Molecular beacon probes used in pcr end-point detection, and systems and kits thereof

Abstract

The application provides a molecular beacon probe used in PCR end-point detection, and a system and a kit thereof.The molecular beacon probe comprises, in sequence, a 5' end stem sequence, a loop sequence, a flexible Spacer structure and a 3' end stem sequence, and has a whole hairpin structure; the 5' end stem sequence and the loop sequence are fully complementary to a target sequence to be detected; the loop sequence and the flexible Spacer structure form a loop structure of the molecular beacon probe; and the flexible Spacer structure is a polyethylene glycol structure.The application introduces the Spacer structure into a short sequence high-specificity probe, breaks through the problems of background control and structure loop formation of traditional molecular beacon in complex mutation detection, and significantly improves detection accuracy and application range.In addition, a fluorescence blocking probe is introduced into the system, the probe system effectively improves the specific recognition ability of enzyme-dependent molecular beacon probes in a high homology background, and makes accurate detection of multiple homologous mutation sites possible.

Description

Technical Field

[0001] This invention relates to the field of digital PCR detection, and more specifically, to molecular beacon probes, systems, and kits used in PCR endpoint detection methods. Background Technology

[0002] In clinical practice, a set of clinically significant genetic biomarkers is often used to assess treatment response and disease progression. For example, in breast cancer, PIK3CA gene mutations are not only a research hotspot but are also gradually becoming important targets for clinical treatment; ESR1 gene mutations are one of the key molecular mechanisms leading to resistance to endocrine therapy in patients with advanced breast cancer. In colorectal cancer, KRAS and NRAS gene mutations are important biomarkers for resistance to anti-EGFR monoclonal antibody therapies (such as cetuximab).

[0003] Liquid biopsy technology, particularly mutation detection based on cell-free DNA (cfDNA), is becoming an important tool in precision medicine. However, detecting low-abundance mutations in cfDNA faces numerous challenges, including insufficient sensitivity, limited throughput, and high cost. Digital PCR technology can significantly improve the detection sensitivity of low-abundance mutations. However, detecting multiple highly homologous gene mutations in a single reaction system is often limited by technical bottlenecks such as background noise, fluorescence interference, and decreased signal-to-noise ratio, which may lead to detection failures or inaccurate results.

[0004] Molecular beacon probe technology is an effective method to solve the above problems. A molecular beacon is a dual-labeled oligonucleotide probe, 25-40 nucleotides in length, with a hairpin-shaped structure consisting of a circular probe sequence and a short, self-complementary stem structure. Its 5' end is labeled with a fluorescent group, and its 3' end with a quencher group. At room temperature, due to the stem-loop structure, the fluorescent and quencher groups are close together, thus suppressing the fluorescence signal. During the PCR annealing phase, if the probe encounters a target DNA sequence that is perfectly complementary to it, it will preferentially bind to the target due to thermodynamic advantages, causing the hairpin structure to open and the fluorescent group to move away from the quencher group, thereby emitting a fluorescence signal. The stem sequence of the molecular beacon is usually not complementary to the target; instead, it is a specially designed short inverted repeat sequence. Its purpose is to maintain the stability of the hairpin structure without interfering with target detection.

[0005] In digital PCR, since it is an endpoint detection method, the release of fluorescence signals usually depends on enzyme digestion. Therefore, traditional molecular beacon probes cannot meet the high requirements for signal intensity and specificity. Enzyme-dependent molecular beacons are needed. Traditional molecular beacons usually adopt a hairpin structure, with the stem formed by complementary sequences at the 5' and 3' ends. However, this stem sequence does not pair with the target sequence and only serves to maintain the closed conformation and suppress the fluorescence signal. This structure performs well in real-time quantitative PCR, but it is not suitable for endpoint detection in digital PCR. Digital PCR requires enzyme-dependent molecular beacons, whose 5' stem region needs to be fully or partially complementary to the target sequence to provide the double-stranded structure required for enzyme recognition and cleavage. However, this structure is difficult to design: 1) The overlap between the stem and the target needs to be precisely controlled: usually, 3-5 bases at the 5' end are complementary to the target sequence to form a cleavage site, but if the design is not appropriate, mismatches can easily occur, affecting recognition efficiency. 2) The total length of the stem needs to be maintained between 5 and 7 bases: This is to balance the stability of the hairpin structure with the dissociation efficiency after target binding, avoiding an overly stable structure that makes opening difficult, or an overly loose structure that causes fluorescence leakage. 3) The design of the 3' end stem sequence is limited: It must be complementary to the 5' end to form a stable closed structure, but if this sequence also partially matches the target, it will seriously interfere with the overall Tm (melting temperature) of the probe, leading to unstable signal or even detection failure.

[0006] In mutation detection, especially for the identification of single nucleotide polymorphisms (SNPs), probe specificity is crucial. To effectively distinguish between perfectly matched and mismatched sequences, the melting temperature difference (ΔTm) between the two should be maximized during the design phase. Ideally, ΔTm should be maintained above 3–5°C to achieve high specificity. A common strategy is to introduce locked nucleic acids (LNAs) to modify gene bases, thereby increasing the overall Tm of the probe. This allows for a shorter probe length without sacrificing thermal stability; it is generally recommended to keep the sequence length within 13–25 bases. This enhances both the specificity of mutation recognition and optimizes the conformational stability of the molecular probe.

[0007] However, when it is necessary to simultaneously identify a group of homologous mutations (such as multiple mutant subtypes at the same gene locus), it is usually necessary to design multiple highly similar but mutually exclusive molecular beacon probes in parallel. This presents the following challenges: 1) Short-sequence probes are difficult to form stable stem-loop structures, especially in the design of 5–7 bp stem pairing structures, which are prone to low loop-closing efficiency due to insufficient spatial rigidity; 2) Decreased probe closure efficiency increases the distance between the fluorophore and the quencher group, resulting in increased background fluorescence signal; 3) When multiple probes are used in parallel, especially when detecting multiple homologous sites, the cumulative effect of background signal is more significant, easily affecting the sensitivity and accuracy of detection. Summary of the Invention

[0008] To address the aforementioned technical issues, this invention introduces a Spacer structure into the molecular beacon detection probe, resolving the contradiction between stem complementarity and structural stability at the design level, thereby enhancing the design freedom and detection performance of enzyme-dependent beacons.

[0009] To overcome these challenges, this invention provides a molecular beacon probe for use in PCR endpoint detection. The molecular beacon probe sequentially comprises a 5' stem sequence, a loop sequence, a flexible spacer structure, and a 3' stem sequence, forming a hairpin structure. A fluorescent group is labeled at the 5' end, and a quencher group is labeled at the 3' end. The complementary sequences of the 5' and 3' stem sequences form a stable stem structure. The 5' stem and loop sequences are completely complementary to the target sequence. The loop sequence and the flexible spacer structure form the loop structure of the molecular beacon probe. The flexible spacer structure is a polyethylene glycol structure.

[0010] In one embodiment, the loop sequence is modified with locked nucleic acid.

[0011] In one embodiment, the number of polyethylene glycol units in the flexible spacer structure is not less than three.

[0012] In one embodiment, the number of polyethylene glycol units in the flexible spacer structure is 3-6.

[0013] In one embodiment, the present invention provides the application of the above-described molecular beacon probe in a digital PCR detection method.

[0014] In one embodiment, the present invention provides a digital PCR detection kit, the kit comprising the aforementioned molecular beacon probes.

[0015] In one embodiment, the present invention provides a multi-mutation site detection probe system based on a digital PCR platform. The probe system is used to detect multiple gene mutations with high homology in a reaction system. The probe system includes a molecular beacon detection probe and a fluorescent blocking probe designed for each gene mutation. The sequence of the fluorescent blocking probe is complementary to the 5-7 base region at the 5' end of each molecular beacon detection probe. The 3' end of the fluorescent blocking probe is labeled with a fluorescence quenching group. The short-chain probe binds to the 5' end of the molecular beacon detection probe in the free state to form a stable secondary blocking structure.

[0016] In one embodiment, the present invention provides a digital PCR detection kit, the kit comprising the above-described multi-mutation site detection probe system.

[0017] This invention innovatively introduces a Spacer flexible connector arm into the molecular beacon detection probe structure, located between the 3' stem sequence of the probe and the 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 stem-loop structure, significantly improving closure efficiency; 2) Effective reduction of background signal: The distance between fluorescence and quenching groups is more stable in the closed-loop state, and fluorescence suppression is more sufficient in the non-binding state; 3) Enhanced probe structure design compatibility: The Spacer reduces the possibility of non-specific pairing between the 3' stem and the target, avoiding interference with the probe Tm, and facilitating multi-probe co-detection design; 4) Improved multiple mutation detection capability: When detecting multiple homologous mutations or multi-site mutations, the Spacer structure enables the probe to maintain more consistent structural stability and background suppression capability, thereby achieving high-throughput and high-sensitivity mutation site resolution detection.

[0018] This invention overcomes the challenges of background control and structural looping in the detection of complex mutations using traditional molecular beacons by introducing a spacer structure into a short, highly specific probe, significantly improving detection accuracy and applicability. Furthermore, the system incorporates a short auxiliary probe sequence (fluorescent blocking probe) with a quencher group labeled at its 3' end, complementary to the 5–7 nt sequence at the 5' end of the main probe, further blocking any unclosed free probe structures. This probe system effectively enhances the specific recognition capability of enzyme-dependent molecular beacon probes in high homology backgrounds, enabling accurate detection of multiple homologous mutation sites, and is particularly suitable for endpoint detection platforms with extremely high signal-to-noise ratio requirements, such as digital PCR. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the structure and working principle of the Spacer molecular beacon probe of the present invention. In the figure, "F" represents a fluorescent group, "Q" represents a quenching group; solid lines represent recognition sequences complementary to the target region (including the 5-end stem sequence); single dashed lines represent the Spacer; double dashed lines represent 3-end stem sequences (complementary to the 5-end stem sequence); short vertical lines represent stem complementary sequences.

[0021] Figure 2 This is a schematic diagram of the fluorescent blocking probe structure and working principle of the present invention. Detailed Implementation

[0022] 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 merely some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. Unless otherwise specified, the following embodiments are all conventional methods in the art.

[0023] Example 1: Multi-mutation site detection probe system based on digital PCR platform of the present invention

[0024] This invention provides a digital PCR detection probe system suitable for a group of homologous mutations (multiple mutant subtypes at a certain site), specifically designed for highly sensitive detection of low-abundance cfDNA mutations in liquid biopsies. This probe system integrates key structural regions such as a molecular beacon hairpin structure, a 3' spacer arm, a 5' complementary fluorescent blocking probe, and LNA (locked nucleic acid) modification, aiming to improve detection specificity, suppress background signals, and enhance applicability and scalability in complex samples.

[0025] like Figure 1 and Figure 2 As shown: The probe system of the present invention comprises the following main components.

[0026] (1) Design of molecular beacon hairpin structure

[0027] The probe body of this invention is based on a classic molecular beacon structure. The molecular beacon probe sequentially comprises a 5' stem sequence, a loop sequence, a flexible spacer structure, and a 3' stem sequence, forming a hairpin structure. A fluorescent group is labeled at the 5' end, and a quencher group is labeled at the 3' end. The complementary sequences of the 5' and 3' stem sequences form a stable stem structure. The 5' stem and loop sequences are completely complementary to the target sequence. The loop sequence and the flexible spacer structure form the loop structure of the molecular beacon probe. The flexible spacer structure is a polyethylene glycol structure. Short complementary sequences at the 5' and 3' ends form a stable stem structure, which is composed of short complementary sequences (5–7 nt) at both ends.

[0028] Unlike traditional molecular beacons, this invention features two key design innovations:

[0029] • Perfect complementarity design of the 5' end to the target: In endpoint detection methods such as digital PCR, the release of fluorescent signals depends on the recognition and digestion of the probe. By making the 5' stem sequence of the probe completely complementary to the target sequence, the binding efficiency and the success rate of the digestion reaction are significantly improved, thereby enhancing the detection sensitivity.

[0030] • Flexible Spacer Structure Introduced in the Loop: This invention incorporates a flexible spacer into the loop structure, which not only helps the probe form a stable hairpin structure but also effectively blocks potential non-specific complementarity between the 3' stem and the target, avoiding interference with the probe's melting temperature (Tm). This design greatly simplifies the stem-loop structure design process and improves structural controllability and detection efficiency.

[0031] The probe is labeled with a fluorescent group at its 5' end and a quencher group at its 3' end. When the target is not recognized, the probe remains closed, and the fluorescence is effectively quenched. Once it binds to the target, the stem-loop structure unwinds, and enzyme digestion generates a fluorescent signal, completing the detection. This structure is particularly suitable for single-molecule mutation recognition and meets the high sensitivity requirements of digital PCR platforms.

[0032] (2) 3' end Spacer introduction strategy and structural optimization

[0033] To further optimize the probe conformation and improve its ability to form hairpin structures in a free state, this invention innovatively introduces a flexible spacer before the 3' stem sequence. This spacer offers the following functional advantages:

[0034] • Enhanced hairpin structure stability and reduced background fluorescence: Spacer improves the overall flexibility of the probe, making it easier to close into a stable stem-loop structure when not bound to the target. This allows the fluorescent group and the quenching group to maintain close contact, effectively suppressing background signals. It is especially suitable for probes with shorter lengths (<25 nt).

[0035] • Removing 3' end design constraints simplifies sequence matching: Due to the presence of the spacer, the 3' end stem sequence is physically blocked and no longer potentially pairs with the target, thus avoiding interference with the probe's Tm value. During the design process, the 5' end is fully paired with the target, and the 3' end stem only needs to be complementary to the reverse sequence of the 5' end, greatly simplifying the design process.

[0036] • Facilitates subsequent hydrolase recognition and reaction: After the probe binds to the target, the 5' end is completely complementary to the target, opening the structure and exposing the enzyme cleavage site, thus improving signal release efficiency. The flexible space provided by the spacer also benefits the hydrolase's performance in the bound state.

[0037] Therefore, the probe structure of the present invention is not only more flexible and efficient in design, but also exhibits higher specificity, stability and detection sensitivity in practical applications, making it particularly suitable for high-precision, endpoint detection-type digital PCR applications.

[0038] The 5' stem and loop sequences, which are completely complementary to the target sequence, are controlled to a total length between 12 and 30 bases. This region carries a sequence that is highly complementary to the mutation site to be detected. It is the core functional region for achieving accurate target identification and determines the specificity and binding selectivity of the probe.

[0039] Stem region: The 3' end of the probe contains a short sequence of 5-7 bases, designed to be inversely complementary to the 5' stem region. When the probe is not bound to the target, the two ends of the stem automatically pair to form a stable hairpin-shaped stem-loop structure, ensuring that the fluorophore and quencher are in close proximity, effectively suppressing background fluorescence, and forming the basis for the closed conformation of the probe;

[0040] Fluorescence and quenching labeling: A fluorescent group (such as FAM, HEX, etc.) is labeled at the 5' end of the probe, and a fluorescent quenching group (such as BHQ, DABCYL, etc.) is labeled at the 3' end. In the closed state, the fluorescent group approaches the quenching group, and the signal is effectively quenched; when the probe recognizes the target, the structure opens, the fluorescent group is released, and emits a detectable signal under the action of enzyme cleavage, realizing the specific release and quantitative detection of the signal;

[0041] The Spacer structure region, located between the 3' stem sequence and the recognition region of the probe, consists of a flexible spacer linker arm based on a polyethylene glycol (PEG) backbone. The introduction of the spacer significantly enhances the overall spatial flexibility of the probe, facilitating the formation of a stable, closed stem-loop structure in the free state, thereby reducing background fluorescence and improving the probe's signal-to-noise ratio. Furthermore, the spacer effectively blocks potential non-specific complementary pairing between the 3' stem sequence and the target, avoiding interference with the probe's melting temperature (Tm) and enhancing design flexibility and stability.

[0042] (3) Short-chain probes with complementary fluorescence blocking at the 5' end (fluorescence blocking probes)

[0043] This invention further innovatively designs a short-chain auxiliary probe whose sequence is complementary to the 5-7 base region at the 5' end of the molecular beacon probe, and whose 3' end is labeled with a fluorescence quenching group. For example... Figure 2 As shown, the short-chain probe can bind to the 5' end of the molecular beacon probe in its free state, forming a stable secondary closed structure, thereby further enhancing the fluorescence suppression ability of the beacon probe when it is not bound to the target, significantly reducing the background signal and improving the overall signal-to-noise ratio.

[0044] The design of this auxiliary probe takes into full account the compatibility with PCR reaction conditions. Due to its low melting temperature (Tm), it will not effectively bind to the target sequence at the annealing temperature of PCR, thus not interfering with the amplification process of the target region. This short-chain probe, through synergy with the beacon probe, optimizes the static conformation of the beacon probe while maintaining its high sensitivity and specificity in the dynamic reaction system, making it suitable for the detection of low-abundance mutations in environments with high background interference.

[0045] The fluorescent blocking probe is a short oligonucleotide chain of 5-7 nucleotides, complementary to the 5' sequence of the beacon probe, with a quenching group labeled at the 3' end. When the host probe is in a free state, this short probe binds to the host probe through base pairing, constructing an auxiliary blocking structure to further enhance the fluorescence signal suppression ability and improve sensitivity.

[0046] (4) LNA-modified bases

[0047] To address technical bottlenecks such as the difficulty in identifying single-base differences and the insignificant melting temperature difference (ΔTm), this invention introduces locked nucleic acid (LNA) modified bases into the recognition sequence of the beacon probe to enhance the binding stability between the probe and the target sequence. The introduction of LNA effectively increases the melting temperature (Tm) while shortening the probe length, thereby significantly increasing the thermodynamic difference between perfect matches and single-base mismatches while maintaining sufficient thermal stability, thus improving recognition resolution and mutation recognition specificity. Traditional molecular beacon probes may experience reduced stem-loop structure formation and stability after probe sequence shortening. This invention introduces a flexible spacer structure in the probe loop region. The addition of the spacer improves the overall conformational flexibility of the probe, enabling it to form a stable stem-loop structure in its free state even with shortened probe length and the introduction of LNA, maintaining a low background signal and ensuring the high efficiency and reliability of the probe in low-abundance mutation detection.

[0048] One to five LNA-modified bases are introduced into the loop sequence, preferably near the mutation recognition site, to improve the thermodynamic binding stability with the target sequence, enhance the melting temperature difference (ΔTm) between perfectly matched and mismatched sequences, and achieve single-base resolution.

[0049] The probe system of this invention has the following significant advantages: It significantly reduces background signal by effectively suppressing non-specific fluorescence release from free probes through a dual-structure design of spacer and fluorescent blocking probes; it has strong multi-mutation co-detection capability, flexible structural design, and adaptability to the simultaneous use of multiple probes, making it suitable for simultaneous detection of multiple highly homologous or different gene mutation sites in a single tube; it has high detection specificity, and combined with LNA technology, it can achieve high-discrimination identification of single-base mutations, especially suitable for accurate quantification of low-frequency mutations; and it is compatible with digital PCR platforms: the probe system is highly compatible with digital PCR platforms and is suitable for molecular diagnosis and efficacy monitoring of trace samples such as ctDNA / cfDNA.

[0050] In summary, the probe system of this invention overcomes the performance bottleneck of traditional molecular beacons in multi-target, high-homology, and multi-probe co-detection applications, especially in the quantitative detection of low-frequency mutations in clinical liquid biopsies, demonstrating significant application value and promising prospects. This invention's probe system is adaptable to co-detection of multiple mutation sites and is suitable for low-abundance plasma cfDNA samples; it employs a molecular beacon structure, avoiding the high background problem of traditional molecular beacon probes; it introduces a spacer to improve the efficiency of probe hairpin structure formation, significantly reducing background signal and improving detection reliability; it is the first to combine a short-chain fluorescent blocking probe design with a 5' complementary sequence and a quenching group, achieving a "double inhibition" effect and effectively reducing the background fluorescence of free probes; and it uses LNA to shorten probe length while simultaneously improving Tm value and specificity, supporting high-sensitivity mutation recognition.

[0051] Example 2: Comparison Test with Different Probes

[0052] This embodiment designs various types of probes to detect the PIK3CA E545K mutation site. The probe designs are as follows: 1) a standard probe with a sequence complementary to the target; 2) an LNA probe with a sequence complementary to the target but containing 5 LNAs to increase Tm and shorten length; 3) an enzyme-dependent molecular beacon probe with the following structure: a 5' stem sequence (completely or partially complementary to the target), a loop sequence (complementary to the target), and a 3' stem sequence (complementary to the 5' end); 4) a spacer molecular beacon probe with the following structure: a 5' stem sequence (completely complementary to the target), a loop sequence (complementary to the target), a spacer, and a 3' stem sequence (complementary to the 5' end). All probes are labeled with a fluorescent group at the 5' end and a quencher group at the 3' end. Primer and probe sequences are shown in Table 1. The detection results of various probes on E545K positive plasmids and wild-type plasmids were compared, and the median background signal and median positive signal were compared to examine the specificity and accuracy of the probes.

[0053] Table 1. Primer and probe sequences

[0054]

[0055] Note: + represents LNA-modified bases; lowercase letters represent 3-terminal stem sequences that do not match the target region; underlined lowercase letters represent 3-terminal stem sequences that match the target; uppercase letters represent loop sequences and 5-terminal stem sequences that match the target region; and the underlined uppercase letters in the middle are mutation sites.

[0056] The detection system was prepared as follows: using 4× SuperMix from Xinyi Manufacturing Technology (Beijing) Co., Ltd., 2 μl of E545K positive plasmid and wild-type plasmid (500 copies / μl) were added to the template respectively, and the background signal, positive signal, and positive copy number were detected:

[0057]

[0058] The digital PCR workflow is as follows:

[0059] A. Microdroplet preparation: Using a droplet generation chip (NewYi Manufacturing Technology (Beijing) Co., Ltd.) and a sample preparation instrument (NewYi Manufacturing Technology (Beijing) Co., Ltd.), add 30 µL of PCR reaction system to the sample well of the droplet generation chip and prepare microdroplets according to the instrument's instruction manual.

[0060] B. PCR Amplification: Place the 8-tube array containing microdroplets into a PCR instrument for amplification. The amplification program is set as follows:

[0061]

[0062] C. Microdroplet Detection: After PCR is completed, place the 8-tube array and the droplet detection chip (New Yi Manufacturing Technology (Beijing) Co., Ltd.) into the chip analyzer (New Yi Manufacturing Technology (Beijing) Co., Ltd.) and perform droplet detection according to the analyzer's instruction manual.

[0063] D. Data Analysis: After PCR amplification, each microdroplet is detected using a microarray analyzer, and the fluorescence signal intensity of the microdroplets is recorded. Droplets containing the target gene will be detected with a corresponding positive fluorescence signal. The fluorescence intensity within the microdroplets is digitized using a fluorescence classification threshold. Microdroplets with strong fluorescence are interpreted as "1" (positive), and microdroplets with weak fluorescence are interpreted as "0" (negative). The number of "1"s and "0"s is counted, and corrected using the Poisson distribution formula, the total copy number of the target gene in the template can be calculated.

[0064] The test results are shown in Table 2.

[0065] 1) Conventional probes are relatively long and have poor specificity, exhibiting non-specific signals against wild-type templates; 2) Probes with 5 added LNAs improve specificity, exhibiting no non-specific signals against wild-type templates and accurately detecting mutants, but the background signal is relatively high; 3) Molecular beacon probes for digital PCR require partial or complete 5' end matching with the target to improve enzyme digestion efficiency, but it is also necessary to balance whether the added 3' end stem sequence affects the probe's specificity. As shown in Table 2, when the 5' end stem sequence in this design is completely matched with the target, its 3' end complementary stem sequence has 3 consecutive bases matching the target (underlined lowercase letters), thus affecting the probe's specificity and exhibiting non-specific signals against wild-type templates; 4) After modifying the stem sequence, two bases are added to the 5' end, not matching the target. At this time, the 3' end stem sequence does not match the target, resulting in better beacon probe specificity, exhibiting no non-specific signals against wild-type templates, and accurately detecting mutant templates, but the background is still relatively high; 5) The background signal of the Spacer molecular beacon probe is lower than that of the above molecular beacon probes, and the signal-to-noise ratio is higher. The detected target sequence copy number was not significantly different from that of the probe, consistent with the expected quantitative results. This indicates that the introduction of the spacer does not affect the probe's identification and amplification efficiency of the target, ensuring the accuracy of the detection results. At the same time, no non-specific signals were detected in the wild-type template, indicating that the probe has good specificity.

[0066] Table 2. Comparison of detection results for different types of probes

[0067]

[0068] Example 3: Spacer Spacing Length Test

[0069] Because the spacer region provides a flexible spatial structure for the molecular beacon probe, facilitating easier pairing and stable formation of a hairpin stem-loop structure between the 3' and 5' stem sequences, the spacer spacer region needs to be constructed using chemical units with high flexibility. This invention uses flexible chains, represented by ethylene glycol (PEG), as the basic unit of the spacer structure to ensure successful closure and maintain a low background signal when the probe is in a free state. To further optimize the performance of the spacer structure, this experiment systematically evaluated the effects of different spacer lengths (i.e., the number of PEG units) on the conformational stability and background signal suppression effect of the beacon probe, screening out suitable spacer region lengths for the spacer molecular beacon probe, providing a basis for the standardized design of subsequent probes.

[0070] Primer and probe sequences are shown in Table 3 below. The reaction system and digital PCR procedure are described in Example 1. Different probes were added, and 2 μl of E545K positive plasmid and wild-type plasmid (500 copies / μl) were added to the template respectively. Background signal, positive signal, and positive copy number were detected.

[0071] Table 3. Primer and probe sequences

[0072]

[0073] Note: + indicates LNA-modified bases; lowercase letters represent 3-terminal stem sequences that do not match the target region, uppercase letters represent loop sequences and 5-terminal stem sequences that match the target region, and the underlined uppercase letters in the middle are mutation sites.

[0074] The detection results are shown in Table 4. The results show that when the number of polyethylene glycol (PEG) units used in the Spacer region is 6 (Spacer 18), 4 (Spacer 12), and 3 (Spacer 9), the background signal of the probe in the free state is significantly lower than that of the group containing only 2 PEG units (Spacer 6). This indicates that when the Spacer length reaches 3 or more PEG units, it can effectively endow the probe with sufficient flexibility, promote the stable closure of the stem-loop structure, thereby significantly reducing the fluorescence background signal in the unbound state and improving the signal-to-noise ratio of the detection system.

[0075] There was no significant difference in the target sequence copy number detected in each group, which was consistent with the expected quantitative results. No positive signal was found in wild-type plasmids, indicating that the change in spacer length provides structural flexibility without affecting the probe's recognition and amplification efficiency of the target, ensuring the consistency of detection sensitivity. At the same time, the probes maintained specificity and there were no non-specific detection signals.

[0076] Table 4. Detection results of molecular beacon probes with different spacer lengths

[0077]

[0078] Example 4: Testing the co-detection effect of multiple homologous sites of the fluorescent blocking probe.

[0079] To verify whether the Spacer probes designed in this invention can achieve joint detection of multiple homologous mutation sites in the same reaction tube, corresponding Spacer probes (Spacer 18 was used in this embodiment) were designed for seven common mutant genotypes (E542K, E545K, E545A, E545G, E545D, Q546E, and Q546R) at sites 542–546 of the PIK3CA gene. Simultaneously, to suppress non-specific fluorescence signals that may be triggered by free probes in the system, a short-chain fluorescent blocking probe was designed, and a BHQ1 quencher group was introduced at its 3' end to effectively block the signal of free probes that have not bound to the target sequence. Primer and probe sequences are shown in Table 5.

[0080] Table 5. Primer and probe sequences

[0081]

[0082] Note: + indicates LNA-modified bases; lowercase letters represent 3-terminal stem sequences that do not match the target region, uppercase letters represent loop sequences and 5-terminal stem sequences that match the target region, and the underlined uppercase letters in the middle are mutation sites.

[0083] The detection system was prepared as shown in Tables 6 and 7 below. Each mutant positive plasmid and wild-type plasmid was added to the template (at a concentration of 500 copies / μl).

[0084] Table 6. Multisite detection system without fluorescent blocking probes

[0085]

[0086] Table 7. Multisite detection system with added fluorescent blocking probes

[0087]

[0088] The digital PCR workflow is described in Example 2. The detection results are shown in Table 8. The Spacer probe strategy allows for accurate detection of multiple mutation sites in the same reaction tube. However, due to the simultaneous presence of multiple Spacer probes, their background signals overlap to some extent, resulting in a higher overall background level in the reaction system. Therefore, a fluorescent blocking probe is introduced to effectively block the non-specific fluorescence signal generated by the free probe, further reducing background noise and significantly improving the signal-to-noise ratio.

[0089] The quantitative detection results for each mutation type were largely consistent with expectations, indicating that the system effectively reduces background signal and improves detection sensitivity without affecting the accuracy of the target copy number. Furthermore, for wild-type templates in this multi-probe system, regardless of whether fluorescent blocking probes were added, only background-level signals were detected, with no positive fluorescent signals observed. These results further validate the good specificity of the mutant probes.

[0090] The high specificity of this system is due to the introduction of an LNA (locked nucleic acid) structure into the probe design, which shortens the probe length and enhances the ability to recognize mismatched bases, preventing the mutant probe from generating non-specific signals when facing wild-type templates. In summary, the "Spacer molecular beacon probe + fluorescent blocking probe" combined probe system designed in this invention can achieve highly specific detection of multiple homologous mutation sites within a single tube, significantly reducing background signal and improving the signal-to-noise ratio, thereby contributing to improved accuracy and interpretability of detection results.

[0091] Table 8. Detection results of multi-probe systems with and without fluorescent blocking probes.

[0092]

[0093] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0094] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A digital PCR assay kit for detecting a mutant genotype of a PIK3CA gene at positions 542-546, characterized by, The kit includes an enzyme-dependent molecular beacon probe, comprising a 5' stem sequence, a loop sequence, a flexible spacer structure, and a 3' stem sequence, forming a hairpin structure. The 5' end is labeled with a fluorescent group, and the 3' end with a quencher group. The complementary sequences of the 5' and 3' stem sequences form a stable stem structure. The 5' stem and loop sequences are completely complementary to the target sequence. The loop sequence and the flexible spacer structure form the loop structure of the molecular beacon probe. The flexible spacer structure is made of polyethylene glycol. The molecular beacon probe sequences included in the kit are as follows: E545K Spacer 18 molecular beacon probe: CTCCTG+CT+T+A+GT+GATTT-Spacer 18-caggag; E542K Spacer 18 molecular beacon probe: CTCAG+T+GATT+T+T+AGAGAG-Spacer 18-actgag; E545A Spacer 18 molecular beacon probe: CTTTCTCCTGC+ G +CAGTGA-Spacer 18-agaaag; E545G Spacer 18 molecular beacon probe: CTCCTG+C+ CCAGTGA-Spacer 18-caggag; E545D Spacer 18 molecular beacon probe: CTTTCTC+CT+G+ A TCAGTGATTTC-Spacer 18-agaaag; Q546E Spacer 18 molecular beacon probe: CTTTCTCC+T+ C+CTCAGTGA-Spacer 18-agaaag, and Q546R Spacer 18 molecular beacon probe: CTTTCTC+C+C GCTCAGTGA-Spacer 18-agaaag; In this context, + represents locked nucleoside (LNA).

2. The use of the digital PCR detection kit of claim 1 in a method for detecting PIK3CA mutations at sites 542–546 based on digital PCR for non-diagnostic purposes.

3. A multi-mutation site detection probe system based on a digital PCR platform, characterized in that, The probe system is used to detect multiple highly homologous gene mutations in a single reaction system. The probe system includes a molecular beacon probe and a fluorescent blocking probe designed for each gene mutation. The sequence of the fluorescent blocking probe is complementary to the 5-7 base region at the 5' end of each molecular beacon probe. The 3' end of the fluorescent blocking probe is labeled with a fluorescence quencher group. The fluorescent blocking probe binds to the 5' end of the molecular beacon probe in its free state, forming a stable secondary blocking structure. The molecular beacon probe sequences included in the probe system are as follows: E545K Spacer 18 molecular beacon probe: CTCCTG+CT+T+A+GT+GATTT-Spacer 18-caggag; E542K Spacer 18 molecular beacon probe: CTCAG+T+GATT+T+T+AGAGAG-Spacer 18-actgag; E545A Spacer 18 molecular beacon probe: CTTTCTCCTGC+ G +CAGTGA-Spacer 18-agaaag; E545G Spacer 18 molecular beacon probe: CTCCTG+C+ CCAGTGA-Spacer 18-caggag; E545D Spacer 18 molecular beacon probe: CTTTCTC+CT+G+ A TCAGTGATTTC-Spacer 18-agaaag; Q546E Spacer 18 molecular beacon probe: CTTTCTCC+T+ C+CTCAGTGA-Spacer 18-agaaag, and Q546R Spacer 18 molecular beacon probe: CTTTCTC+C+C GCTCAGTGA-Spacer 18-agaaag; In this context, + represents locked nucleoside (LNA).

4. A digital PCR detection kit, characterized in that, The kit includes the multi-mutation site detection probe system as described in claim 3.

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