering the sites of E545K, E545A, E545G, E545D, Q546E, and Q546R) are shown in Table 1. Tm and ΔTm are obtained through OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0053] As shown in Table 1 above, Tube A is mainly used to detect various mutation types at the 545–546 sites of the PIK3CA gene. The results in Example 1 showed that when the common molecular beacon detection probe was used to detect mutations at homologous sites, the background signal was relatively high, and for some mutant probes, the ΔTm was low, and there was non-specific signal for the wild type. Therefore, in this example, to improve the efficiency and specificity of mutant site detection, LNA probes were used to improve specificity. By shortening the probe sequence to 14–25 bp, while enhancing the base pairing stability, the overall Tm value was maintained above 64 °C, and the ΔTm was effectively increased to more than 5 °C, enhancing the mismatch recognition ability. The primer and probe information used is shown in Table 3 below.

[0054] Table 3. Primer and Probe Information for Tube A

[0055] Note: ΔTm is the Tm difference between the mutant probe matching the mutant template and mismatching with the wild-type template; + represents locked nucleic acid LNA; lowercase letters are the 3'-end stem sequences that do not match the target; the capital letters with a horizontal line at the 3'-end represent the 3'-end stem sequences that match the target; the underlined bases in the middle represent the mutation sites.

[0056] For the preparation of the reaction system and the operation steps of digital PCR detection, refer to Example 1. A simulated sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (the concentrations of mutant and wild-type were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and together with the wild-type genome as templates, a comparative test was carried out between the common molecular beacon detection probe and the LNA molecular beacon detection probe. The detection results of the probes are shown in Tables 4 - 5 below. Among them, the background signal of the common molecular beacon detection probe was high (Table 4), and there was signal of the wild-type template on the mutant probe, and there was also signal of E545K on other mutant probes, so the wild type and mutant templates could not be distinguished, and E545K and other mutants could not be distinguished; the LNA molecular beacon detection probe could detect all 6 mutation sites, the copy number was in line with expectations, the specificity was good, the mutant type and wild type could be distinguished, and E545K could be distinguished, but the background signal in the FAM channel was relatively high and the signal-to-noise ratio was low.

[0057] Table 4. Detection Results of Common Molecular Beacon Detection Probe

[0058] Note: Non-specific detection signals are indicated by bold italic numbers.

[0059] Table 5. Detection Results of LNA Molecular Beacon Detection Probe

[0060] Further optimize the LNA molecular beacon detection probe system, reduce the background signal of the reaction system, improve the signal-to-noise ratio, use the molecular beacon detection probe modified with LNA, and introduce the flexible Spacer18 group. The Spacer structure is placed in the loop region of the probe, which can block the potential non-specific complementary pairing between the 3'-end stem and the target, avoid interfering with Tm, and enhance the stability and specificity of the formation of the hairpin structure. The primer probe sequences are shown in Table 6.

[0061] Table 6. Primer Probe Sequences

[0062] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template matching and mismatching with the wild-type template; + represents locked nucleic acid LNA; lowercase letters are the 3'-end stem sequences that do not match the target; the capital letters with a horizontal line at the 3'-end represent the 3'-end stem sequences that match the target; Spacer 18 is 6 polyethylene glycol (PEG) units; the underlined bases in the middle part represent the mutation sites.

[0063] For the preparation of the reaction system and the operation steps of digital PCR detection, refer to Example 1. A simulated sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (the concentrations of mutant and wild-type were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and together with the wild-type genome as a template, the probe test was carried out. The detection results of the probe are shown in Table 7 below. After using the Spacer probe, the background signal of the system was further reduced, the signal-to-noise ratio was improved, and each mutant template and wild-type template were specifically detected, and the copy numbers were in line with the expected results.

[0064] Table 7. Detection Results of Spacer Probe

[0065] To further reduce the background signal of the amplification reaction system, the present invention also designed a fluorescence quenching probe. This probe is a short molecule probe complementary to the 5'-end sequence of the beacon probe, and 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: when the molecular beacon detection probe is in the free unclosed state, the fluorescence quenching probe effectively quenches the fluorescence by complementary binding to the 5'-end sequence, thereby further blocking the non-specific background signal generated by the free unclosed probe in the system. The sequences are shown in Table 8.

[0066] Table 8. Fluorescence Quenching Probe Sequences

[0067] For the preparation of the reaction system and the operation steps of digital PCR detection, refer to Example 1. A simulated sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (the concentrations of mutant and wild-type were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and together with the wild-type genome as the template, probe tests were carried out. The detection results of the probes are shown in Table 9 below. After using the fluorescence-blocked probe, the background signal of the FAM channel in the system was reduced again, further improving the signal-to-noise ratio, and each mutant template and wild-type template were specifically detected, and the copy numbers were in line with the expected results.

[0068] Table 9. Detection Results of Fluorescence-Blocked Probes

[0069] (2) Selection of Reaction Conditions for Tube B The primers and probes used in Tube B (covering the sites of E542K, C420R, H1047R, H1047L, and H1047Y) are shown in Table 5. Similarly, Tm and ΔTm were obtained through OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0070] As shown in Table 10, Tube B is mainly used to detect multiple mutation types at positions 542, 420, and 1047 of the PIK3CA gene, for genotyping detection of different positions. Since H1047R is the most common site clinically, genotyping detection is also carried out, while H1047L and H1047Y are not genotyped. Based on Example 1, the ΔTm of the common molecular beacon detection probe for 1047Y was low, resulting in non-specific signals for the wild-type template. Therefore, LNA modification was added to the probe, the length was shortened, ΔTm was increased, and the specificity of the probe was improved. The primer and probe information used in Tube B is shown in Table 10 below.

[0071] Table 10. Primer and Probe Information for Tube B

[0072] Note: ΔTm is the Tm difference between the mutant probe matching the mutant template and mismatching with the wild-type template; + represents locked nucleic acid LNA; lowercase letters are the 3'-end stem sequences that do not match the target; the underlined bases in the middle represent the mutation sites.

[0073] For the preparation of the system and digital PCR operation, refer to the description in the optimization experiment of Example 1 above.

[0074] A simulated sample with a 10% mutation rate was prepared using a mutant plasmid and fragmented wild-type genomic DNA (the mutant and wild-type concentrations were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and together with the wild-type genome as a template, probe screening was carried out. The detection results are shown in Tables 11-12 below. Among them, the common molecular beacon detection probe had a high background signal (Table 11), and the wild-type template had a signal on the mutant probe, so the wild-type and mutant templates could not be distinguished; the LNA molecular beacon detection probe could detect all 5 mutation sites, the copy number met the expectations, and the specificity was good, and the mutant and wild-type could be distinguished, but the background signal of the ROX channel was on the high side and the signal-to-noise ratio was low.

[0075] Table 11. Detection Results of Common Molecular Beacon Detection Probes

[0076] Note: Non-specific detection signals are represented by bold italic numbers.

[0077] Table 12. Detection Results of LNA Molecular Beacon Detection Probes

[0078] The LNA molecular beacon detection probe system was further optimized to reduce the background signal of the reaction system and improve the signal-to-noise ratio. The molecular beacon detection probe modified with LNA was used, and a flexible Spacer18 group was introduced. The Spacer structure was placed in the loop region of the probe, which could block the potential non-specific complementary pairing between the 3'-end stem and the target, avoid interfering with Tm, and enhance the stability and specificity of the formation of the hairpin structure. The primer probe sequences are shown in Table 13.

[0079] Table 13. Spacer Probe Sequences of Tube B

[0080] Note: ΔTm is the Tm difference between the mutant probe matching the mutant template and mismatching with the wild-type template; + represents locked nucleic acid LNA; lowercase letters are the 3'-end 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 sites.

[0081] The preparation of the reaction system and the operation steps of digital PCR detection are shown in Example 1. A simulated sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (the concentrations of mutant and wild-type were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and it was used as a template together with the wild-type genome for probe testing. The detection results of the probe are shown in Table 14 below. After using the Spacer probe, the background signal of the system was further reduced, the signal-to-noise ratio was increased, and each mutant template and wild-type template were specifically detected, and the copy number was consistent with the expected results.

[0082] Table 14. Detection results of Spacer probe

[0083] To further reduce the background signal of the amplification reaction system, the present invention also designed a fluorescence quenching probe. This probe is a short molecule probe that is 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 detection probe is in a free and unclosed state, the fluorescence quenching probe effectively quenches fluorescence by complementary binding to the 5′-end sequence, thereby further blocking the non-specific background signal generated by the free and unclosed probes in the system. The sequence is: SEQ ID NO. 46: gcatca (labeled with BHQ1 at the 3′-end). After adding the fluorescence quenching probe, the ROX, HEX, and CY5 background signals of the reaction system were all further reduced.

[0084] The preparation of the reaction system and the operation steps of digital PCR detection are shown in Example 1. A simulated sample with a 10% mutation rate was prepared using mutant plasmids and fragmented wild-type genomic DNA (the concentrations of mutant and wild-type were 50 and 500 copies / µL respectively, and 2 µL was used for each system), and it was used as a template together with the wild-type genome for probe testing. The detection results of the probe are shown in Table 15 below. After using the fluorescence quenching probe, the background signal in the FAM channel of the system was reduced again, further increasing the signal-to-noise ratio, and each mutant template and wild-type template were specifically detected, and the copy number was consistent with the expected results.

[0085] Table 15. Detection results after adding the fluorescence quenching probe

[0086] Example 3 Optimization of reaction system components and reaction conditions In the above embodiments, the nucleic acids used for screening primers and probes and optimizing their combinations are plasmid nucleic acids and fragmented genomic DNA, and the added amount of nucleic acid template in the system is 2 μL. In the high-sensitivity blood PIK3CA mutation detection, to meet the detection requirements of low-abundance mutations in clinical tests, nucleic acids need to be extracted from a large-volume plasma sample, and as large a volume of template as possible should be added in the amplification reaction to improve the detection sensitivity.

[0087] In this embodiment, 2 mL of simulated plasma sample (incorporating 10 copies of various mutant plasmid DNAs) is used for nucleic acid extraction, and the large-volume simulated plasma sample is prepared and divided into small portions for standby. During the extraction process, a small volume of 40 μL is used for elution to increase the nucleic acid concentration. Subsequently, as large a volume of nucleic acid template as possible is added to the amplification system to enhance the detection sensitivity. In this embodiment, 20 μL of nucleic acid template is added, and the system components in Example 1 are used as a control to optimize the system.

[0088] The optimization measures include: 1) Adding BSA (bovine serum albumin) component to improve the stability of the amplification reaction. 2) To improve the dispersion of nucleic acids in droplets and the amplification efficiency, the extracted nucleic acids are pretreated: heated at 80 °C for 5 minutes to denature double-stranded nucleic acids into single-stranded nucleic acids, thereby improving the uniformity of the distribution of target copies in droplets. The heating treatment also helps to remove potential residual PCR reaction inhibitors and improve the overall detection sensitivity and reliability.

[0089] The reaction system of Tube A is prepared as follows:

[0090] The reaction system of Tube B is prepared as follows:

[0091] The digital PCR operation is as described in Example 1. The templates include preheating pretreatment and non-preheating pretreatment. The detection results are shown in the following table. It can be seen that when nucleic acids are extracted from a large volume (2 mL) of plasma and the large-volume sample loading amount of the template is 20 μL, adding BSA can stably detect low-copy positive mutations, while low-copy positive mutations cannot be stably detected without adding BSA; in addition, after the nucleic acid template is preheated, the copy number is further increased. The template sample loading amount of the kit of the present invention is 20 μL. When the template concentration is 0.4 ng / μL, the template sample loading amount is 6 ng. Through preheating pretreatment, the wild-type copy number of about 3000 - 5000 copies can be obtained. When the mutant copy number is greater than 3 copies, a positive result is reported, and at this time, the requirement of a sensitivity of 0.1% is met.

[0092]

[0093] Example 4 Composition of the Detection Kit of the Present Invention This kit contains a total of 2 reaction solutions. Among them, reaction solution A detects E545K (labeled with ROX fluorescence), E545A, E545G, E545D, Q546E, Q546R (all labeled with FAM fluorescence, not typed), and wild-type 545-546 (labeled with HEX fluorescence as the internal standard gene) of the PIK3CA gene; reaction solution B detects E542K (labeled with CY5.5 fluorescence), C420R (labeled with FAM fluorescence), H1047R (labeled with CY5 fluorescence), H047L and H1047Y (labeled with ROX fluorescence, not typed), and wild-type H1047 (labeled with HEX fluorescence as the internal standard gene) of the PIK3CA gene. Three nucleic acid reaction solutions (reagent A, reagent B, and reagent C), a negative control (DEPC-treated water), and a positive control (a mixture of each mutant plasmid) are packaged together and accompanied by a product instruction manual to obtain the kit for 11 mutation hotspots of the PIK3CA gene in the present invention. The compositions of the premix, detection solution, negative control, and positive control are shown in Table 16 below.

[0094] Table 16. Components of the PIK3CA Gene Mutation Detection Kit

[0095] Example 5 Verification of Clinical Samples of the Kit (1) Reagent preparation: According to the number of test samples, negative controls, and positive controls, take out the PCR reaction solution and calculate the number of aliquots n of each reaction solution = number of samples + positive control + negative control. According to the required number of samples n, add 7.5 μL of PCR reagent A and 3 μL of PCR reagent B (or C) to the PCR reaction tubes respectively, mix well, and centrifuge briefly for later use.

[0096] (2) Sample treatment: The sample is 2 ml of anticoagulated plasma. A commercial extraction kit is used to extract plasma-free DNA, and after extraction, TE buffer is used to elute the DNA.

[0097] Add 10.5 μl of PCR reaction solution B or C and 19.5 μl of the free DNA to be tested into the reaction tube, and perform droplet generation, PCR amplification, and droplet detection according to the operations in Example 1 above. After the PCR amplification reaction is completed, genotype determination is performed according to the target copy number. The principles for result determination are as follows: ① The copy numbers of all targets and the wild-type copy number in the positive control are both greater than 50 copies; ② The copy numbers of all targets in the negative control group are 0, and the wild-type copy number is not higher than 5 copies; ③ If the above two conditions are simultaneously met, the experiment is determined to be successful, and typing is performed according to the copy numbers of all targets in the sample to be tested. The interpretation criteria are as follows: When the copy number of a certain 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, then the target is positive; if the copy numbers of all targets are not higher than 3 copies, and the copy number of the internal standard gene (wild-type + mutant copy number) is greater than 500 copies, then the sample is negative; if the copy number of the internal standard gene is less than 500 copies, the test result is invalid, and there may be amplification inhibition or too low nucleic acid concentration, and nucleic acid needs to be extracted again for testing.

[0098] The test results are shown in the following table. A total of 11 clinical samples were tested, including 5 positive samples with different mutations and 6 negative samples. The results obtained using the kit of the present invention were consistent with the NGS sequencing results, indicating that the composition of the present invention can well detect the PIK3CA mutations in plasma free DNA. Moreover, the kit of the present invention can accurately give the mutation rate of the sample, which is convenient for clinical dynamic monitoring of the change in mutation abundance.

[0099]

[0100] It should be understood that the present invention disclosed herein is not limited to the specific methods, protocols, and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, the scope of which is limited only by the appended claims.

[0101] 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. These equivalents are also included in the appended claims.

Claims

1. Digital PCR detection kit for detecting multiple mutation sites of PIK3CA gene, characterized in that, The detection of the kit is achieved through two digital PCR reaction tubes, tube A and tube B, for detecting 11 mutation sites of the PIK3CA gene. Among them, tube A contains the detection of sites E545K, E545A, E545G, E545D, Q546E, and Q546R, and tube B contains the detection of sites E542K, C420R, H1047R, H1047L, and H1047Y. Each site is detected using its respective spacer molecular beacon detection probe. The molecular beacon detection probe sequentially includes a 5'-terminal stem sequence, a loop sequence, a flexible Spacer structure, and a 3'-terminal stem sequence, presenting an overall hairpin structure. The 5'-end is labeled with a fluorescent group, and the 3'-end is labeled with a quenching group. The complementary sequences of the 5'-terminal stem sequence and the 3'-terminal stem sequence form a stable stem structure. The 5'-terminal 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.

2. The digital PCR detection kit according to claim 1, wherein The loop sequence is modified with locked nucleic acid.

3. The digital PCR detection kit according to claim 1, wherein The number of polyethylene glycol units in the flexible Spacer structure is not less than three.

4. The digital PCR detection kit according to claim 1, wherein The number of polyethylene glycol units in the flexible Spacer structure is 3 - 6.

5. The digital PCR detection kit according to claim 4, wherein The number of polyethylene glycol units in the flexible Spacer structure is 6.

6. The digital PCR detection kit according to claim 5, wherein Among them, tube A includes the spacer molecular beacon detection probe SEQ ID NO. 27 for E545K, the spacer molecular beacon detection probe SEQ ID NO. 28 for E545A, the spacer molecular beacon detection probe SEQ ID NO. 29 for E545G, the spacer molecular beacon detection probe SEQ ID NO. 30 for E545D, the spacer molecular beacon detection probe SEQ ID NO. 31 for Q546E, and the spacer molecular beacon detection probe SEQ ID NO. 32 for Q546R.

7. The digital PCR detection kit according to claim 6, characterized in that, Among them, tube A also includes the wild-type detection probe SEQ ID NO. 33 for E545, the upstream primer SEQ ID NO. 13 for E542 - 546, and the downstream primer SEQ ID NO. 14 for E542 - 546.

8. The digital PCR detection kit according to claim 6, wherein The A tube further includes a fluorescence quenching probe B1 for blocking the spacer molecular beacon detection probes for E545K, E545A, and E545G, and its sequence is SEQ ID NO. 34; a fluorescence quenching probe B2 for blocking the spacer molecular beacon detection probes for Q546E, Q546R, and E545D, and its sequence is SEQ ID NO. 35; the sequence of the fluorescence quenching probe is complementary to the 6-base region at the 5' end of each of the above molecular beacon detection probes, and the 3' end of the fluorescence quenching probe is labeled with a fluorescence quenching group. The fluorescence quenching probe binds to the 5' end in the free state of the molecular beacon detection probe to form a stable secondary blocking structure.

9. The digital PCR detection kit according to claim 5, wherein The B tube further includes a spacer molecular beacon detection probe SEQ ID NO. 40 for H1047R, a spacer molecular beacon detection probe SEQ ID NO. 41 for H1047L, a spacer molecular beacon detection probe SEQ ID NO. 42 for H1047Y, a spacer molecular beacon detection probe SEQ ID NO. 44 for C420R, and a spacer molecular beacon detection probe SEQ ID NO. 45 for E542K.

10. The digital PCR detection kit according to claim 9, wherein The B tube further includes a fluorescence quenching probe B3 for blocking the spacer molecular beacon detection probes for H1047R, H1047L, and H1047Y, and its sequence is SEQ ID NO. 46; the sequence of the fluorescence quenching probe is complementary to the 6-base region at the 5' end of each of the above molecular beacon detection probes, and the 3' end of the fluorescence quenching probe is labeled with a fluorescence quenching group. The fluorescence quenching probe binds to the 5' end in the free state of the molecular beacon detection probe to form a stable secondary blocking structure.

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