Wild type gene amplification repression oligonucleotide sequence, composition and application of wild type gene amplification repression oligonucleotide sequence

Abstract

The invention discloses a wild type gene amplification repression oligonucleotide sequence, a composition and application of the wild type gene amplification repression oligonucleotide sequence. The invention also discloses a competitive binding oligonucleotide sequence element which is competitively bound with a mutant allele site with the wild-type gene amplification repression oligonucleotide sequence, and when the competitive binding oligonucleotide sequence element is used together with the wild-type gene amplification repression oligonucleotide sequence disclosed by the invention, the mutant allele site is selectively bound with the mutant allele site. On the basis of the existing widely applied amplification blocker gene mutation detection method, the mutation enrichment efficiency can be greatly improved, the detection sensitivity of the amplification blocker gene mutation detection method is improved, and particularly, low-frequency and ultralow-frequency gene mutation can be stably and reliably detected in liquid biopsy commonly used in tumor precision medicine at present.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a wild-type gene amplification suppressor oligonucleotide sequence, a composition and an application thereof. Background Art

[0002] Amplification-retarded mutation detection PCR (ARMS PCR), also known as allele-specific oligonucleotide PCR, was proposed and validated by Newton et al. in 1989. It is designed to detect single-point mutation polymorphisms in known sequences. Early ARMS detection systems used PCR and gel electrophoresis analysis of the target amplicon, identifying the single nucleotide polymorphism (SNP) of the target using normal and mutant primers. The technical principle is based on allele-specific extension: PCR amplification initiated from the 3' end of the primer is performed. Once a mismatch is intentionally introduced at the 3' end of the primer as the forward primer for the mutant gene in the test sample, extension of the DNA single strand can only occur when the 3' end base of the allele-specific primer is perfectly complementary to the base at the mutation site in the test sample. However, due to the mismatch between the wild-type test sample allele and the 3' end base of the primer, extension cannot occur.

[0003] Ahlawat et al. developed an ARMS PCR assay based on four primers (two outer primers and two inner primers) to genotype Fec gene polymorphisms in goats. Hamzeh improved the four-primer ARMS-PCR system and increased the number of mismatches of the ARMS primers relative to the wild-type 3' end to two, improving the specificity and convenience of SNP typing. Ntziora developed a detection method combining the ARMS PCR system with the Light Cycler molecular beacon system, which detected mutations associated with antiviral resistance in chronic hepatitis B virus (HBV) with 100% sensitivity. Cross-validation with first-generation sequencing confirmed the stability of the method. Jiang et al. disclosed an improved ARMS primer structure (Super-ARMS) and its use method, which improved the reaction specificity of ARMS-PCR. Chen et al. used Super-ARMS and digital PCR to detect ESR1 gene mutations in plasma ctDNA from 207 breast cancer patients, confirming that Super-ARMS PCR has similar detection sensitivity to ddPCR and the convenience of conventional ARMS-PCR. Cao et al. compared the consistency of Super-ARMS and digital PCR in detecting the drug-resistant EGFR T790M mutation in ctDNA extracted from patients with non-small cell lung cancer after EGFR-TKI treatment. They confirmed that the Super-ARMS detection system has a detection sensitivity close to that of digital PCR. Feng et al. analyzed the consistency of EGFR mutation detection in plasma ctDNA from 79 patients with non-small cell lung cancer using Super-ARMS and digital PCR. The results showed that the consistency of Super-ARMS and digital PCR detection reached 91.1%.

[0004] The aforementioned public research reveals that the Super-ARMS detection system, optimized based on ARMS-PCR, has further improved both sensitivity and ease of use. The "Human EGFR Mutation Gene Detection Kit (Multiplex Fluorescence PCR Method)" approved in 2018, with acceptance number CSZ1700057, is available on the CDME (https: / / www.cmde.org.cn, National Medical Products Administration Medical Device Technical Review Center). The kit's sensitivity targets a minimum 95% detection rate for each mutation site, with a minimum detection limit of 0.2%-0.8% mutation ratio at a DNA concentration of 15 ng / reaction. Genetic mutation is a key hallmark of cancer, and most currently available small molecule targeted drugs are designed to target specific gene mutations. Therefore, mutation detection in tumor samples is a crucial tool for disease classification and treatment planning.

[0005] To develop more accurate treatment plans, a more comprehensive picture of tumor DNA mutations is needed. Liquid biopsies offer advantages over tissue biopsies, such as ease of sampling and the ability to overcome the heterogeneity of cancer tissues. However, the concentration of tumor DNA in blood is far lower than in tissue, typically below 1%, or even 1‰ or less. Furthermore, given the relatively low adoption of digital PCR, a widely used absolute quantification tool in clinical settings, its testing costs remain high. Therefore, a detection technology with higher sensitivity, ease of use, and clinical applicability is urgently needed.

[0006] It can be found that the basic principle of the current ARMS-PCR technology, including the original ARMS-PCR and several improved ARMS-PCR technologies reported in recent years, is to utilize the 3'-5' exonuclease activity of Taq DNA polymerase without proofreading activity. Based on this, primers with mismatches at the 3' end will extend at a slower rate than primers with perfect 3' matches. When the number of mismatched bases reaches a certain number, the 3' terminal base cannot be extended due to the difficulty in forming a phosphodiester bond, the reaction is terminated, and no amplified fragment of a specific length is obtained, indicating that the template DNA does not have a mutation corresponding to the 3' end of the primer; if the PCR result can obtain an amplified fragment of a specific length, it indicates that the template DNA has a mutation corresponding to the 3' end of the primer, because it is impossible to determine whether the wild-type template exists in the PCR product sequence through subsequent sequencing, and it is not possible to fully determine whether the primer with a certain number of mismatched bases at the 3' end does not anneal or extend with the template at all, its essence is also a mutation enrichment detection system. In the widely used Super-ARMS technology, a modified ARMS-PCR, the 5' end of a conventional ARMS mismatch primer (completely complementary to the mutation) is connected to the 3' end of a second sequence complementary to the mismatch primer via a steric spacer, achieving a palindromic structure at room temperature. In a PCR reaction, this structure generates a PCR reaction annealing structure that competes with the palindrome only when the mismatch primer collides with a site with a mutation during molecular motion. This means that the mismatch primer binds to the mutant template for PCR extension. If it collides with the wild-type template, the TM value is higher under perfect match compared to N mismatches with the wild-type. Therefore, the mismatch primer will preferentially reform the palindrome with the second sequence instead of undergoing PCR extension with the wild-type template, enabling more specific detection of mutant molecules.

[0007] Analyzing ARMS technology, we find that mismatched primers can only inhibit one single strand (denoted as the ARMS positive strand) where the wild-type site is located, while the other single strand (denoted as the ARMS negative strand) is not inhibited during amplification (the wild-type and mutant types are amplified simultaneously by the downstream ARMS primers without mismatches). This results in an inability to efficiently enrich the mutant molecules during the detection process, reducing the detection rate. Amplification-blocking mutation detection technology has many application advantages, such as fast speed, low cost, and high flexibility. However, currently, both conventional amplification-blocking mutation detection and optimized amplification-blocking mutation detection have limited inhibitory effects on the wild-type background through mismatched primers bound to single strands, and are unable to further improve detection sensitivity. Therefore, they cannot solve the application scenario of tumor liquid biopsy, which is increasingly accepted by doctors and patients. Summary of the Invention

[0008] The present invention aims to provide a novel wild-type gene amplification suppressor oligonucleotide sequence, composition, application thereof, and a method for detecting amplification-blocking gene mutations based thereon, thereby significantly improving the detection sensitivity of such methods. The wild-type gene amplification suppressor oligonucleotide sequence, composition, application thereof, and amplification-blocking gene mutation detection methods based thereon all act on the negative strand of the ARMS.

[0009] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides a wild-type gene amplification suppressor oligonucleotide sequence element, which includes a nucleotide chain S1 of a first sequence region and a nucleotide chain S2 of a second sequence region, wherein the 3' end of the first sequence region is connected to the 5' end of the second sequence region; at least the connection region between the first sequence region and the second sequence region contains a thiolation modification; wherein the first sequence region does not match or bind to the wild-type site, mutation site, and upstream and downstream sequences of the wild-type site and mutation site of the genome to be detected; and the second sequence region is completely complementary to the wild-type genotype base sequence of the gene site to be detected.

[0010] As a preferred embodiment, the first sequence region's 3'-end 1-4 bases and the second sequence region's 5'-end 1-5 bases contain at least one thiolation modification, and the thiolation modification exists between any two adjacent bases.

[0011] As a preferred embodiment, the first sequence region contains thiolation modifications between the last base at the 3' end and the first base at the 5' end of the second sequence region, between the first base at the 5' end and the second base at the 5' end of the second sequence region, between the second base at the 5' end and the third base at the 5' end of the second sequence region, and between the third base at the 5' end and the fourth base at the 5' end of the second sequence region.

[0012] In some embodiments, the base located in the second sequence region that is completely complementary to the wild-type allele at the mutation site to be detected contains a locked nucleic acid modification.

[0013] In some embodiments, the physical coordinates of the base in the second sequence region that is completely complementary to the wild-type allele at the mutation site to be detected are set to 0, the base with the physical coordinate set to 0 contains a locked nucleic acid modification, and in the upstream and downstream sequences of the base, 10 bases within the physical coordinate range of -5 to -1 and 1 to 5 are arbitrarily selected for locked nucleic acid modification.

[0014] As a preferred embodiment, the number of locked nucleic acid modifications is 2-4.

[0015] As a preferred embodiment, the bases with physical coordinates -2 and 2 contain locked nucleic acid modifications.

[0016] In some embodiments, the 3' end of the wild-type gene amplification suppressor oligonucleotide sequence element contains a first blocking modification. The first blocking modification includes, but is not limited to, 3' terminal phosphorylation, 3' terminal amino modification, Cn steric hindrance modification (n=3, 6, 9, 12, 18, etc.), inverted dT modification, and 3' terminal base modification with a dideoxynucleotide. In the present invention, the first blocking modification is used to prevent extension of the 3' end by DNA polymerase.

[0017] In some embodiments, the first sequence region does not match or bind to any position of the genomic nucleotide sequence of the species in which the mutant gene to be detected is located.

[0018] In some embodiments, the length of the first sequence region is 10-13 bases.

[0019] As a preferred embodiment, the nucleotide sequence of the first sequence region is 5'-GACTGATACTG-3' (SEQ ID NO.16) or 5'-TCTCGGTACTC-3' (SEQ ID NO.17), more preferably 5'-GACTGATACTG-3'. Of course, it should be understood that the nucleotide sequence of the first sequence region can also be other sequences, as long as the nucleotide sequence of the first sequence region does not match or combine with the wild-type site, mutation site, and upstream and downstream sequences of the wild-type site and mutation site of the genome to be detected. The genomic sequences of different species are different. Based on the characteristics of the nucleotide sequence of the first sequence region (the nucleotide sequence of the first sequence region does not match or combine with the wild-type site, mutation site, and upstream and downstream sequences of the wild-type site and mutation site of the genome to be detected), a variety of nucleotide sequences can be generated when designing the first sequence region for the genome to be detected of different species. Therefore, an exhaustive enumeration is not made here.

[0020] In some embodiments, the length of the second sequence region is 18-25 bases. Of course, it should be understood that the lengths of the second sequence regions designed for the genomes to be tested of different species may be the same or different.

[0021] As a preferred embodiment, the 3' end of the second sequence region is complementary to the upstream primer (ARMS-qPCR forward primer) in the ARMS-qPCR amplification reaction system that carries a mismatch with the wild-type mutation detection site over 6-8 bases.

[0022] The wild-type gene amplification suppressor oligonucleotide sequence element of the present invention can perfectly match and complementarily bind to the upstream and downstream sequences of the wild-type allele of the target gene to be detected site. When the Taq enzyme-mediated PCR is subsequently extended to the 5' end of the sequence element, the properties of the S1 sequence and the thiolation modification between the S1 and S2 sequences can prevent the Flap endonuclease activity of the Taq enzyme, thereby preventing the sequence element from being cleaved by the enzyme, thereby keeping the sequence element firmly bound to the wild-type template, further inhibiting the single strand that cannot be inhibited by the ARMS mismatch primer (i.e., the ARMS negative strand) from generating new template products that may be bound by the ARMS mismatch primer, thereby significantly reducing the wild-type background.

[0023] For the detection of single nucleotide polymorphisms (SNP) sites, the wild-type gene amplification suppressor oligonucleotide sequence element is designed as follows: the S2 sequence in the sequence element covers the SNP site to be detected and its upstream and downstream sequences, and the site to be detected is designed at the nucleotide center or near-center position of the S2 sequence of the sequence element, that is, the nucleotide sequence of the sequence element is N, and the site to be detected is located at the position of N / 2±2~3, and the base of the sequence element corresponding to the detection site and the bases separated by 1 base upstream and downstream thereof must be designed as locked nucleic acid modified forms (the position to be detected is set to 0, and the positions separated by 1 base should be -2 and 2), and the S1 sequence is a universal sequence that does not match the wild / mutated site of the gene to be detected and its upstream and downstream sequences and has no specific binding in the sample species to be detected; the thiolation modification between the S1-S2 sequences is as described above. In the early design of the present invention, consideration was given to the potential risk that the base complementarity between the design and the ARMS amplification primers might lead to a reduction in the amount of effective ARMS primers, thereby affecting the amount of subsequent PCR products. Subsequent experiments verified that the wild-type amplification suppressor nucleotide sequence element detected at the single nucleotide polymorphism site would not produce competitive binding with the ARMS primers under the PCR reaction conditions set by the present invention, thereby affecting the amplification effect, and would not affect the overall amplification efficiency and effect.

[0024] For the detection of insertion or deletion (Ins&Del) polymorphic sites, the wild-type gene amplification suppressor oligonucleotide sequence element is designed as follows: the S2 sequence in the sequence element covers the wild-type sequence corresponding to the insertion or deletion site to be detected (that is, there is no insertion sequence polymorphism in the region or there is no deletion sequence polymorphism in the region), and the design ensures that when the subtypes of deletion or insertion to be detected exist at the same time (the situation where there is no simultaneous existence in real-world clinical samples), the wild-type gene amplification suppressor oligonucleotide sequence element cannot bind to various insertion and deletion subtypes; the wild-type gene amplification suppressor oligonucleotide sequence element is designed with 3-5 base-spaced locked nucleic acid modifications around its center to improve the affinity binding between the sequence element and the wild type, and the S1 sequence is a universal sequence designed by the present invention in the sample species to be detected (for example, humans in the embodiment of the present invention, homo Sapiens); the thiolation modification between the S1-S2 sequences is as described above; in the early design of the present invention, consideration was given to the potential risk that the design for indels and the base complementarity of the ARMS amplification primers might lead to a reduction in the amount of effective ARMS primers, thereby affecting the amount of subsequent PCR products. Subsequent experiments verified that the wild-type amplification suppressor nucleotide sequence element involving indel detection of N bases (N≥5) would not produce competitive binding with the ARMS primers that would affect the amplification effect under the PCR reaction conditions set by the present invention, and would not affect the overall amplification efficiency and effect.

[0025] The second aspect of the present invention provides a composition comprising a competitive binding oligonucleotide sequence element and the wild-type gene amplification suppressing oligonucleotide sequence element described in the first aspect of the present invention, wherein the competitive binding oligonucleotide sequence element and the wild-type gene amplification suppressing oligonucleotide can competitively bind to the same DNA chain; the competitive binding oligonucleotide sequence element and the wild-type gene amplification suppressing oligonucleotide element competitively bind to the upstream or downstream of the mutation site to be detected (excluding the gene site to be detected); the competitive binding oligonucleotide sequence element and the upstream or downstream sequence of the mutation site to be detected (excluding the gene site to be detected) are reverse complementary.

[0026] In some embodiments, the competitive binding oligonucleotide sequence is 15-25 bp in length.

[0027] In some embodiments, the 3' end of the competitive binding oligonucleotide sequence element overlaps with a sequence at the 5' end of the nucleotide chain S2 of the second sequence region, or the 5' end of the competitive binding oligonucleotide sequence element overlaps with a sequence at the 3' end of the nucleotide chain S2 of the second sequence region. In some embodiments, the overlapping sequence is 5-10 bases in length.

[0028] In some embodiments, the 3' end of the competitive binding oligonucleotide sequence element contains a second blocking modification.

[0029] The first blocking modification and the second blocking modification described in the present invention are the same or different.

[0030] As a preferred embodiment, the second blocking modification includes but is not limited to: 3' terminal phosphorylation modification, 3' terminal amino modification, Cn steric hindrance modification (n = 3, 6, 9, 12, 18, etc.), Inverted dT modification and 3' terminal base was dideoxynucleotide modification.

[0031] The competitive binding oligonucleotide sequence element provided by the present invention can perfectly match and complementarily bind to upstream or downstream sequences that do not include the mutant allele of the target gene test site. It is placed in the same reaction system as the wild-type gene amplification suppressor oligonucleotide sequence element. Since the test site on the wild-type gene amplification suppressor oligonucleotide sequence element is complementary to the wild-type template and the locked nucleic acid is modified at a position away, it is sensitive to the TM change of the mismatch (mutant). When the mutant allele is present, the competitive binding oligonucleotide sequence element sequence preferentially binds to the mutant template and is cleaved by the 5'-3' exonuclease of the Taq enzyme when the Taq enzyme-mediated PCR is extended to the 5' end, thereby ensuring the normal extension of the mutant allele type and ensuring that a limited number of mutant templates are detected more efficiently.

[0032] In the present invention Figure 1 The schematic diagram shows the structures of the wild-type gene amplification suppressing oligonucleotide sequence element and the competitive binding oligonucleotide sequence element of the present invention.

[0033] The third aspect of the present invention provides a wild-type gene amplification retardation detection system, which comprises the composition described in the second aspect of the present invention, and further comprises an ARMS forward primer, an ARMS reverse primer, and a fluorescent probe.

[0034] In some embodiments, the wild-type gene amplification retardation detection system further comprises DNA polymerase, PCR buffer, and dNTPs. Preferably, the DNA polymerase is Taq enzyme.

[0035] The fourth aspect of the present invention provides a method for improving mutation enrichment in amplification retardation gene mutation detection, which comprises adding the template to be detected to the wild-type gene amplification retardation detection system described in the third aspect of the present invention and performing PCR amplification.

[0036] The present invention adds the above-mentioned wild-type gene amplification suppressing oligonucleotide sequence element and competitive binding oligonucleotide sequence element to a real-time fluorescence quantitative PCR reaction tube for amplification-blocking gene mutation detection, forming a real-time fluorescence quantitative PCR reaction system composed of five (types of) oligonucleotides: ARMS forward primer (primer group), ARMS reverse primer, fluorescent probe, wild-type gene amplification suppressing oligonucleotide sequence element, and competitive binding oligonucleotide sequence element.

[0037] In some embodiments, in the wild-type gene amplification retardation detection system, the final molar concentration of the wild-type gene amplification suppressing oligonucleotide sequence element or the competitive binding oligonucleotide sequence element in the wild-type gene amplification retardation detection system is 0.2 μM-0.8 μM, preferably 0.4 μM.

[0038] The fifth aspect of the present invention provides the use of the wild-type gene amplification suppressing oligonucleotide sequence element described in the first aspect of the present invention, the composition described in the second aspect of the present invention, and the wild-type gene amplification retardation detection system described in the third aspect of the present invention in the preparation of products for detecting gene mutations.

[0039] In some embodiments, the product for detecting gene mutations is an amplification-retarded gene mutation detection product, which is suitable for ARMS-PCR or Super-ARMS technology.

[0040] The sixth aspect of the present invention provides a method for detecting gene mutations, which comprises adding a template to be detected to the wild-type gene amplification retardation detection system described in the third aspect of the present invention, and performing PCR amplification.

[0041] In some embodiments, in the wild-type gene amplification retardation detection system, the final molar concentration of the wild-type gene amplification suppressing oligonucleotide sequence element or the competitive binding oligonucleotide sequence element in the amplification retardation gene mutation detection system is 0.2 μM-0.8 μM, preferably 0.4 μM.

[0042] The present invention Figure 2 A schematic diagram showing the principle of improving the sensitivity of conventional amplification-retarded gene mutation detection methods according to the present invention.

[0043] Beneficial effects of the present invention:

[0044] The present invention discloses a novel wild-type gene amplification suppressor oligonucleotide sequence element, composition, and application thereof. The present invention also discloses a competitive binding oligonucleotide sequence element that competitively binds to mutant allele sites with the wild-type gene amplification suppressor oligonucleotide sequence element. When used together with the novel wild-type gene amplification suppressor oligonucleotide sequence element of the present invention, the competitive binding oligonucleotide sequence element can significantly improve the efficiency of mutation enrichment and the detection sensitivity of the amplification-blocking gene mutation detection method based on the existing widely used amplification-blocking gene mutation detection method. In particular, it can achieve stable and reliable detection of low-frequency and ultra-low-frequency gene mutations in liquid biopsies currently commonly used in precision oncology medicine.

[0045] In the present invention, the Blocker sequence that inhibits the ARMS negative chain of the wild-type template (that is, the wild-type gene amplification suppressor oligonucleotide sequence) is designed to be a Blocker sequence with a Flap sequence (that is, the first sequence region). The key design is that the thiolation modification between several bases at the junction of the Flap sequence and the target sequence matching region (that is, the second sequence region) prevents the endonuclease activity of the Taq enzyme, ensuring that the Taq enzyme cannot endonuclease the Blocker sequence during the reaction, thereby achieving stable binding of the Blocker sequence to the wild-type template. At the same time, the locked nucleic acid design at the base of the mutation site of the Blocker sequence increases the TM value of the wild-type Blocker sequence. Only when the Blocker sequence binds to the ARMS negative chain carrying the mutation, due to the low tolerance of the locked nucleic acid for mismatches, the competitive binding oligonucleotide sequence element will preferentially bind to the ARMS negative chain carrying the mutation. The competitive binding oligonucleotide sequence element will be cut by the Taq enzyme when the downstream ARMS primer extends to the first base at its 5' end to form an amplification product, thereby achieving efficient enrichment of the mutant template and greatly reducing the situation where the detection sensitivity caused by the wild-type background cannot fully meet the actual clinical needs of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shown is a schematic structural diagram of the wild-type gene amplification suppressing oligonucleotide sequence element and the competitive binding oligonucleotide sequence element of the present invention;

[0047] Figure 2 A schematic diagram showing the principle of improving the sensitivity of conventional amplification-retarded gene mutation detection methods according to the present invention;

[0048] Figure 3 Shown are the amplification curves of the EGFR T790M mutation site test reaction system and the control reaction system;

[0049] Figure 4Shown are the amplification curves of the EGFR p.E746_A750delELREA site test reaction system and the control reaction system;

[0050] Figure 5 Shown are the amplification curves of the EGFR p.D770_N771insSVD site test reaction system and the control reaction system. DETAILED DESCRIPTION

[0051] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. The examples and features in the examples of this application may be combined with each other unless there is a conflict.

[0052] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0053] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in this technical field.

[0054] Based on the guidance of the technical solution design principles in the invention content part of the present invention, this application designs multiple specific embodiments for illustration. Of course, based on the guidance of the technical solution design principles, other specific embodiments other than the embodiments mentioned in this application can also be designed.

[0055] Example 1

[0056] This example uses the human AI-Edigene EGFR p.T790M Reference Standard Plus from Cobigenes. The standard is in the form of genomic DNA. The EGFR T790M mutation corresponds to the Cosmic Catalogue of Somatic Mutations in Cancer (COSM6240), with a mutation frequency of 50% ± 5%.

[0057] The specific operation mode of this embodiment is as follows:

[0058] 1. Design wild-type gene amplification inhibitory oligonucleotide sequence elements and competitive binding oligonucleotide sequence elements for human EGFR gene T790M as follows:

[0059] Wild-type gene amplification suppression oligonucleotide sequence element (PCO79, SEQ ID NO.1): 5'-GACTGATACTG*G*T*G*CAGCTCATC L AC L GC L AGCTCA-3'PO4 - ;

[0060] Competitive binding oligonucleotide sequence element (FCO79, SEQ ID NO. 2):

[0061] 5'-CCTCACCTCCACCGTGCAG-3'PO4 - ;

[0062] The ARMS amplification primer and probe sequences are as follows:

[0063] The upstream amplification primer is the ARMS mismatch primer sequence (FAR79, SEQ ID NO.3): 5'-CCGAAGGGCATGAGCTTCA-3',

[0064] Downstream amplification primer sequence (RAR79, SEQ ID NO.4): 5'-TCCCTCCAGGAAGCCTACG-3',

[0065] Fluorescent probe sequence (AAR79, SEQ ID NO.5):

[0066] 5'(5,6FAM)-CCCAGCAGGCGGCACACG-3'BHQ1.

[0067] The sequences in this example are from left to right from the 5' end to the 3' end. "*" indicates a thiolation group between bases, the superscript "L" indicates a locked nucleic acid modification of the corresponding base to the lower left, "PO4-" indicates a 3' phosphorylation modification, 5,6FAM is a fluorescent group modification of the fluorescent probe, and BHQ1 is a quencher modification of the fluorescent probe. Unless otherwise specified, the abbreviations used in other examples will follow this example.

[0068] 2. Dilution of the standard as follows:

[0069] Genomic DNA (gDNA) was extracted from peripheral blood mononuclear cells (PBMCs) collected from whole blood buffy coats of three healthy volunteers. After negative EGFR T790M detection by ddPCR, the samples were mixed into a healthy human gDNA pool and the nucleic acid concentration was quantified using Qubit2.0 (ThermoFisher). The pool was then used to dilute a standard with a fixed EGFR T790M mutation frequency to a lower mutation frequency.

[0070] The EGFR T790M mutation frequency standard with a 50% mutation frequency was diluted with the above-mentioned healthy human mixed tube gDNA (200 μL total volume, large volume dilution method) to mutation frequencies of 2%, 1%, 0.5%, 0.2%, and 0.1%, and the mass concentration of each diluted mutation frequency standard was kept consistent by adding Tris-EDTA solution (TE); the actual mutation frequency of the diluted standard with a mutation frequency of 0.5% was 0.483% by ddPCR detection, which was within the error tolerance range (CV ≤ 5%), and the downstream mutation frequency standard was further diluted according to this actual quantitative result.

[0071] 3. Fluorescence real-time quantitative PCR detection:

[0072] Using the diluted mutation frequency standard gDNA as a template, prepare the fluorescent real-time quantitative PCR test reaction system and control reaction system according to Table 1-1 and Table 1-2, and execute the PCR reaction program in Table 2 on the RocGene Archime X6:

[0073] Table 1-1 Reaction system (test)

[0074]

[0075]

[0076] Table 1-2 Reaction system (control)

[0077] Components Volume (μL) 2×AceQ Universal U+Probe Master Mix V2 (Vazyme, Q513) 12.5 FAR79 (SEQ ID NO. 3) (10 μM) 0.5 RAR79 (SEQ ID NO. 4) (10 μM) 0.5 AAR79 (SEQ ID NO. 5) (10 μM) 0.5 Template DNA X(10ng) <![CDATA[Double-distilled water (ddH2O)]]> 11-X Total components 25

[0078] Table 2 PCR reaction conditions

[0079]

[0080]

[0081] 4. Results and Analysis

[0082] The baseline and threshold settings for qPCR reactions were automatically generated using Archimed Analyzer v2.0.2, the software system that comes with the RocGene Archimed machine. The Ct values ​​of the amplification curves for each diluted mutation frequency sample were obtained and collated.

[0083] from Figure 3 As can be seen from the amplification curves and the results in Table 3, after adding the wild-type gene amplification suppressor oligonucleotide sequence element and the competitive binding oligonucleotide sequence element in this example, the Ct values ​​of the diluted samples with four mutation frequencies of 1%, 0.5%, 0.2%, and 0.1% of EGFR T790M were all 3-6 cycles earlier than those of the conventional ARMS-qPCR reaction system without these two sequence elements. For the ultra-low frequency mutation 0.1% VAF sample, the coefficient of variation of the test system was less than 1%, and the average Ct was 35.15, indicating that the system has stable detection capabilities for this mutation frequency and has potential applicability for stable detection of 0.1%. The NTC did not peak, while the EGFR T790M strong positive control (10% VAF) peaked normally, with a standard S-shaped curve (not shown in this example).

[0084] Table 3 Ct values ​​and coefficient of variation of the EGFR T790M mutation site test reaction system and the control reaction system

[0085]

[0086] Note: The Ct value of the wells where no Ct value was detected in the software was recorded as 45.

[0087] Example 2

[0088] This example uses the human AI-Edigene EGFR p.E746_A750delELREA Reference Standard Plus (CBP10334) from Cobigenes. The standard is genomic DNA (gDNA). The EGFR p.E746_A750delELREA mutation corresponds to the Cosmic Catalogue of Somatic Mutations in Cancer (COSM6225). The mutation frequency is 100%.

[0089] The specific operation mode of this embodiment is as follows:

[0090] 1. Design wild-type gene amplification suppression oligonucleotide sequence elements and competitive binding oligonucleotide sequence elements for human EGFR gene p.E746_A750delELREA as follows:

[0091] Wild-type gene amplification suppression oligonucleotide sequence element (PCO19-1, SEQ ID NO.6): 5'-GACTGATACTG*T*T*G*GC L TTTCGGAGATGTTGC L TTCL TC L TT-3'PO4 - ;

[0092] Competitive binding oligonucleotide sequence element (FCO19, SEQ ID NO.7):

[0093] 5'-TCGAGGATTTCCTTGTTGGCTT-3'PO4 - ;

[0094] The ARMS amplification primer and probe sequences are as follows:

[0095] The upstream amplification primer is the ARMS mismatch primer sequence (FAR19-1, SEQ ID NO.8): 5'-TCCCGTCGCTATCAAGACATC-3',

[0096] Downstream amplification primer sequence (RAR19, SEQ ID NO.9): 5'-TGGGCCTGAGGTTCAGAG-3',

[0097] Fluorescent probe sequence (AAR19, SEQ ID NO.10):

[0098] 5'(5,6FAM)-CCTCGATGGTGAGTTTCTGCTTTGCT-3'BHQ1.

[0099] The sequences in this example are from left to right from the 5' end to the 3' end. "*" indicates a thiolation group between bases, the superscript "L" indicates a locked nucleic acid modification of the corresponding base to the lower left, "PO4-" indicates a 3' phosphorylation modification, 5,6FAM is a fluorescent group modification of the fluorescent probe, and BHQ1 is a quencher modification of the fluorescent probe. Unless otherwise specified, the abbreviations used in other examples will follow this example.

[0100] 2. Dilution of the standard as follows:

[0101] Genomic DNA (gDNA) was extracted from peripheral blood mononuclear cells (PBMCs) collected from whole blood buffy coats of three healthy volunteers. After negative detection of EGFR p.E746_A750delELREA by ddPCR, the gDNA was mixed into a tube of healthy human gDNA pool and the nucleic acid concentration was quantified using Qubit2.0 (Thermo Fisher). The pool was then used to dilute a standard with a fixed EGFR p.E746_A750delELREA mutation frequency to a lower mutation frequency.

[0102] The EGFR p.E746_A750delELREA mutation standard with a 100% mutation frequency was diluted with the healthy human mixed tube gDNA (200 μL total volume, large-volume dilution method) to mutation frequencies of 2%, 1%, 0.5%, 0.2%, and 0.1%. Tris-EDTA solution (TE) was added to maintain the mass concentration of each diluted mutation frequency standard. The actual mutation frequency of the diluted standard with a 1% mutation frequency was 0.961% by ddPCR, which was within the error tolerance range (CV ≤ 5%). The subsequent mutation frequency standards were further diluted according to this actual quantitative result.

[0103] 3. Fluorescence real-time quantitative PCR detection:

[0104] Using the diluted mutation frequency standard gDNA as a template, prepare the fluorescent real-time quantitative PCR test and control reaction systems according to Table 4-1 and Table 4-2, and execute the PCR reaction program in Table 5 on the RocGene Archime X6:

[0105] Table 4-1 Reaction system (test)

[0106]

[0107]

[0108] Table 4-2 Reaction system (control)

[0109] Components Volume (μL) 2×AceQ Universal U+Probe Master Mix V2 (Vazyme, Q513) 12.5 FAR19-1 (SEQ ID NO. 8) (10 μM) 0.5 RAR19 (SEQ ID NO. 9) (10 μM) 0.5 AAR19 (SEQ ID NO. 10) (10 μM) 0.5 Template DNA X(10ng) <![CDATA[Double-distilled water (ddH2O)]]> 11-X Total components 25

[0110] Table 5 PCR reaction conditions

[0111]

[0112]

[0113] 4. Results and Analysis

[0114] The baseline and threshold settings for qPCR reactions were automatically generated using Archimed Analyzer v2.0.2, the software system that comes with the RocGene Archimed machine. The Ct values ​​of the amplification curves for each diluted mutation frequency sample were obtained and collated.

[0115] from Figure 4As shown in the amplification curves and the results in Table 6, after the addition of the wild-type gene amplification suppressor oligonucleotide sequence element and the competitive binding oligonucleotide sequence element in this example, the Ct values ​​of diluted samples at four mutation frequencies of 1%, 0.5%, 0.2%, and 0.1% of EGFR p.E746_A750delELREA were all 2.3-9.8 cycles earlier than those of a conventional ARMS-qPCR reaction system without these two sequence elements. For the ultra-low mutation frequency 0.1% VAF sample, the coefficient of variation of the test system was <2%, and the average Ct was 35.11, indicating that the system has stable detection capabilities for this mutation frequency and has potential applicability for stable detection of 0.1%. While the NTC did not peak, the strong positive control for EGFR p.E746_A750delELREA (10% VAF) peaked normally, with a standard S-shaped curve (not shown in Example 2).

[0116] Table 6 Ct values ​​and coefficient of variation of the EGFR p.E746_A750delELREA site test reaction system and the control reaction system

[0117]

[0118] Note: The Ct value of the wells where no Ct value was detected in the software was recorded as 45.

[0119] Example 3

[0120] This example uses the human AI-Edigene EGFR p.D770_N771insSVD Reference Standard Plus from Cobigenes. The standard is genomic DNA (gDNA). The EGFR p.D770_N771insSVD mutation corresponds to the Cosmic Catalogue of Somatic Mutations in Cancer (COSM13428), with a mutation frequency of 100%.

[0121] The specific operation mode of this embodiment is as follows:

[0122] 1. Design wild-type gene amplification inhibitory oligonucleotide sequence elements and competitive binding oligonucleotide sequence elements for human EGFR gene p.D770_N771insSVD as follows:

[0123] Wild-type gene amplification suppression oligonucleotide sequence element (PCO20-1, SEQ ID NO.11): 5'-GACTGATACTG*G*G*C*ACAC L GTGGGGGTT L GTL -3'PO4 - ;

[0124] Competitive binding oligonucleotide sequence element (FCO20, SEQ ID NO.12):

[0125] 5'-CCAGCAGGCGGCACAC-3'PO4 - ;

[0126] The ARMS amplification primer and probe sequences are as follows:

[0127] The upstream amplification primer is the ARMS mismatch primer sequence (FAR20-1, SEQ ID NO.13): 5'-TGGCCAGCGTGGCGAG-3',

[0128] Downstream amplification primer sequence (RAR20, SEQ ID NO.14): 5'-GAAGGGCATGAGCTGCGT-3',

[0129] Fluorescent probe sequence (AAR20, SEQ ID NO.15):

[0130] 5'(5,6FAM)-CATCTGCCTCACCTCCACCGTGC-3'BHQ1.

[0131] The sequences in this example are from left to right from the 5' end to the 3' end. "*" indicates a thiolation group between bases, the superscript "L" indicates a locked nucleic acid modification of the corresponding base to the lower left, "PO4-" indicates a 3' phosphorylation modification, 5,6FAM is a fluorescent group modification of the fluorescent probe, and BHQ1 is a quencher modification of the fluorescent probe. Unless otherwise specified, the abbreviations used in other examples will follow this example.

[0132] 2. Dilution of the standard as follows:

[0133] Genomic DNA (gDNA) was extracted from peripheral blood mononuclear cells (PBMCs) collected from whole blood buffy coats of three healthy volunteers. After negative detection of EGFR p.D770_N771insSVD by ddPCR, the gDNA was mixed into a tube of healthy human gDNA pool and the nucleic acid concentration was quantified using Qubit2.0 (Thermo Fisher). The pool was then used to dilute a standard with a fixed mutation frequency of EGFR p.D770_N771insSVD to a lower mutation frequency.

[0134] The EGFR p.D770_N771insSVD mutation frequency standard with a 100% mutation frequency was diluted with the above-mentioned healthy human mixed tube gDNA (200 μL total volume, large volume dilution method) to mutation frequencies of 2%, 1%, 0.5%, 0.2%, and 0.1%, and the mass concentration of each diluted mutation frequency standard was kept consistent by adding Tris-EDTA solution (TE); the actual mutation frequency of the diluted standard with a 1% mutation frequency was 0.954% by ddPCR detection, which was within the allowable error range (CV≤5%), and the downstream mutation frequency standard was further diluted according to this actual quantitative result.

[0135] 3. Fluorescence real-time quantitative PCR detection:

[0136] Using the diluted mutation frequency standard gDNA as a template, the fluorescent real-time quantitative PCR test and control reaction systems were prepared with reference to Tables 7-1 and 7-2. The PCR reaction program in Table 5 (same as in Example 2) was performed on a RocGene Archime X6:

[0137] Table 7-1 Reaction system (test)

[0138]

[0139]

[0140] Table 7-2 Reaction system (control)

[0141] Components Volume (μL) 2×AceQ Universal U+Probe Master Mix V2 (Vazyme, Q513) 12.5 FAR20-1 (SEQ ID NO. 13) (10 μM) 0.5 RAR20 (SEQ ID NO. 14) (10 μM) 0.5 AAR20 (SEQ ID NO. 15) (10 μM) 0.5 Template DNA X(10ng) <![CDATA[Double distilled water (ddH2O)]]> 11-X Total components 25

[0142] 4. Results and Analysis

[0143] The baseline and threshold settings for qPCR reactions were automatically generated using Archimed Analyzer v2.0.2, the software system that comes with the RocGene Archimed machine. The Ct values ​​of the amplification curves for each diluted mutation frequency sample were obtained and collated.

[0144] from Figure 5As shown in the amplification curves and the results in Table 8, after adding the wild-type gene amplification suppressor oligonucleotide sequence element and the competitive binding oligonucleotide sequence element, the Ct values ​​of diluted samples at four mutation frequencies of 1%, 0.5%, 0.2%, and 0.1% for EGFR p.D770_N771insSVD were all improved by 2.5-10 cycles compared to a conventional ARMS-qPCR reaction system without these two sequence elements. For the ultra-low mutation frequency 0.1% VAF sample, the coefficient of variation of the test system was less than 2%, with an average Ct of 34.93, indicating that the system has stable detection capabilities for this mutation frequency and has potential applicability for stable detection of 0.1%. While the NTC peak did not occur, the strong positive control for EGFR p.D770_N771insSVD (10% VAF) showed a normal peak, with the curve exhibiting a standard S-shaped curve (not shown in Example 3).

[0145] Table 8 Ct values ​​and coefficient of variation of the EGFR p.D770_N771insSVD site test reaction system and the control reaction system

[0146]

[0147] Note: The Ct value of the wells where no Ct value was detected in the software was recorded as 45.

[0148] From the above description, it can be seen that the above three embodiments of the present invention all achieve the following technical effects: the new wild-type gene amplification suppressing oligonucleotide sequence elements and competitive binding oligonucleotide sequence elements disclosed in the present invention are added to the conventional amplification blocking gene mutation detection system, and the amplification of the wild-type detection site contained in the DNA chain bound by the non-amplification blocking gene mutation detection primer is suppressed, and the mutation is efficiently enriched. The enrichment efficiency is greatly improved compared with the conventional amplification blocking gene mutation detection method, especially for ultra-low frequency mutations of 0.1% and 0.2%. The method disclosed in the present invention, for the first time in the conventional amplification blocking gene mutation detection, realizes the simultaneous inhibition of the positive and negative DNA chains where the wild-type detection site is located in the detection site, avoiding the false negative caused by the sensitivity of the conventional amplification blocking gene mutation detection system itself in the liquid biopsy. At the same time, it has simple operation, the same reaction time as the conventional amplification blocking gene mutation detection, and the cost of detecting low-frequency mutations is greatly reduced compared with the existing ddPCR. It is worth mentioning that the haploid genome equivalent copy number corresponding to the DNA input amount of the ultra-low allele mutation frequency in the preferred embodiment of the present invention follows the Poisson distribution during sampling. Therefore, for a 95% detection possibility, there is an equal probability that no template molecule can be obtained. This method has been verified by repeated calibration of standards and it was found that the coefficient of variation between replicate wells for the detection of ultra-low frequency mutations is generally less than 2%, suggesting that the mutation enrichment ability of the two oligonucleotide sequences disclosed in the present invention under the addition may be fully reflected in the presence of 1-2 copies in the reaction system.

[0149] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A wild-type gene amplification suppressor oligonucleotide sequence element, comprising a nucleotide chain S1 of a first sequence region and a nucleotide chain S2 of a second sequence region, wherein the 3' end of the first sequence region is connected to the 5' end of the second sequence region; At least the connecting region between the first sequence region and the second sequence region contains a thiolation modification; in, The first sequence region does not match or bind to the wild-type site, mutation site, and upstream and downstream sequences of the wild-type site and mutation site of the genome to be detected; the second sequence region is completely complementary to the wild-type genotype base sequence of the gene site to be detected.

2. The wild-type gene amplification suppressor oligonucleotide sequence element according to claim 1, wherein The position of the thiolation modification is between any two adjacent bases among the 1st to 4th bases from the 3' end of the first sequence region and the 1st to 5th bases from the 5' end of the second sequence region; Preferably, there is a thiolation modification between the last base at the 3' end of the first sequence region and the first base at the 5' end of the second sequence region, between the first base at the 5' end and the second base at the 5' end of the second sequence region, between the second base at the 5' end and the third base at the 5' end of the second sequence region, and between the third base at the 5' end and the fourth base at the 5' end of the second sequence region.

3. The wild-type gene amplification suppressor oligonucleotide sequence element according to claim 1 or 2, wherein: The bases in the second sequence region that are completely complementary to the wild-type allele at the mutation site to be detected contain locked nucleic acid modifications.

4. The wild-type gene amplification suppressor oligonucleotide sequence element according to claim 3, wherein: The physical coordinate of the base in the second sequence region that is completely complementary to the wild-type allele at the mutation site to be detected is set to 0, and at least one base among the 10 bases in the physical coordinate range of -5 to -1 and 1 to 5 contains a locked nucleic acid modification; Preferably, the bases with physical coordinates -2 and 2 contain locked nucleic acid modifications.

5. The wild-type gene amplification suppressor oligonucleotide sequence element according to any one of claims 1 to 2 or claim 4, wherein: The 3' end of the second sequence region contains a first blocking modification, which is selected from one or more of the following modifications: phosphorylation modification, amino modification, Cn steric hindrance modification, inverted dT modification, and 3' terminal base being a dideoxynucleotide modification.

6. A composition comprising a competitive binding oligonucleotide sequence element and a wild-type gene amplification suppressing oligonucleotide sequence element according to any one of claims 1 to 5, wherein the competitive binding oligonucleotide sequence element and the wild-type gene amplification suppressing oligonucleotide element can competitively bind to the same DNA strand; in, The competitive binding oligonucleotide sequence element is reversely complementary to the upstream nucleotide sequence or downstream nucleotide sequence of the gene mutation site to be detected, and does not bind to the gene mutation site to be detected.

7. The composition according to claim 6, wherein The 3' end of the competitive binding oligonucleotide sequence element overlaps with a sequence at the 5' end of the nucleotide chain S2 of the second sequence region, or the 5' end of the competitive binding oligonucleotide sequence element overlaps with a sequence at the 3' end of the nucleotide chain S2 of the second sequence region; Preferably, the overlapping sequences are 5-10 bases in length.

8. The composition according to claim 6 or 7, wherein The 3' end of the competitive binding oligonucleotide sequence element contains a second blocking modification, which is selected from one or more of the following modifications: phosphorylation modification, amino modification, Cn steric hindrance modification, inverted dT modification and 3' terminal base was a dideoxynucleotide modification.

9. A wild-type gene amplification retardation detection system, comprising the composition according to any one of claims 6 to 8, and further comprising an ARMS forward primer, an ARMS reverse primer, and a fluorescent probe.

10. Use of the wild-type gene amplification suppressing oligonucleotide sequence element according to any one of claims 1 to 5, the composition according to any one of claims 6 to 8, and the wild-type gene amplification retardation detection system according to claim 9 in the preparation of a product for detecting gene mutations.

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