Molecular beacon probe used in PCR end point detection method, and system and kit thereof

Abstract

The invention provides a molecular beacon probe used in a PCR (Polymerase Chain Reaction) end point detection method as well as a system and a kit thereof, the molecular beacon probe sequentially comprises a 5'end stem sequence, a loop sequence, a flexible Spacer structure and a 3 'end stem sequence, and the whole molecular beacon probe is of a hairpin structure; the 5'end stem sequence and the loop sequence are completely complementary to a to-be-detected target sequence, and 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. 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. Besides, a fluorescent closed probe is also introduced into the system, and the probe system effectively improves the specific recognition capability of the enzyme digestion dependent molecular beacon probe under a high homologous background, so that accurate detection of a plurality of homologous mutation sites becomes possible.

Description

Technical Field

[0001] The present invention relates to the field of digital PCR detection, and more particularly, to molecular beacon probes used in PCR end-point detection methods, as well as systems and kits thereof. Background Art

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

[0003] Liquid biopsy technology, especially the detection of mutations based on cell-free DNA (cfDNA) in plasma, is becoming an important tool in precision medicine. However, detecting low-abundance mutations in cfDNA faces many challenges, mainly including insufficient detection sensitivity, limited throughput, and high costs. Using digital PCR technology can significantly improve the detection sensitivity of low-abundance mutations. At the same time, detecting multiple highly homologous gene mutations in a reaction system is often limited by technical bottlenecks such as background noise, fluorescence interference, and signal-to-noise ratio decline, which may lead to detection failure or inaccurate results.

[0004] Molecular Beacon probe technology is a method that can effectively solve the above problems. A molecular beacon is a dual-labeled oligonucleotide probe with a length of 25 to 40 nucleotides, and its structure is hairpin-shaped, consisting of a circular probe sequence and a short self-complementary stem structure. Its 5' end is labeled with a fluorophore, and its 3' end is labeled with a quencher. At room temperature, due to the formation of the stem-loop structure of the probe, the fluorophore and the quencher are close to each other, thus inhibiting the fluorescence signal. When in the annealing stage of PCR, if the probe encounters a target DNA sequence that is completely complementary to it, according to the thermodynamic advantage, the probe will preferentially bind to the target, resulting in the opening of the hairpin structure, and the fluorophore will move away from the quencher, thus emitting a fluorescence signal. The stem sequence of the molecular beacon usually does not complementarily pair with the target but is a specially designed short inverted repeat sequence, the purpose of which is to maintain the stability of the hairpin structure without interfering with the target detection.

[0005] In digital PCR technology, since it is an end-point detection method, the release of fluorescence signals usually depends on enzymatic cleavage reactions. Therefore, traditional molecular beacon probes cannot meet its high requirements for signal intensity and specificity. Enzyme cleavage-dependent molecular beacons need to be used. Traditional molecular beacons usually adopt a hairpin structure, and its stem is formed by complementary sequences at the 5' end and 3' end. However, this stem sequence does not pair with the target sequence and only serves to maintain the closed conformation and inhibit fluorescence signals. This structure performs well in real-time fluorescence quantitative PCR but is not suitable for the end-point detection method of digital PCR. Digital PCR requires enzyme cleavage-dependent molecular beacons, and the 5' end 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 overlapping region 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 an enzyme cleavage site. However, if the design is improper, it is easy to cause mismatches and affect the recognition efficiency. 2) The total length of the stem needs to be maintained between 5-7 bases to balance the stability of the hairpin structure and the dissociation efficiency after binding to the target, avoiding difficulties in opening due to overly stable structures or fluorescence leakage due to overly loose structures. 3) The design of the 3' end stem sequence is restricted. It must be complementary to the 5' end to form a stable closed structure. However, once this sequence also partially matches the target, it will seriously interfere with the overall Tm (melting temperature) of the probe, resulting in unstable signals or even detection failure.

[0006] In mutation detection, especially for the recognition of single nucleotide polymorphisms (SNPs), the specificity of the probe is crucial. To effectively distinguish between perfectly matched and single-base mismatched sequences, the melting temperature difference (ΔTm) between the two should be increased as much as possible during design. Ideally, ΔTm should be maintained above 3-5 °C to achieve highly specific recognition. A commonly used strategy is to introduce locked nucleic acid (LNA) modifications to gene bases to increase the overall Tm of the probe. At the same time, the probe length can be shortened without sacrificing thermal stability. It is usually recommended to control the sequence length within 13-25 bases. This not only enhances the specificity of mutation recognition but also optimizes the conformational stability of the molecular probe.

[0007] However, when it is necessary to simultaneously recognize a group of homologous mutations (such as multiple mutation subtypes at the same gene locus), multiple highly similar but mutually exclusive molecular beacon probes usually need to be designed in parallel. The following challenges will be faced at this time: 1) Short-sequence probes are difficult to form stable stem-loop structures. Especially in the design of a 5-7 bp stem pairing structure, it is easy to have low closed-loop efficiency due to insufficient spatial rigidity. 2) The decrease in the probe closed-loop efficiency will increase the distance between the fluorophore and the quencher group, resulting in an increase in background fluorescence signals. 3) When multiple probes are used in parallel, especially when detecting multiple homologous sites, the cumulative effect of background signals is more significant, which is extremely likely to affect the detection sensitivity and interpretation accuracy. Summary of the Invention

[0008] To solve the above technical problems, the present invention introduces a Spacer structure into the molecular beacon detection probe, which solves the contradiction between stem complementarity and structural stability at the design level, and improves the design freedom and detection performance of enzyme cleavage-dependent beacons.

[0009] To overcome these challenges, the present invention provides a molecular beacon probe used in a PCR end-point detection method. The molecular beacon probe sequentially includes a 5'-end stem sequence, a loop sequence, a flexible Spacer structure, and a 3'-end stem sequence, and has an overall hairpin structure. A fluorescent group is labeled at the 5'-end, and a quenching group is labeled at the 3'-end. 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 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 molecular beacon probe in a digital PCR detection method.

[0014] In one embodiment, the present invention provides a digital PCR detection kit, which includes the above molecular beacon probe.

[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 highly homologous gene mutations in one reaction system. The probe system includes a molecular beacon detection probe and a fluorescence blocking probe designed for each gene mutation respectively. The sequence of the fluorescence blocking probe is complementary to the 5-7 base region at the 5'-end of each molecular beacon detection probe described above. A fluorescence quenching group is labeled at the 3'-end of the fluorescence blocking probe. This short-chain probe binds to the 5'-end of the molecular beacon detection probe in its free state to form a stable secondary closed structure.

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

[0017] 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) Improve spatial flexibility: enabling short-sequence probes to efficiently form stable hairpin-shaped stem-loop structures, significantly enhancing the closing efficiency; 2) Effectively reduce background signals: the distance between the fluorescence and quenching groups is more stable in the closed-loop state, and fluorescence suppression is more sufficient in the unbound state; 3) Enhance the compatibility of 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) Improve the ability to detect multiple mutations: when detecting multiple homologous mutations or multi-site mutations, the Spacer structure enables the probe to maintain more consistent structural stability and background suppression ability, thereby achieving high-throughput and high-sensitivity detection and resolution of mutation sites.

[0018] By introducing the Spacer structure into short-sequence highly specific probes, the present invention breaks through the problems of background control and loop formation in the detection of complex mutations by traditional molecular beacons, significantly improving the detection accuracy and scope of application. In addition, a short-chain auxiliary probe sequence (fluorescence-blocking probe) is introduced into the system, with a quenching group labeled at its 3'-end and complementary to the 5–7 nt sequence at the 5'-end of the main probe to further block 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is a schematic diagram of the Spacer molecular beacon probe structure and working principle of the present invention, where "F" in the figure represents the fluorescent group and "Q" represents the quenching group; the solid line represents the recognition sequence complementary to the target region to be detected (including the stem sequence at the 5'-end); the single dashed line represents the Spacer; the double dashed line represents the stem sequence at the 3'-end (complementary to the stem sequence at the 5'-end); the short vertical line represents the complementary sequence of the stem. Figure 2 It is a schematic diagram of the structure and working principle of the fluorescence-blocking probe of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] 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 them. 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 scope of protection of this application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0022] Example 1 Digital PCR Detection Probe System for Multiple Mutation Sites Based on the Present Invention The present invention provides a digital PCR detection probe system suitable for a group of homologous mutations (multiple mutation subtypes exist at a certain site), which is designed specifically for the highly sensitive detection of low-abundance mutations in cfDNA in liquid biopsy. This probe system integrates key structural regions such as the molecular beacon hairpin structure, 3'-end Spacer spacer arm, 5'-end complementary fluorescence quenching probe, and LNA (locked nucleic acid) modification, aiming to improve detection specificity, suppress background signals, and enhance applicability and scalability in complex samples.

[0023] As Figure 1 and Figure 2 shown: The probe system of the present invention includes the following main components.

[0024] (1) Design of Molecular Beacon Hairpin Structure The probe body of the present invention is based on the classic molecular beacon structure. The molecular beacon 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; a fluorescent group is labeled at the 5'-end, and a quenching group is labeled at the 3'-end. 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 probe; the flexible Spacer structure is a polyethylene glycol structure. The short complementary sequences at the 5'-end and 3'-end form a stable stem structure, which is composed of short complementary sequences (5–7 nt) at both ends.

[0025] Different from traditional molecular beacons, the present invention has made two key innovations in design: • Complete complementary design of the 5'-end with the target: In end-point detection methods such as digital PCR, the release of fluorescence signals depends on the recognition and enzymatic cleavage of the probe. By making the 5'-end stem sequence of the probe completely complementary to the target sequence, the binding efficiency and the success rate of the enzymatic cleavage reaction are significantly improved, thereby enhancing the detection sensitivity.

[0026] • Flexible Spacer structure introduced in the loop region: In the loop region structure of the present invention, a flexible Spacer is added, which not only helps the probe to form a stable hairpin structure, but also effectively blocks the possible non-specific complementarity between the 3'-end stem and the target, avoiding interference with the melting temperature (Tm) of the probe. This design greatly simplifies the design process of the stem-loop structure and improves the structural controllability and detection efficiency.

[0027] The 5'-end of the probe is labeled with a fluorescent group, and the 3'-end is labeled with a quenching group. When the target is not recognized, the probe remains in a closed state and the fluorescence is effectively quenched; once it binds to the target, the stem-loop structure unfolds, and enzymatic cleavage generates a fluorescent signal to complete the detection. This structure is particularly suitable for single-molecule mutation recognition and meets the high-sensitivity requirements of the digital PCR platform.

[0028] (2)3'-end Spacer introduction strategy and structure optimization To further optimize the probe conformation and improve its ability to form a hairpin structure in the free state, the present invention innovatively introduces a flexible spacer (Spacer) before the 3'-end stem sequence. This Spacer has the following functional advantages: • Enhance the stability of the hairpin structure and reduce background fluorescence: The Spacer improves the overall flexibility of the probe, making it easier to close into a stable stem-loop structure when the target is not bound, so that the fluorescent group and the quenching group remain in close contact, effectively suppressing the background signal, and is particularly suitable for probes with a short length (<25 nt).

[0029] • Remove the 3'-end design constraints and simplify the sequence matching problem: 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 Tm value of the probe. 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.

[0030] • Facilitate subsequent recognition and reaction of hydrolase: After the probe binds to the target, due to the complete complementarity of the 5'-end with the target, the structure opens to facilitate the exposure of the cleavage site and improve the signal release efficiency. The flexible space provided by the Spacer is also conducive to the function of the hydrolase in the bound state.

[0031] Therefore, the probe structure of the present invention is not only more flexible and efficient in terms of design, but also shows higher specificity, stability and detection sensitivity in practical applications, and is particularly suitable for high-precision, end-point detection digital PCR applications.

[0032] The 5'-end stem sequence and loop sequence that are completely complementary to the target sequence to be measured, with the total length controlled between 12 and 30 bases. This region carries a sequence with high complementarity to the mutation site to be detected, which is the core functional region for achieving precise target recognition and determines the specificity and binding selectivity of the probe; Stem structure region: The 3'-end of the probe contains a short sequence with a length of 5-7 bases, which is designed to form an inverse complementary relationship with the 5'-end stem region. When the probe does not bind to the target, the double-ended stems automatically pair to form a stable hairpin-like stem-loop structure, ensuring that the fluorophore and quencher are in close proximity, effectively suppressing the background fluorescence, and constituting the basis for the closed conformation of the probe; Fluorescence and quenching labeling: A fluorophore (such as FAM, HEX, etc.) is labeled at the 5'-end of the probe, and a fluorescence quencher (such as BHQ, DABCYL, etc.) is labeled at the 3'-end. In the closed state, the fluorophore is close to the quencher, and the signal is effectively quenched; when the probe recognizes the target, the structure opens, the fluorophore is released and emits a detectable signal under the action of enzymatic cleavage, realizing the specific release and quantitative detection of the signal; Spacer structure region: Located between the 3'-end stem sequence and the recognition region of the probe, a flexible Spacer linker arm based on a polyethylene glycol (PEG) backbone is inserted. The introduction of Spacer significantly enhances the overall spatial flexibility of the probe, helps the probe to more easily form a stable closed stem-loop structure in the free state, thereby reducing the background fluorescence and improving the signal-to-noise ratio of the probe. In addition, Spacer effectively blocks the possible non-specific complementary pairing between the 3'-end stem sequence and the target, avoiding interference with the melting temperature (Tm) of the probe and enhancing the flexibility and stability of the design.

[0033] (3)Short-chain probe with 5'-end complementary fluorescence quenching (fluorescence quenching probe) The present 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 a fluorescence quencher is labeled at the 3'-end. As Figure 2 shown, this short-chain probe can bind to the 5'-end of the molecular beacon probe in the free state of the molecular beacon probe, forming a stable secondary closed structure, thereby further enhancing the fluorescence suppression ability of the beacon probe when it does not bind to the target, significantly reducing the background signal and improving the overall signal-to-noise ratio.

[0034] The design of this auxiliary probe fully considers the compatibility of PCR reaction conditions. Since its melting temperature (Tm) is relatively low, it will not bind effectively to the target sequence at the annealing temperature of PCR, so it will not interfere with the amplification process of the target region. This short-chain probe, through its synergistic effect with the beacon probe, not only optimizes the static conformation of the beacon probe but also maintains its high sensitivity and high specificity in the dynamic reaction system, and is suitable for the detection of low-abundance mutations in high-background interference environments.

[0035] The fluorescence-blocking probe is a short-chain oligonucleotide with a length of 5 to 7 nucleotides, which is complementary to the 5'-end sequence of the beacon probe. The 3'-end of this short-chain probe is labeled with a quenching group. When the main probe is in the free state, this short-chain probe binds to the main probe through base pairing to construct an auxiliary blocking structure, further enhancing the fluorescence signal inhibition ability and improving the sensitivity.

[0036] (4)LNA-modified bases Aiming at the technical bottlenecks such as the difficulty in identifying single-base differences and the insignificant melting temperature difference (ΔTm), the present 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 can effectively increase its melting temperature (Tm) while shortening the probe length, thereby significantly increasing the thermodynamic difference between perfect match and single-base mismatch under the premise of maintaining sufficient thermal stability, and improving the recognition resolution and the specificity of mutation recognition. The formation and stability of the stem-loop structure may be affected after the shortening of the probe sequence in the traditional molecular beacon probe. The present invention introduces a flexible Spacer structure into the loop region of the probe. The addition of Spacer improves the flexibility of the overall conformation of the probe, so that even under the conditions of shortening the probe length and introducing LNA, the probe can still form a stable stem-loop structure in the free state, maintaining a low background signal, thereby ensuring the high efficiency and reliability of the probe in the detection of low-abundance mutations.

[0037] Introduce 1 to 5 LNA-modified bases into the loop sequence, preferably designed near the mutation recognition site, to improve the thermodynamic binding stability with the target sequence, enhance the melting temperature difference (ΔTm) between the perfect match and the mismatch sequence, and achieve single-base resolution ability.

[0038] The probe system of the present invention has the following significant advantages: the background signal is significantly reduced. Through the dual-structure design of Spacer and fluorescence-blocking probe, the non-specific fluorescence release of the free probe is effectively inhibited; it has strong multi-mutation co-detection ability, flexible structure design, suitable for multiple probes to be used simultaneously, and is suitable for detecting multiple highly homologous or different gene mutation sites in a single tube; it has high detection specificity. Combining with LNA technology, it can achieve high-discrimination recognition of single-base mutations, especially suitable for the precise quantification of low-frequency mutations, and is compatible with the digital PCR platform: the probe system is highly compatible with the digital PCR platform and is applicable to molecular diagnosis and efficacy monitoring of trace samples such as ctDNA / cfDNA.

[0039] In summary, the probe system of the present invention solves the performance bottlenecks of traditional molecular beacons in multi-target, high homology, and co-detection applications of multiple probes. Especially in the quantitative detection of low-frequency mutations in clinical liquid biopsies, it has great application value and broad prospects for promotion. The probe system of the present invention is adapted to co-detect multiple mutation sites and is suitable for low-abundance plasma cfDNA samples; it adopts the molecular beacon structure to avoid the high-background problem of traditional molecular beacon probes; it introduces a Spacer to improve the formation efficiency of the probe hairpin structure, significantly reduces the background signal, and improves the detection reliability; for the first time, it combines the design of a short-chain fluorescent blocking probe with a 5' complementary sequence + quenching group to achieve a "double inhibition" effect, effectively reducing the background fluorescence of free probes; and LNA shortens the probe length, while increasing the Tm value and specificity, supporting highly sensitive mutation recognition.

[0040] Example 2 Comparative Test of Different Probes In this example, various types of probes were designed to detect the PIK3CA E545K mutation site. The designs of various probes are as follows: 1) Ordinary probe, with a structure of a sequence complementary to the target; 2) LNA probe, with a structure of a sequence complementary to the target but containing 5 LNAs to increase the Tm and shorten the length; 3) Enzyme cleavage-dependent molecular beacon probe, with a structure including: a 5'-end (fully or partially complementary to the target) stem sequence - a loop (complementary to the target) sequence - a 3'-end (complementary to the 5'-end) stem sequence; 4) Spacer molecular beacon probe, with a structure as follows: a 5'-end (fully complementary to the target) stem sequence - a loop (complementary to the target) sequence - Spacer - a 3'-end (complementary to the 5'-end) stem sequence. These probes are all labeled with a fluorescent group at the 5'-end and a quenching group at the 3'-end. The primer probe sequences are shown in Table 1. The detection results of various probes for the E545K positive plasmid and the wild-type plasmid were compared, the median background signal and the median positive signal were compared, and the specificity and accuracy of the probes were also investigated.

[0041] Table 1. Primer Probe Sequences

[0042] Note: + represents an LNA-modified base; lowercase letters represent the 3'-end stem sequence that does not match the target region, underlined lowercase letters represent the 3'-end stem that matches the target, capital letters represent the loop sequence and the 5'-end stem sequence that match the target region, and the underlined capital letters in the middle are the mutation sites.

[0043] The detection system was prepared as follows. Using the 4× SuperMix from Novogene Bioinformatics Technology Co., Ltd., 2 μl of the E545K positive plasmid and the wild-type plasmid (500 copies / μl) were added as templates respectively to detect the background signal, positive signal, and positive copy number:

[0044] The digital PCR workflow is as follows: A. Microdroplet preparation: Use a droplet generation chip (manufactured by Singleron Biotechnologies Co., Ltd.) and a sample preparation instrument (manufactured by Singleron Biotechnologies Co., Ltd.). Add 30 µL of the PCR reaction system to the sample wells of the droplet generation chip, and perform microdroplet preparation according to the operating instructions of the preparation instrument.

[0045] B. PCR amplification: Place the 8-strip tube containing microdroplets on a PCR instrument for amplification. The amplification program is set as follows:

[0046] C. Microdroplet detection: After PCR is completed, place the 8-strip tube and the droplet detection chip (manufactured by Singleron Biotechnologies Co., Ltd.) into a chip analyzer (manufactured by Singleron Biotechnologies Co., Ltd.), and perform droplet detection according to the operating instructions of the analyzer.

[0047] D. Data analysis: After PCR amplification, each microdroplet is detected by the chip analyzer, and the fluorescence signal intensity of the microdroplets is recorded. Droplets containing the target gene to be detected will be detected with corresponding positive fluorescence signals. The fluorescence intensity inside the microdroplets is digitized through a fluorescence classification threshold. Microdroplets with stronger fluorescence are judged as "1" (positive), and microdroplets with weaker fluorescence are judged as "0" (negative). Count the number of "1"s and "0"s, and correct through the Poisson distribution formula to calculate the total copy number of the target gene in the template.

[0048] The detection results are shown in Table 2.

[0049] 1) Ordinary probes have a relatively long length and poor specificity, showing non-specific signals for wild-type templates; 2) Probes with 5 additional LNAs have improved specificity, showing no non-specific signals for wild-type templates and being able to accurately detect mutants, but with relatively high background signals; 3) Molecular beacon probes for digital PCR require partial or complete matching of the 5'-end with the target to improve cleavage efficiency, but the 3'-end stem sequence added also needs to be balanced to avoid affecting the probe specificity. As shown in Table 2, when the 5'-end stem sequence of the probe designed in this study is completely matched with the target, 3 consecutive bases of its 3'-end complementary stem sequence match the target (underlined lowercase letters), thus affecting the probe specificity and showing non-specific signals for wild-type templates; 4) After modifying the stem sequence, two bases are added to the 5'-end and do not match the target. At this time, the 3'-end stem sequence does not match the target. The beacon probe has good specificity, showing no non-specific signals for wild-type templates and being able to accurately detect mutant templates, but still with relatively high background; 5) The background signal of the Spacer molecular beacon probe is lower than that of the above-mentioned molecular beacon probes, and the signal-to-noise ratio is higher. The copy number of the detected target sequence has no significant difference from that of the above-mentioned probes and is consistent with the expected quantitative results, indicating that the introduction of Spacer does not affect the recognition and amplification efficiency of the probe for the target, ensuring the accuracy of the detection results. At the same time, when detecting wild-type templates, there are no non-specific signals, indicating that the probe has good specificity.

[0050] Table 2. Comparison of detection results of different types of probes

[0051] Example 3. Spacer spacer length test Since the Spacer region provides a flexible spatial structure for the molecular beacon probe, facilitating the easier pairing and stable formation of a hairpin-like stem-loop structure between the 3'-end stem sequence and the 5'-end stem sequence, the Spacer spacer needs to be constructed with chemical units having high flexibility. In this invention, a flexible chain represented by ethylene glycol (PEG) is used as the basic unit of the Spacer structure to ensure that when the probe is in the free state, it can be closed smoothly and maintain a low background signal. 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 of the beacon probe and the background signal suppression effect, and screened out the spacer length suitable for the Spacer molecular beacon probe to provide a basis for the subsequent standardized design of the probe.

[0052] The primer probe sequences are shown in Table 3 below. The reaction system and digital PCR procedure refer to Example 1. Different probes were added respectively, and 2 μl of E545K positive plasmid and wild-type plasmid (500 copies / μl) were added to the template respectively to detect the background signal, positive signal and positive copy number.

[0053] Table 3. Primer and Probe Sequences

[0054] Note: + represents LNA-modified bases; lowercase letters represent the 3'-stem sequences that do not match the target region, uppercase letters represent the loop sequences that match the target region and the 5'-stem sequences, and the underlined uppercase letters in the middle are the mutation sites.

[0055] The detection results are shown in Table 4. It can be seen from the results 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 signals of the probes in the free state are all significantly lower than those in the group containing only 2 PEG units (Spacer 6). This indicates that when the Spacer length reaches 3 PEG units or more, 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.

[0056] There was no significant difference in the copy number of target sequences detected in each group, which was consistent with the expected quantitative results. There were no positive signals in the wild-type plasmids, indicating that while the change in Spacer length provides structural flexibility, it does not affect the recognition and amplification efficiency of the probe for the target, ensuring the consistency of detection sensitivity. At the same time, the probes all maintained specificity and there were no non-specific detection signals.

[0057] Table 4. Detection Results of Molecular Beacon Probes with Different Length Spacers

[0058] Example 4. Test on the Co-detection Effect of Multiple Homologous Sites of Fluorescence Quenched Probes To verify whether the Spacer probes designed in the present invention can achieve the combined detection of multiple homologous mutation sites in the same reaction tube, corresponding Spacer probes (Spacer 18 is used in this example) were designed for 7 common mutant genotypes at the 542–546 sites of the PIK3CA gene (including E542K, E545K, E545A, E545G, E545D, Q546E, and Q546R). At the same time, to inhibit the non-specific fluorescence signals that may be caused by free probes in the system, a short-chain fluorescence quenched probe was designed, and a BHQ1 quenching group was introduced at its 3' end to effectively quench the signals of free probes that did not bind to the target sequence. The primer and probe sequences are shown in Table 5.

[0059] Table 5. Primer and Probe Sequences

[0060] Note: + represents LNA-modified bases; lowercase letters represent the 3'-stem sequence that does not match the target region, uppercase letters represent the loop sequence that matches the target region and the 5'-stem sequence, and the underlined uppercase letters in the middle are the mutation sites.

[0061] The detection system was prepared as shown in Tables 6 and 7, and the templates were added with each mutant positive plasmid and wild-type plasmid (with concentrations of 500 copies / μl), respectively: Table 6. Multisite Detection System without Fluorescent Blocking Probes

[0062] Table 7. Multisite Detection System with Added Fluorescent Blocking Probes

[0063] The digital PCR procedure is shown in Example 2. The detection results are shown in Table 8. The Spacer probe strategy can achieve accurate detection of multiple mutation sites in the same reaction tube. However, due to the simultaneous presence of multiple Spacer probes, there is a certain degree of signal superimposition in their background signals, resulting in a relatively high overall background level in the reaction system. Therefore, fluorescent blocking probes were introduced to effectively block the non-specific fluorescent signals generated by free probes, further reducing the background noise and significantly improving the signal-to-noise ratio.

[0064] The quantitative detection results of each mutation type were basically consistent with the expectations, indicating that while effectively reducing the background signal and improving the detection sensitivity, this system does not affect the accuracy of the target copy number. In addition, for the wild-type template in this multi-probe system, whether or not fluorescent blocking probes were added, only background-level signals were detected, and no positive fluorescent signals appeared. The detection results further verified the good specificity of the mutant probes.

[0065] The highly specific detection results of this system are due to the introduction of the LNA (locked nucleic acid) structure in the probe design, which shortens the probe length and enhances the recognition ability of mismatched bases, enabling the mutant probes to not generate non-specific signals when facing wild-type templates. In summary, the "Spacer molecular beacon probe + fluorescent blocking probe" combined probe system designed in the present invention can achieve highly specific detection of multiple homologous mutation sites in a single tube, significantly reduce the background signal, improve the signal-to-noise ratio, and thus contribute to improving the accuracy and interpretability of the detection results.

[0066] Table 8. Probe Detection Results of the Multi-Probe System with and without Fluorescent Blocking Probes

[0067] It should be understood that the invention disclosed herein is not limited solely to the particular methods, procedures, and materials described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0068] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. The molecular beacon probe used in the PCR end-point detection method is characterized in that The molecular beacon probe sequentially includes a 5'-terminal stem sequence, a loop sequence, a flexible Spacer structure, and a 3'-terminal stem sequence, and has an overall hairpin structure; a fluorescent group is labeled at the 5'-end, a quenching group is labeled at the 3'-end, and 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 probe; the flexible Spacer structure is a polyethylene glycol structure.

2. The molecular beacon probe according to claim 1, wherein The loop sequence is modified with locked nucleic acid.

3. The molecular beacon probe according to claim 1, characterized in that, The number of polyethylene glycol units in the flexible Spacer structure is not less than three.

4. The molecular beacon probe according to claim 1, characterized in that, The number of polyethylene glycol units in the flexible Spacer structure is 3-6.

5. Use of the molecular beacon probe according to any one of claims 1-4 in a digital PCR detection method.

6. A digital PCR detection kit, characterized in that, The kit includes the molecular beacon probe according to any one of claims 1-4.

7. 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 reaction system. The probe system includes a molecular beacon detection probe according to any one of claims 1-4 and a fluorescence blocking probe designed for each gene mutation respectively. The sequence of the fluorescence blocking probe is complementary to a 5-7 base region at the 5'-end of each of the above molecular beacon detection probes. A fluorescence quenching group is labeled at the 3'-end of the fluorescence blocking probe. This short-chain probe binds to the 5'-end in the free state of the molecular beacon detection probe to form a stable secondary closed structure.

8. A digital PCR detection kit, characterized in that, The kit includes the multi-mutation site detection probe system according to claim 7.

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