Multi-strand displacement mediated qPCR technology and application thereof in tumor SNVs diagnosis

Abstract

The invention relates to a multi-strand displacement mediated qPCR technology for ultrasensitive detection of tumor-related SNVs and a fluorescent probe design scheme, and belongs to the technical field of molecular diagnosis. According to the method, a functional nucleic acid chain combination system containing primers, an inhibition chain, a rescue chain and a universal reporter probe (URP) is constructed, and a bidirectional Toehold-mediated chain displacement reaction is utilized, so that high-specificity distinguishing of mutant type and wild type targets is realized. Wherein the inhibition chain is selectively combined with a wild-type target to reduce the amplification efficiency of the wild-type target, the rescue chain specifically recognizes a mutant-type target and releases a medium primer under the action of polymerase, and a URP hairpin structure is triggered to dissociate to output a fluorescence signal. The interaction among functional nucleic acid chains is finely regulated through thermodynamic parameter difference, and ultrahigh-sensitivity detection of SNVs with the abundance as low as 0.01% can be realized.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a multi-strand displacement-mediated qPCR technology and fluorescent probe design scheme for ultrasensitive detection of tumor-related SNVs. Background Technology

[0002] ctDNA contains a variety of genetic mutations associated with tumor drivers and drug resistance, including single nucleotide variants (SNVs), insertions, and deletions. SNVs are the most common form of mutation, caused by changes in a single nucleotide, widely distributed throughout the human genome, and closely related to tumor development and progression. Detection of tumor-related SNVs not only provides crucial information for tumor diagnosis and molecular subtyping but can also be used to evaluate the efficacy of targeted therapies and monitor drug resistance mechanisms, thus providing strong support for precision medicine.

[0003] Currently, commonly used SNV detection technologies in clinical practice mainly include next-generation sequencing (NGS), digital PCR (dPCR), and qPCR. NGS platforms, leveraging the advantages of high-throughput parallel sequencing, can simultaneously detect hundreds to thousands of gene loci. High-depth sequencing based on unique molecular tags can detect mutation abundance as low as 0.1%. However, NGS results are affected by various factors such as sample quality, library quality, and sequencing depth, requiring highly skilled professionals and relying on complex bioinformatics analysis processes. Furthermore, its high cost and long testing cycle limit its application in real-time diagnosis and dynamic monitoring. In contrast, dPCR, with its ultra-high sensitivity of 0.01%–0.001%, has significant advantages in the dynamic monitoring of known mutations and the precise detection of trace targets. This technology, based on the single-molecule template amplification principle, can achieve absolute quantification of targets without a standard curve, demonstrating unique advantages in real-time acquisition of low-abundance ctDNA characteristics in patient samples, especially in SNV detection. However, existing dPCR systems are limited by issues such as the number of fluorescence channels, signal crosstalk, microreaction capacity, and the complexity of multi-channel signal analysis, making it difficult to efficiently and accurately detect multiple targets in a single reaction.

[0004] In contrast, qPCR technology, due to its short detection cycle, low cost, and widespread platform availability, has been widely used in various clinical testing scenarios and has shown promising prospects in tumor MRD monitoring and efficacy evaluation. However, the presence of abundant wild-type DNA in clinical samples greatly limits the specificity of qPCR in detecting low-abundance SNVs. Constructing an efficient and highly selective PCR system to distinguish between mutant and wild-type templates remains one of the key challenges of qPCR in ctDNA detection.

[0005] To address the aforementioned issues, representative PCR technologies currently include Amplification Arrestor Mutant System-PCR (ARMS-PCR) and Blocker Replacement Amplification-PCR (BDA-PCR). ARMS-PCR, as a traditional molecular detection method, primarily relies on the efficient discrimination of primer 3'-end mismatches by Taq DNA polymerase to selectively amplify mutant alleles. Primer amplification efficiency reaches its peak when the primer perfectly matches the mutant DNA template; however, a base mismatch at the 3' end between the primer and the wild-type DNA template significantly reduces amplification efficiency. Therefore, primers matching the mutant allele can selectively amplify the mutant gene while inhibiting the amplification of wild-type alleles. However, in practical applications, wild-type templates may still experience low-level amplification, and the lack of sufficient selectivity of fluorescent probes leads to interference from WT background signals, limiting its specificity and sensitivity in detecting low-abundance SNVs, thus failing to meet the needs of early tumor detection.

[0006] BDA-PCR introduces a repressor strand that specifically binds to the wild-type template. By utilizing the competitive binding and substitution process between primers and the repressor strand between WT and MT templates, it significantly reduces the amplification efficiency of the wild-type template, thus achieving selective enrichment of the MT template. This design can achieve a 1000-fold enrichment effect for mutant amplification products. However, the presence of the repressor strand also reduces primer binding efficiency, thus affecting the amplification of mutant alleles, causing the amplification of SNVs (heterozygous mutations) to lag behind that of SNPs (homozygous mutations). Therefore, BDA-PCR alone is insufficient to overcome the 0.1% detection sensitivity, often requiring additional detection methods for auxiliary analysis. The allele-specific-BDA (As-BDA) system combines BDA technology with allele-specific TaqMan probes, possessing both targeted enrichment and selective recognition functions, and can improve the detection sensitivity of SNVs to 0.01%. However, the system still suffers from an increase in WT background signal, mainly due to insufficient balance between the inhibitory chain and the signal probe, as well as false triggering of the TaqMan probe. The resulting non-specific amplification signal can lead to deviations in the interpretation of low-abundance SNVs, affecting the accuracy of clinical diagnosis and treatment decisions. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a multiple strand substitution-mediated qPCR detection system and a fluorescent probe design scheme for constructing a tumor-associated single nucleotide variant (SNV) specific detection system. This invention relates to an ultrasensitive and highly specific detection method and design scheme for tumor-associated SNVs. The detection method and design scheme provided by this invention can meet the needs of precision diagnosis and treatment decisions in oncology, achieving accurate detection and identification of multiple SNVs, and has advantages such as high sensitivity, high specificity, and flexible design.

[0008] To achieve the objectives of this invention, in a first aspect, this invention provides a functional nucleic acid strand combination mode based on multiple strand displacement reaction for constructing an ultrasensitive detection system for SNVs. This functional nucleic acid strand combination includes primers, a repressor strand, a rescue strand, and a universal reporter probe (URP). The primers are rigorously screened to efficiently amplify the target gene fragment; the repressor strand selectively binds to wild-type targets, hindering the hybridization rate of the primers and rescue strand with wild-type DNA, thereby reducing the amplification efficiency and signal output efficiency of the wild-type target; the rescue strand selectively recognizes mutant targets, and only the rescue strand that hybridizes with mutant targets can be correctly cleaved by Taq DNA polymerase, releasing the mediator primer. Subsequently, the mediator primer hybridizes with and extends the single-stranded region of the universal reporter probe, disrupting the hairpin structure of the universal reporter probe and generating a fluorescent signal. If the rescue strand is not correctly cleaved, when the released mediator primer hybridizes with the URP, a 3'-end mismatch will occur, preventing it from being extended by the polymerase, resulting in the URP failing to generate a significant fluorescent signal.

[0009] Secondly, based on the functional nucleic acid strand combination described in the first aspect, the present invention provides a design scheme for a functional nucleic acid strand:

[0010] (1) Primer design principle: The forward primer (FP) is located upstream of the mutation site, with the 3' end of the primer spaced 1 to 8 nt away from the mutation site; the reverse primer (RP) is designed based on the position of the forward primer to ensure that the final length of the amplicon is 80 to 140 bp. The remaining design principles are no different from those of conventional primer design.

[0011] (2) Repression chain design: The repressor chain is completely complementary to the wild-type target in the reaction system, and has one or more base mismatches with the mutant target. The 3' end of the repressor chain is blocked with poly-T or A. The 5' end of the repressor chain partially overlaps with the forward primer, and the 3' end partially overlaps with the rescue chain. The recommended length is 20-40 nt, the Tm value is 62℃-66℃, and the Toehold length is 5-10 nt.

[0012] (3) Rescue strand design concept: The rescue strand is structurally divided into a probe region and a mediator primer region. The probe region is completely complementary to the mutant target and has a base mismatch with the wild-type target, serving as the target-specific recognition region. Its length depends on the specific target, with a recommended Tm value of 58℃~68℃. The specific recognition ability of the probe region can be adjusted by modifying the 5'-end binding site, changing the 3'-end length, or artificially introducing mismatches. The mediator primer region is partially complementary to the single-stranded region of the universal reporter probe. During PCR, it can be cleaved by the 5'-flap endonuclease activity of Taq DNA polymerase, thus freeing itself from the rescue strand. It then hybridizes and extends with the universal reporter probe, opening the hairpin structure of the universal reporter probe or separating the fluorescent and quenching groups labeled on the molecular beacon, thereby generating fluorescence. The recommended length of the mediator primer region is 14~22nt, with a Tm value of 58~64℃.

[0013] (4) General reporter probe design concept: URPs can be designed as molecular beacons or hairpin probes. The design of its own secondary structure ensures that the fluorescence signal is quenched when no target is present. Only when the target is present in the system and the rescue strand is correctly cleaved can the correct mediator primer hybridize with the URP and extend, thereby generating a fluorescence signal. The recommended length of URP is 45-60 nt, and the Tm value is 72-85 °C.

[0014] Thirdly, based on the functional nucleic acid chain combination described in the first aspect of the present invention, a real-time fluorescence quantitative PCR detection technology for tumor-associated SNVs is provided for the specific detection of SNVs.

[0015] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0016] (1) The present invention can design and combine primers, rescue strands, repressor strands and URPs in a reasonable manner, and design functional nucleic acid strand combinations for different SNV sites as needed to construct a multiple strand substitution-mediated qPCR system to realize the detection and identification of low abundance SNVs with a sensitivity ≤0.01%.

[0017] (2) Based on classic qPCR, this invention introduces a rescue strand to achieve selective recognition of mutant targets, and simultaneously introduces a repressor strand to selectively inhibit wild-type targets, significantly improving the detection performance and design flexibility of the qPCR reaction system. Compared with clinical detection methods such as next-generation sequencing and digital PCR, the detection method of this invention greatly simplifies the operation process, reduces the requirements for testing personnel and equipment, and is easier to promote clinically. Compared with commonly used clinical qPCR technologies, the multiple strand substitution-mediated qPCR detection system provided by this invention offers flexible design, and its sensitivity and specificity are comparable to, or even superior to, commonly used clinical qPCR technologies. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the multi-strand displacement-mediated qPCR detection system.

[0019] Figure 2 This is a specificity test result for detecting 0.1% Kras G12A mutations using multiple strand substitution-mediated qPCR technology.

[0020] Figure 3 This is a sensitivity test result for detecting Kras G12A mutations using multistrand substitution-mediated qPCR technology.

[0021] Figure 4 This is a specificity test result for detecting 0.1% EGFR L858R mutation using multiple strand substitution-mediated qPCR technology.

[0022] Figure 5 This is an amplification curve for detecting EGFR L858R mutations in 0.01%–10% of individuals using multiple strand displacement-mediated qPCR technology. Detailed Implementation

[0023] To clarify the technical solution and advantages of the present invention, the present invention will be clearly and thoroughly described below through preferred embodiments. The following embodiments are only used to exemplify and further explain the content of the present invention, and are not intended to limit the present invention. The embodiments shown in this application are some embodiments, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by researchers and those skilled in the art without significant creative effort are within the scope of protection of the present invention. Furthermore, unless otherwise specified, the embodiments and feature vectors in the embodiments of this application can be arbitrarily combined with each other.

[0024] Preferred embodiment 1

[0025] The inventors designed a bidirectional strand substitution-mediated qPCR technology during their research for the accurate detection of SNVs. For example... Figure 1As shown, in the qPCR system, the inventors fixed the forward primer position 1-6 nt upstream of the mutation site and designed a highly selective rescue strand downstream of the forward primer to specifically recognize the mutation site. During the PCR cycle, the 5'-flap endonuclease activity of Taq DNA polymerase cleaves the rescue strand and releases the mediator primer. Subsequently, the mediator primer hybridizes with the URP and extends under the action of Taq DNA polymerase, opening the hairpin structure of the URP and releasing a fluorescent signal. In this process, to reduce signal interference caused by non-specific amplification of the wild-type target, the inventors introduced a suppressor strand based on the suppressor probe substitution amplification (BDA) technology. This suppressor strand competes with the primer and rescue strand for binding to the wild-type target. The thermodynamic parameters between the primer, rescue strand, and suppressor strand are used to finely regulate the equilibrium of the bidirectional strand substitution reaction, achieving accurate recognition of the mutant target.

[0026] The inventors designed and screened a set of optimal combinations of functional nucleic acid strands using the aforementioned bidirectional strand substitution-mediated qPCR technology for the ultrasensitive detection of Kras-G12A. The functional nucleic acid strand sequences are shown in Table 1, and the detection results are shown in [Table 1]. Figure 2 .

[0027] Table 1. Functional nucleic acid strand sequences of the Kras G12A detection system

[0028]

[0029] In this embodiment, the universal fluorescent reporter probe is a hairpin fluorescent reporter probe, with a quencher group BHQ1 labeled at the 5' end, a FAM fluorescent group labeled in the middle, and an Aminolinker block at the 3' end. In the absence of a mutant target, the URP forms a hairpin structure, with the quencher group and fluorescent group positioned close together, resulting in no fluorescent signal. In the presence of a mutant target, the mediator primer hybridizes with the single-stranded region of the URP. After polymerase extension, the resulting single-stranded product hybridizes complementary to the URP, forming a longer linear double-stranded structure, disrupting the URP hairpin structure, and generating a fluorescent signal.

[0030] In this embodiment, the preferred final concentration of the functional nucleic acid strand is 400 nM for the forward or reverse primer, 400 nM for the rescue strand, 1500 nM for the repressor strand, and 100 nM for the URP. The specific system is shown in Table 2. The target is 0.1% Kras-G12A (prepared by mixing 1 fM MT and 1 pMWT target in equal volumes) and 1 pM Kras-WT plasmid. Each batch is tested in quadruplicates, with 15 μL of detection system per well, for a total volume of 63 μL. In some other embodiments, the rescue strand concentration can be 100 nM to 600 nM, and the repressor strand concentration can be 1000 nM to 1600 nM. The PCR premix used in the reaction system is AceQ Universal U+Probe Master Mix V2 produced by Nanjing Novizan Biotechnology Co., Ltd., with an initial concentration of 2× and a final concentration of 1× in the reaction system.

[0031] Table 2. Concentration ratio of functional nucleic acid chains in the Kras-G12A detection system

[0032]

[0033]

[0034] In this embodiment, the PCR amplification program was: 37℃×5min, 95℃×5min, 60×(95℃×15s, 60℃×30s, plate read), with the FAM channel selected as the fluorescence channel. The fluorescence signal intensity was collected once per cycle. After calculation by the instrument's software, an amplification curve was generated based on the fluorescence signal intensity, and the Cq value was output. The real-time quantitative PCR instrument model was: CFX96 Touch system (Bio-Rad Laboratories, Hercules, CA).

[0035] Analysis of the detection results in this embodiment shows that the primer-rescue strand-inhibition strand combination of this system can achieve specific recognition of mutant targets, while wild-type targets maintain the background signal level.

[0036] In this embodiment, the sensitivity and detection performance stability of the reaction system were tested by configuring targets at abundance levels of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, and 10%. The different abundance configurations followed the same preparation method as the 0.1% KrasG12A target, using 0.1 fM, 1 fM, 10 fM, and 100 fM Kras G12A plasmids mixed with 1 pM wild-type targets, respectively. The detection results are shown in […]. Figure 3 The results in the figure show that the bidirectional strand substitution-enhanced qPCR technology described in this embodiment can achieve a detection sensitivity as low as 0.01%, and can stably detect targets at different abundance levels.

[0037] The above results demonstrate that the rescue strand accurately identifies mutant targets and, in collaboration with URP, efficiently outputs fluorescence signals. Simultaneously, the detection results of this example show that the inhibitor strand effectively reduces the amplification efficiency of wild-type targets and significantly decreases the binding ratio between primers and wild-type targets, thereby ensuring effective binding of primers to low-concentration mutant targets against a 1pM wild-type DNA background, thus guaranteeing high sensitivity of the detection system.

[0038] Preferred embodiment 2

[0039] In this embodiment, based on the fundamental principles of multiple strand substitution reactions and the functional nucleic acid strand design scheme described in the invention, a set of functional nucleic acid strands was designed and screened for EGFR L858R for efficient detection. The functional nucleic acid strand sequences are shown in Table 3.

[0040] Table 3. Functional nucleic acid strand sequences of the EGFR L858R detection system

[0041]

[0042] In this embodiment, the secondary structure of the URP is similar to that of the reporter probe involved in Preferred Embodiment 1, i.e., a stem-loop structure, but its 5'-end labeling group is BHQ2, the middle part of the sequence (i.e., the end of the hairpin stem) is labeled with a HEX fluorescent group, and the 3'-end is still blocked with Aminolinker. Its mechanism of action is similar to that of the URP described in Preferred Embodiment 1.

[0043] In this embodiment, the final concentrations of the forward primer, reverse primer, and rescue strand remained at 400 nM, the repressor strand concentration was adjusted to 1200 nM, and the URP concentration was adjusted to 150 nM. The concentrations of each component are shown in Table 4. The target was 0.1% EGFR L858R (prepared by mixing equal volumes of 1 fM MT and 1 pMWT target) and 1 pM EGFR-WT plasmid. Four replicates were performed per batch, with each well containing 15 μL of the detection system, for a total volume of 63 μL. In some other embodiments, the rescue strand concentration could be 100 nM–600 nM, and the repressor strand concentration could be 1000 nM–1600 nM. The PCR premix used in the reaction system was AceQ Universal U+Probe Master Mix V2 produced by Nanjing Novizan Biotechnology Co., Ltd., with an initial concentration of 2× and a final concentration of 1× in the reaction system.

[0044] Meanwhile, in this embodiment, referring to the 0.01% to 10% preparation scheme in Example 1, EGFR L858R targets with different abundance levels were prepared to test the detection performance of the EGFR L858R detection system.

[0045] Table 4. Concentration ratio of functional nucleic acid chains in the EGFR L858R detection system

[0046] reagent components Concentration or volume 2×PCR master mix V2 1× forward primer 400nM reverse primer 400nM Chain of Salvation 400nM Inhibition chain 1200nM URP 150nM Target: 0.1% L858R or 1 pMWT 1μL Ultrapure water Add water to bring the volume to 63μL

[0047] In this embodiment, the PCR amplification program was: 37℃×5min, 95℃×5min, 60×(95℃×15s, 60℃×30s, plate read), with the HEX channel selected as the fluorescence channel. The fluorescence signal intensity was collected once per cycle. After calculation by the instrument's software, an amplification curve was generated based on the fluorescence signal intensity, and the Cq value was output. The real-time quantitative PCR instrument model was: CFX96 Touch system (Bio-Rad Laboratories, Hercules, CA).

[0048] In this embodiment, the detection results for 0.1% EGFR L858R are shown below. Figure 4 The detection results for different abundance gradients from 0.01% to 10% are shown in [the table below]. Figure 5 Experimental results show that the EGFR L858R detection system achieved stable detection of mutant targets at different concentration levels, with a detection sensitivity as low as 0.01%. The results of this embodiment further confirm the scientific validity and practicality of accurately detecting tumor-related SNVs by utilizing the principle of multiple strand substitution reaction, employing highly specific rescue strands to efficiently identify mutant targets, and using the most suitable inhibitory strand to inhibit primer binding, thereby achieving precise detection of tumor-related SNVs.

[0049] As can be seen from the above embodiments, the multiple strand substitution-assisted qPCR detection method of the present invention has the advantages of high sensitivity, strong specificity, customizable design, and high-throughput detection potential. Furthermore, the interaction between the functional nucleic acid strands involved in the qPCR detection system of the present application does not generate non-specific signals, resulting in low background signals and improved accuracy of detection results.

[0050] The scope of protection of this invention is not limited to the preferred embodiments described above. Any equivalent substitutions of known components or technologies, reasonable adjustments to feature combinations, and variations conceivable based on the inventive concept, all within the scope of the technical concept of this invention, should be included within the scope of protection of this invention.

Claims

1. A multi-chain displacement reaction system, characterized in that, The system is constructed based on the principle of multiple strand substitution reaction and includes primers, a rescue strand, and a repressor strand. The rescue strand is structurally divided into a probe region and a mediator primer region. The probe region is completely complementary to the mutant target, enabling specific recognition of the mutant target sequence. It then hybridizes with a universal reporter probe through the mediator primer region, mediating the output of a fluorescence signal. The repressor strand is completely complementary to the wild-type target sequence but has a one-base mismatch with the mutant target. It can competitively inhibit the hybridization reaction between the primer, the rescue strand, and the wild-type target sequence, thereby achieving efficient enrichment and accurate identification of the mutant target.

2. A qPCR detection method mediated by bidirectional strand displacement reaction, characterized in that, The primers and rescue strands trigger the Toehold-mediated strand displacement reaction from the 5'-end and 3'-end of the repressor strand, respectively. The equilibrium of the strand displacement reaction is regulated by the difference in thermodynamic parameters between the primer / rescue strand / repressor strand-mutant target and the primer / rescue strand / repressor strand-wild-type target.

3. A multiple strand displacement-mediated qPCR detection system constructed according to the reaction principle described in claims 1 and 2 is applied to the detection of tumor-related SNVs, characterized in that... By leveraging the synergistic interaction of primers, rescue strands, and repressor strands, precise identification of mutant targets can be achieved. At the same time, the design of coupling mediator primers and universal reporter probes enables efficient output of fluorescence signals.

4. The functional nucleic acid strand design scheme provided by the multi-strand substitution-mediated qPCR detection system according to claims 1 and 2, characterized in that, Based on the Teohold-mediated strand displacement reaction principle and the thermodynamic parameter differences that finely regulate the interactions between functional nucleic acid strands.

5. The universal fluorescent reporter probe sequences numbered SEQ-5 and SEQ-10 and the modified group labeling positions provided by the multiple strand displacement-mediated qPCR detection system according to claims 1 and 2 are characterized in that, The hairpin structure design improves the signal-to-noise ratio and signal output efficiency of the detection system.

6. The primer sequences, rescue strand sequences, and repressor strand sequences numbered SEQ-1 to 4, SEQ-6 to 9 provided by the multiple strand displacement-mediated qPCR detection system according to claims 1 and 2 are characterized in that, Based on the mutant and wild-type sequences of KrasG12A and EGFR L858R, rescue strands that can specifically identify the mutation sites are designed through precise thermodynamic parameter control, and a suitable repressor strand sequence is matched to achieve efficient identification of KrasG12A and EGFR L858R mutations.

7. A design scheme and detailed parameters for multiple functional nucleic acid strands based on the principle of multiple strand substitution reaction, characterized in that, By leveraging the differences in sequence, structure, and thermodynamic parameters of primers, rescue strands, repressor strands, mediator primers, and universal reporter probes, and balancing the interactions between functional nucleic acid strands, an efficient multiplex substitution reaction system and qPCR detection platform are constructed to achieve highly sensitive and specific detection of tumor-related SNVs.

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