Double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application of double-stranded nucleic acid detection composition

Abstract

The invention discloses a double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application of the double-stranded nucleic acid detection composition, belongs to a composition for detecting nucleic acid in the technical field of biology and a use method of the composition, and aims to solve the technical problem of how to efficiently and sensitively detect double-stranded nucleic acid. The double-stranded nucleic acid detection composition based on the lambda exonuclease and the deoxyribozyme comprises a double-stranded nucleic acid detection probe, the lambda exonuclease and a reporter molecule, wherein the double-stranded nucleic acid detection probe comprises a long single-stranded DNA named as a recognition chain R and n short single-stranded DNAs named as Dz; the recognition chain R contains a target binding domain and n Dz binding domains which are sequentially and adjacently arranged in the direction from 5'to 3 '; the 5'end of the recognition chain R is modified with a functional group, when the Dz is a single chain, the Dz is deoxyribozyme capable of enzyme digestion of the reporter molecule, and the 5 'end of the Dz is subjected to sulfo-modification. The kit has high sensitivity and specificity, and the detection limit can be as low as a fly-friction level.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to compositions for nucleic acid detection and their use methods, and particularly to a double-stranded DNA detection composition based on λ exonuclease and deoxyribozyme and its application. Background Art

[0002] In modern medicine, nucleic acid detection technology has been increasingly widely used, especially in the diagnosis and monitoring of infectious diseases. The direct detection of double-stranded nucleic acids is of great significance because it can provide more accurate pathogen identification and disease progression assessment. By enabling early diagnosis and personalized treatment, double-stranded nucleic acid detection can not only reduce the misdiagnosis rate but also provide a reliable basis for clinical decision-making. The evolution of this technology has a profound impact on the field of public health. Especially during the outbreak of an epidemic, rapid and accurate detection methods are crucial for timely implementation of corresponding public health measures.

[0003] In recent years, room-temperature nucleic acid detection technology has gradually attracted attention. Its main advantage lies in the fact that it does not require complex temperature control equipment and can be carried out at room temperature, thus greatly reducing the technical threshold and cost of detection. Room-temperature nucleic acid detection methods effectively simplify the experimental process, making nucleic acid detection more feasible in resource-limited environments, especially in remote areas and developing countries. In particular, the application of the "one-pot approach" enables multiple experimental steps to be completed in the same reaction system, not only improving the convenience of operation but also reducing the risks of sample handling and cross-contamination. By promoting this method, wider screening and monitoring can be achieved, thereby enhancing public health safety and the ability to respond to potential epidemics.

[0004] λ exonuclease (λ exo) is an enzyme encoded by bacteriophage λ and is mainly involved in DNA repair and recombination processes. This enzyme has exonuclease activity and can catalyze the hydrolysis step by step from the 5'-end of double-stranded DNA, releasing 5'-mononucleotides. Due to its high efficiency and specificity, λ exonuclease is widely used in molecular biology research and has become a commonly used exonuclease. A specific probe modified with functional groups can directly target and identify the target double-stranded target with the assistance of λ exonuclease. When the probe binds to the target, it triggers the release of functional single-stranded DNA, which can carry its own fluorescence signal or achieve signal amplification through the cleavage characteristics of deoxyribozyme (DNAzyme). Deoxyribozyme is a class of DNA molecules with catalytic functions that can catalyze a variety of chemical reactions. Some of these DNAzymes, as RNA-cleaving enzymes, can specifically cleave RNA molecules. By using a substrate containing specific RNA as a reporter molecule, the multi-round cleavage characteristics of DNAzyme can achieve signal amplification. This property makes DNAzyme have important application potential in biological detection and molecular diagnosis, significantly improving the sensitivity and accuracy of detection. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to detect double-stranded nucleic acids efficiently and sensitively.

[0006] To solve the above technical problem, the present invention first provides a composition for detecting double-stranded nucleic acids based on λ exonuclease and deoxyribozyme, comprising a double-stranded nucleic acid detection probe, λ exonuclease, and a reporter molecule; The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target is contained in the sample to be tested; the double-stranded nucleic acid detection probe comprises a single-stranded DNA named recognition strand R, and n single-stranded DNAs named Dz connected to the recognition strand R through base complementary pairing; the recognition strand R contains a target binding domain and n Dz binding domains arranged adjacent to each other in sequence from the 5'-end to the 3'-end; the n Dzs bind to the recognition strand R through the n Dz binding domains to form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; the 5'-end of the recognition strand R is modified with a functional group to achieve the binding of the double-stranded nucleic acid detection probe to λ exonuclease; the Dz is a deoxyribozyme that can cleave the reporter molecule, and the 5'-end of the Dz is modified to prevent the Dz from being degraded by λ exonuclease; The reporter molecule is a specific substrate for the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quenching group.

[0007] Before being cleaved by the Dz, the quenching group on the reporter molecule quenches the fluorescence signal of the fluorescent group, and after being cleaved by the Dz, the quenching group does not quench the fluorescence signal of the fluorescent group.

[0008] After the double-stranded nucleic acid detection probe recognizes the double-stranded target, it is digested by λ exonuclease, and the released Dz enzyme cleaves the reporter molecule, thereby releasing and amplifying the fluorescence signal.

[0009] In the above composition, the double-stranded target is double-stranded DNA and / or double-stranded DNA-RNA complex.

[0010] In the above composition, the Dz can be any deoxyribozyme that can cleave a specific substrate. Generally, it consists of a core catalytic domain and two substrate-binding domains located on both sides of the core catalytic domain. The core catalytic domain has the activity of cleaving a specific phosphodiester bond, and the substrate-binding domain is used to specifically bind the specific substrate. The phosphodiester bond is formed by ribonucleotides or deoxyribonucleotides.

[0011] In the above composition, the specific substrate is a DNA-RNA chimera or RNA.

[0012] In the above composition, the 5'-end of the Dz is modified by thiolation, specifically, the 3-5 positions of the 5'-end are thiolated.

[0013] In the above composition, the deoxyribozyme can specifically be selected from any one of 10-23 DNAzyme, 8-17 DNAzyme, 17E DNAzyme (a variant of 8-17 DNAzyme), Mg5 DNAzyme (a variant of 8-17 DNAzyme), 39E DNAzyme, EtNa DNAzyme, Ce13d DNAzyme, etc.

[0014] The Dz can be any deoxyribozyme that can cleave a specific substrate, specifically, it can be 10-23 DNAzyme, 8-17 DNAzyme, 17E DNAzyme (a variant of 8-17 DNAzyme), Mg5 DNAzyme (a variant of 8-17 DNAzyme), and other DNAzymes, such as any one of 39E DNAzyme, EtNa DNAzyme, Ce13d DNAzyme, etc. (see Figure 1 (A)).

[0015] The 10-23 DNAzyme (English name: 10-23 DNAzyme) is described in the non-patent literature "Lan T, Lu Y. Metal ion-dependent DNAzymes and their applications as biosensors. Interplay between Metal Ions and Nucleic Acids, 2011: 217-248."

[0016] The 10-23 DNA enzyme is as follows Figure 1 As shown in (A) of Figure 1 , the core catalytic domain sequence is 5'-GGMTAGHNDNNNCGD-3', as shown in positions 2-16 of SEQ ID No:1 in the sequence listing, with a length of 15 nucleotides. Among them, the letter M represents A or C, H represents A, C or T, N represents A, C, G or T, and D represents A, G or T. The nucleotide tightly linked to the 5' end of the core catalytic domain of the 10-23 DNA enzyme is a purine deoxyribonucleotide, and R in the figure represents purine deoxyribonucleotide A or G. The 10-23 DNA enzyme cleaves the phosphodiester bond between pyrimidine ribonucleotide and purine ribonucleotide. In the figure, Y represents pyrimidine ribonucleotide U or C, and R represents purine ribonucleotide A or G.

[0017] The described 8-17 DNA enzyme (English name 8-17 DNAzyme) is described in the non-patent literature "Schlosser K, Li Y. A versatile endoribonuclease mimic made of DNA: characteristics and applications of the 8–17 RNA‐cleaving DNAzyme. ChemBioChem, 2010, 11(7): 866-879.".

[0018] The 8-17 DNA enzyme is as follows Figure 1 As shown in (B) of Figure 1 , the core catalytic domain sequence of the 8-17 DNA enzyme is 5'-NNNNAGY(N)NNNNCGN(N)-3', with a length of 14-16 nucleotides, where (N) is either one nucleotide N or no nucleotide, that is, the core catalytic domain sequence of the 8-17 DNA enzyme is specifically any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', 5'-NNNNAGYNNNNCGN-3'. The letter N represents A, C, G or T, and Y represents pyrimidine deoxyribonucleotide T or C. The 8-17 DNA enzyme cleaves the phosphodiester bond between deoxyribonucleotide and ribonucleotide. In the figure, N represents deoxyribonucleotide A, C, G or T, and rN represents ribonucleotide A, C, G or U.

[0019] The described 17E DNAzyme (English name: 17E DNAzyme) is an 8-17 DNAzyme variant, which is described in the non-patent literature "Zhou, W.; Zhang, Y.; Ding, J.; Liu, J., In vitro selection in serum: RNA-cleaving DNAzymes for measuring Ca 2+ and Mg 2+ . Acs Sensors 2016, 1 (5), 600-606.".

[0020] The described Mg5 DNAzyme (English name: Mg5 DNAzyme) is an 8-17 DNAzyme variant, which is described in the non-patent literature "Brown, A. K.; Li, J.; Pavot, C. M.-B.; Lu, Y., A lead-dependent DNAzyme with a two-step mechanism. Biochemistry 2003, 42 (23), 7152-7161.".

[0021] The described 39E DNAzyme (English name: 39E DNAzyme) is described in the non-patent literature "Wu, P.; Hwang, K.; Lan, T.; Lu, Y., A DNAzyme-gold nanoparticle probe for uranyl ion in living cells. Journal of the American Chemical Society 2013, 135 (14), 5254-5257.".

[0022] The described EtNa DNAzyme (English name: EtNa DNAzyme) is described in the non-patent literature "Zhou, W.; Saran, R.; Chen, Q.; Ding, J.; Liu, J., A new Na + ‐dependent RNA‐cleaving DNAzyme with over 1000‐fold rate acceleration by ethanol. ChemBioChem 2016, 17 (2), 159-163.".

[0023] The described Ce13d DNAzyme (English name: Ce13d DNAzyme) is recorded in the non-patent literature "Zhou, W.; Zhang, Y.; Huang, P.-J. J.; Ding, J.; Liu, J., A DNAzyme requiring two different metal ions at two distinct sites. Nucleic acids research 2016, 44(1), 354-363."

[0024] In the above composition, n is a natural number from 1 to 10 (n is 1, or n is a natural number from 2 to 10).

[0025] In the above composition, the molar ratio of the recognition strand R to the Dz can be 1: n.

[0026] In the above composition, the functional group can be any one of a phosphate group, a carboxyl group, an amino group, and a thiol group.

[0027] In the above composition, the length of the thiolation modification is 3 to 5 nucleotides.

[0028] In the above composition, the quenching group can be optionally BHQ1; the fluorescent group can be optionally FAM.

[0029] In the above composition, specifically, the composition can be any one set of P1-P7: P1: Using the double-stranded composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 2 and a single-stranded DNA with the sequence of SEQ ID No: 4, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No: 6; P2: Using the double-stranded composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 3 and a single-stranded DNA with the sequence of SEQ ID No: 5; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No: 7; P3. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No:8 and the single-stranded DNA with the sequence of SEQ ID No:9 or the single-stranded RNA with the sequence of SEQ ID No:10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No:11 and two single-stranded DNAs with the sequence of SEQ ID No:4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:6. P4. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No:8 and the single-stranded DNA with the sequence of SEQ ID No:9 or the single-stranded RNA with the sequence of SEQ ID No:10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No:12 and a single-stranded DNA with the sequence of SEQ ID No:4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:6. P5. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No:8 and the single-stranded DNA with the sequence of SEQ ID No:9 or the single-stranded RNA with the sequence of SEQ ID No:10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No:13 and a single-stranded DNA with the sequence of SEQ ID No:4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:6. P6. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No:8 and the single-stranded DNA with the sequence of SEQ ID No:9 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No:14 and a single-stranded DNA with the sequence of SEQ ID No:4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:6. P7. Using the double-strand composed of the single-stranded DNA of positions 1-45 of SEQ ID No:15 and the single-stranded DNA that is reverse complementary to positions 1-45 of SEQ ID No:15 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No:15 and a single-stranded DNA with the sequence of SEQ ID No:4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:6.

[0030] The present invention also provides the above double-stranded nucleic acid detection probe.

[0031] The present invention also provides a method for detecting whether a sample to be tested contains a target double-stranded nucleic acid. The method includes reacting the double-stranded nucleic acid detection probe with the sample to be tested in the presence of λ exonuclease and the reporter molecule, detecting the fluorescence signal of the reaction system, and determining whether there is a target double-stranded nucleic acid in the sample to be tested according to the fluorescence signal.

[0032] In the above method, determining whether there is a target double-stranded nucleic acid in the sample to be tested according to the fluorescence signal may specifically be that if the fluorescence signal changes (significantly changes), then the sample to be tested contains the target double-stranded nucleic acid; if the fluorescence signal does not change (significantly change), then the sample to be tested does not contain the target double-stranded nucleic acid.

[0033] The present invention also provides a product, which is composed of X1 and X2; X1 is the composition or the double-stranded nucleic acid detection probe, and X2 is the reagent and / or instrument required for detection.

[0034] In the above product, the instrument may be a real-time fluorescence detector.

[0035] In the above product, the reagent required for detection also needs to contain a metal ion that enables Dz to have deoxyribozyme activity as a cofactor. For example, the cofactor of the 10-23 DNAzyme can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Ba 2+ and Sr 2+ etc.; the cofactor of the 8-17 DNAzyme can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ etc.; the cofactor of the 17E DNAzyme (8-17 DNAzyme variant) can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ etc.; the cofactor of the Mg5 DNAzyme (8-17 DNAzyme variant) can be selected from Mg 2+ , Mn 2+ , Ca 2+ , Pb 2+ and Zn 2+ etc.; the cofactor of the 39E DNAzyme can be UO 2+ ; the cofactor of the EtNa DNAzyme can be selected from Na + , Ca 2+ ; the cofactor of the Ce13d DNAzyme can be Na + .

[0036] To solve the above technical problems, the present invention also provides the use of the composition, the double-stranded nucleic acid detection probe, the method, and the product in the preparation of nucleic acid detection reagents.

[0037] The present invention discloses a double-stranded nucleic acid detection composition based on λ exonuclease and deoxyribozyme and its application. The double-stranded nucleic acid detection composition includes a double-stranded nucleic acid detection probe. The main body of the double-stranded nucleic acid detection probe is a single-stranded DNA modified with functional groups such as a phosphate group, a carboxyl group, and an amino group at the 5' end. It can specifically recognize and target a target double-stranded nucleic acid (i.e., a double-stranded target, such as double-stranded DNA, double-stranded DNA-RNA complex) with the assistance of λ exonuclease (λ exonuclease). After the probe binds to the target, the release of the functional single-stranded DNA is triggered. The functional single-stranded DNA is a deoxyribozyme, which can cleave a specific substrate with a quenched fluorescent group multiple times, thereby realizing the release and amplification of the fluorescent signal and significantly enhancing the intensity of the fluorescent signal. A remarkable feature of this detection method is that it can detect dsDNA in clinical samples (such as cfDNA in blood, pathogen DNA, DNA in tissue samples and body fluid samples, etc.) without pre-amplification of the analyte, and the detection limit can be as low as femtomolar level. The present invention has high sensitivity and specificity, is widely applicable to the fields of molecular biology, clinical diagnosis and other related fields, and has important scientific research and clinical application values. Description of the Drawings

[0038] Figure 1 Schematic diagram and fluorescence-time trajectory diagram for the specific detection of double-stranded nucleic acid and signal amplification by the type I detection probe constructed in Example 1. Among them, Figure 1 (A) is the schematic diagram of the 10-23 DNAzyme cited in Example 1. The upper strand is the specific substrate, and the lower strand is the 10-23 DNAzyme. The 10-23 DNAzyme contains the nucleotide sequence of 5'-RGGMTAGHNDNNNCGD-3' (see SEQ ID No: 1), where the letter R represents purine deoxyribonucleotide A or G, the letter M represents A or C, the letter H represents A, C or T, the letter N represents A, C, G or T, and the letter D represents A, G or T; the core catalytic domain sequence of the 10-23 DNAzyme is 5'-GGMTAGHNDNNNCGD-3', as shown in the 2nd to 16th positions of SEQ ID No: 1 in the sequence listing, with a length of 15 nucleotides; the 10-23 DNAzyme cleaves the phosphodiester bond between pyrimidine ribonucleotide and purine ribonucleotide. In the figure, the letter Y represents pyrimidine ribonucleotide U or C, and the letter R represents purine ribonucleotide A or G. Figure 1(B) is the schematic diagram of the 8-17 DNAzyme cited in Example 1. The upper strand is the specific substrate, and the lower strand is the 8-17 DNAzyme. The core catalytic domain sequence of the 8-17 DNAzyme is 5'-NNNNAGY(N)NNNNCGN(N)-3', with a length of 14-16 nucleotides, where (N) is either a nucleotide N or no nucleotide, that is, the core catalytic domain sequence of the 8-17 DNAzyme is specifically any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', 5'-NNNNAGYNNNNCGN-3'. The letter N represents A, C, G, or T, and Y represents the pyrimidine deoxynucleotide T or C; the 8-17 DNAzyme cleaves the phosphodiester bond between deoxynucleotides and ribonucleotides. In the figure, N represents deoxynucleotide A, C, G, or T, and rN represents ribonucleotide A, C, G, or U. Figure 1 (C) is a schematic diagram of using the type I detection probe formed by the binding of the recognition strand R and Dz in a 1:1 ratio to detect double-stranded DNA and release Dz equal to the amount of R. Figure 1 (D) and Figure 1 (F) are respectively a schematic diagram of the cleavage cycle of the reporter molecule S1 by Dz1 (10-23 DNAzyme) released by the reaction of the probe P1 with the double-stranded target and a fluorescence-time trajectory diagram generated for the two double-stranded targets of the DNA double-stranded target and the DNA-RNA complex target. Figure 1 (E) and Figure 1 (G) are respectively a schematic diagram of the cleavage cycle of the reporter molecule S2 by Dz2 (8-17 DNAzyme) released by the reaction of the probe P2 with the double-stranded target and a fluorescence-time trajectory diagram generated for the two double-stranded targets of the DNA double-stranded target and the DNA-RNA complex target.

[0039] Figure 2 is a schematic diagram and a fluorescence-time trajectory diagram of the type II detection probe constructed in Example 2 for specifically detecting double-stranded DNA and signal amplification. Among them, Figure 2 (A) is a schematic diagram of the cleavage cycle of the reporter molecule S1 by Dz1 (10-23 DNAzyme) released in a quantity n (n = 2 in Example 3) times that of R by the reaction of the type II detection probe P3 formed by the binding of the recognition strand R and Dz in a 1:n (n = 1-10, n = 2 in Example 3) ratio with double-stranded DNA. Figure 2 (B) is a fluorescence-time trajectory diagram and a bar chart of the type II detection probe P3 for detecting double-stranded DNA.

[0040] Figure 3Schematic diagram and fluorescence-time trace diagram for detection in Example 3 using probe P for detecting double-stranded nucleic acids. Among them, Figure 3 (A) in Figure 3 shows the modifications carried out on the 5'-end of the recognition strand R of the probe for detecting double-stranded nucleic acids using different functional groups; Figure 3 (B) in Figure 3 is a schematic diagram for detection using probe P with the recognition strand R modified with different groups; Figure 3 (C) in Figure 3 is the fluorescence-time trace diagram generated by detecting probe P with the recognition strand R modified with different groups.

[0041] Figure 4 Fluorescence-time trace diagram and bar chart for detecting non-small cell lung cancer-related genes using type I double-stranded nucleic acid detection probe P1 in Example 4. Detailed implementation manners

[0042] The present invention will be further described in detail below in conjunction with the specific implementation manners. The given examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0043] In the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged.

[0044] In the experimental methods in the following examples, unless otherwise specified, they are all conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] The nucleic acid sequences in the following examples are synthesized by Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai).

[0046] The 1×λ exonuclease buffer in the following examples is a product of New England Biolabs (NEB) with the catalog number M0262S, and this reagent contains exonuclease (λ exonuclease).

[0047] Example 1 1. Design of type I double-stranded nucleic acid detection probe and signal amplification system The type I double-stranded nucleic acid detection probe and signal amplification system provided in this example includes a double-stranded nucleic acid detection composition based on λ exonuclease and deoxyribozyme.

[0048] The composition includes a double-stranded nucleic acid detection probe, λ exonuclease, and a reporter molecule; The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target is contained in the sample to be detected; the double-stranded nucleic acid detection probe comprises a single-stranded DNA named recognition strand R, and n single-stranded DNAs named Dz that are connected to the recognition strand R through base complementary pairing; the recognition strand R contains a target binding domain and n Dz binding domains that are adjacent to each other in sequence from the 5'-end to the 3'-end; the n Dzs are combined with the recognition strand R through the n Dz binding domains to form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; a functional group is modified at the 5'-end of the recognition strand R to realize the binding of the double-stranded nucleic acid detection probe to lambda exonuclease; the Dz is a deoxyribozyme that can cleave the reporter molecule, and the 5'-end of the Dz is modified to prevent the Dz from being degraded; The reporter molecule is a specific substrate of the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quenching group. Before being cleaved by the Dz, the quenching group on the reporter molecule quenches the fluorescence signal of the fluorescent group, and after being cleaved by the Dz, the quenching group does not quench the fluorescence signal of the fluorescent group.

[0049] In this embodiment, n = 1, and the molar ratio of the recognition strand R to the Dz is 1:1. Hereinafter, the double-stranded nucleic acid detection probe with the molar ratio of the recognition strand R to the Dz being 1:1 is referred to as type I double-stranded nucleic acid detection probe.

[0050] The thiolation modification can be performed on 3 to 5 nucleotides, and specifically 3 nucleotides are modified in this embodiment.

[0051] The modification group is any one of a phosphate group, a carboxyl group, an amino group, and a thiol group. Carboxylation modification is adopted in this embodiment.

[0052] The Dz can specifically be selected from any one of 10-23 DNAzyme, 8-17 DNAzyme, 17E DNAzyme (variant of 8-17 DNAzyme), Mg5DNAzyme (variant of 8-17 DNAzyme), 39E DNAzyme, EtNa DNAzyme, Ce13d DNAzyme, etc. In this embodiment, two type I detection probes named P1 and P2 are specifically designed, wherein 10-23 DNAzyme is adopted for P1 and 8-17 DNAzyme is adopted for P2: Detection probe P1: It is used to detect the DNA double-stranded target composed of T1 and T2 in Table 1, or to detect the DNA-RNA complex target composed of T1 and T3 in Table 1. It is composed of a recognition strand R1 and a short single-stranded Dz1. The short single-stranded Dz1 can cleave the specific substrate S1, and the cited DNAzyme is as Figure 1The 10-23 DNAzyme shown in (A) of. After the detection probe P1 recognizes the double-stranded target, the recognition strand R1 is digested by λ exonuclease (λ exonuclease) to release an equal amount of 10-23 DNAzyme (as short single-stranded Dz1) as the recognition strand R1. The 10-23 DNAzyme enters the cycle of cleaving the specific substrate S1 as a reporter molecule to generate and amplify the signal.

[0053] The sequence of R1 is shown as SEQ ID No:2 in the sequence listing, with a length of 72 nucleotides, and its first position is modified with a carboxylic acid group. R1 is composed of a target binding domain (positions 1-45) and a Dz binding domain (positions 46-72) connected in the 5'-3' direction; wherein the target binding domain is reverse complementary to positions 21-65 of one strand T1 in the double-stranded target, and the Dz binding domain is reverse complementary to Dz1.

[0054] Dz1 uses a 10-23 DNAzyme, and its schematic diagram is as Figure 1 shown in (A) of. The 10-23 DNAzyme is composed of a core catalytic domain and two substrate binding domains located on both sides of the core catalytic domain. The core catalytic domain sequence of the 10-23 DNAzyme is 5'-GGMTAGHNDNNNCGD-3', as shown in positions 2-16 of SEQ ID No:1 in the sequence listing, with a length of 15 nucleotides, where the letter M represents A or C, H represents A, C or T, N represents A, C, G or T, and D represents A, G or T. The nucleotide tightly connected to the 5' end of the core catalytic domain of the 10-23 DNAzyme is a purine deoxyribonucleotide, and R in the figure represents the purine deoxyribonucleotide A or G. The 10-23 DNAzyme cleaves the phosphodiester bond between a pyrimidine ribonucleotide and a purine ribonucleotide, where Y in the figure represents the pyrimidine ribonucleotide U or C, and R represents the purine ribonucleotide A or G. In this example, the core catalytic domain sequence of the 10-23 DNAzyme is GGCTAGCTACAACGA (as shown in positions 6-21 of SEQ ID No:4). There is a 6 nt substrate binding domain on each side of the catalytic domain sequence for the recognition and binding of the specific substrate S1; the 5' end of Dz1 is thiophosphorylated. The specific sequence of the 10-23 DNAzyme used in this example is shown as SEQ ID No:4 in the sequence listing, with a length of 27 nucleotides. The 10-23 DNAzyme is reverse complementary to positions 46-72 of R1; positions 1-6 of the 10-23 DNAzyme are reverse complementary to positions 8-13 of S1, and positions 22-27 are reverse complementary to positions 1-6 of S1; positions 1-3 of the 10-23 DNAzyme are thiophosphorylated.

[0055] The 10-23 DNA enzyme can cleave a specific substrate S1, and S1 is a DNA-RNA chimera with a sequence as shown in SEQ ID No: 6 in the sequence listing. S1 is 13 nucleotides in length and contains a cleavage recognition sequence of Dz1, purine ribonucleotide and pyrimidine ribonucleotide rGrU. The 5'-terminal T is modified with FAM, and the 3'-terminal A is modified with BHQ1.

[0056] Detection probe P2: used to detect the DNA double-stranded target composed of T1 and T2 in Table 1, or used to detect the DNA-RNA complex target composed of T1 and T3 in Table 1. It consists of a recognition strand R2 and a short single-stranded Dz2. Dz2 can specifically cleave a specific substrate S2, and the cited DNA enzyme is the 8-17 DNA enzyme shown in (B) of Figure 1 After the detection probe P2 recognizes and accesses the double-stranded target, the recognition strand R2 is digested by λ exonuclease (λ exonuclease) and releases an equal amount of 8-17 DNA enzyme (as the short single-stranded Dz2) as the recognition strand R2. The 8-17 DNA enzyme enters the cycle of cleaving S2 as a reporter molecule to generate and amplify the signal.

[0057] The sequence of R2 is as shown in SEQ ID No: 3 in the sequence listing, with a length of 78 nucleotides. Its 1st position is modified with a carboxylic acid group. R2 is formed by connecting a target-binding domain (positions 1-45) and a Dz-binding domain (positions 46-78) in the 5'-3' direction; wherein the target-binding domain is reverse complementary to positions 21-65 of one strand T1 in the double-stranded target, and the Dz-binding domain is reverse complementary to the Dz2.

[0058] Dz2 uses an 8-17 DNA enzyme, and its schematic diagram is as shown in Figure 1As shown in (B) of Figure [not provided]. The 8-17 DNAzyme consists of a core catalytic domain and two substrate-binding domains located on both sides of the core catalytic domain. The core catalytic domain sequence of the 8-17 DNAzyme is 5’-NNNNAGY(N)NNNNCGN(N)-3’, with a length of 14 - 16 nucleotides, where (N) represents either a nucleotide N or no nucleotide, that is, the core catalytic domain sequence of the 8-17 DNAzyme is specifically one of 5’-NNNNAGYNNNNNCGNN-3’, 5’-NNNNAGYNNNNCGNN-3’, 5’-NNNNAGYNNNNNCGN-3’, 5’-NNNNAGYNNNNCGN-3’. The letter N represents A, C, G, or T, and Y represents the pyrimidine deoxyribonucleotide T or C. The 8-17 DNAzyme cleaves the phosphodiester bond between deoxyribonucleotides and ribonucleotides. In the figure, N represents the deoxyribonucleotide A, C, G, or T, and rN represents the ribonucleotide A, C, G, or U. The core catalytic domain sequence of the 8-17 DNAzyme used in this example is TCCGAGCCGGTCGAA (as shown at positions 10 - 24 of SEQ ID No:5). There is a 9 nt substrate-binding domain on each side of the catalytic domain sequence for the recognition and binding of the specific substrate S2; the 5' end of Dz2 is thiophosphorothioate modified. The specific sequence of the 8-17 DNAzyme used in this example is as shown in SEQ ID No:5 in the sequence listing, with a length of 33 nucleotides. The 8-17 DNAzyme is reverse complementary to positions 46 - 78 of R2; positions 1 - 9 of the 8-17 DNAzyme are reverse complementary to positions 12 - 20 of S2, and positions 25 - 33 are reverse complementary to positions 1 - 9 of S2; positions 1 - 3 of the 8-17 DNAzyme are thiophosphorothioate modified.

[0059] The 8-17 DNAzyme can cleave the specific substrate S2, and S2 is a DNA-RNA chimera with a sequence as shown in SEQ ID No:7 in the sequence listing. S2 has a length of 20 nucleotides and contains a cleavage recognition sequence of Dz2 (the catalytic center is TCCGAGCCGGTCGAA) with purine ribonucleotide and pyrimidine deoxyribonucleotide rAdG. The 5'-terminal A is modified with FAM, and the 3'-terminal G is modified with BHQ1.

[0060]

[0061]

[0062] Note: In Table 1, * represents a phosphorothioate backbone linkage, specifically indicating that the non-bridging oxygen atom on the phosphodiester bond between two adjacent nucleotides is replaced by a sulfur atom to form a phosphorothioate group, and the structural formula is Formula (1); the nucleotides linked by the phosphorothioate group are as shown in Formula (2): Formula (1); Formula (2).

[0063] Figure 1 Figure (C) shows a reaction schematic diagram of the type I detection probe for recognizing and processing double-stranded targets (DNA double-stranded targets and DNA-RNA complex targets): The target binding domain of the recognition strand R recognizes T1 in the double-stranded target and binds to it to form double-stranded DNA with a 5'-COOH terminus. Under the action of λ exonuclease, an enzymatic digestion reaction occurs, and the double-stranded DNA with a 5'-COOH terminus is digested, releasing Dz.

[0064] The released Dz ( Figure 1 represented as 10-23 DNAzyme in Figure (D), Figure 1 represented as 8-17 DNAzyme in Figure (E)) has deoxyribozyme activity in the presence of magnesium ions and can cleave the substrate (specific substrate S, the substrate of 10-23 DNAzyme is S1, and the substrate of 8-17 DNAzyme is S2), generating a fluorescence signal, and the fluorescence signal is amplified through cycling.

[0065] 2. Preparation of the type I double-stranded nucleic acid detection probe and signal amplification system 2.1 Preparation of the type I detection probe Prepare the type I detection probe P1: Synthesize and modify single-stranded DNA according to Table 1 to obtain R1 and Dz1.

[0066] In a reaction buffer environment of 1 × λ exonuclease buffer, at room temperature, add R1 and Dz1 to synthesize the type I detection probe P1, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of R1 is 1 μM, and the concentration of Dz1 is 1 μM. Apply an annealing program to ensure the correct binding of R1 and Dz1 to obtain the type I double-stranded nucleic acid detection probe P1.

[0067] The annealing program is set as follows: 90 °C, 2 min; 80 °C, 2 min; 70 °C, 2 min; 60 °C, 2 min; 50 °C, 2 min; 40 °C, 2 min; 37 °C, 2 min; keep at 4 °C.

[0068] Prepare the type I detection probe P2: Synthesize and modify single-stranded DNA according to Table 1 to obtain R2 and Dz2.

[0069] The reaction buffer environment is 1 × λ exonuclease buffer. At room temperature, add R2 and Dz2 to synthesize the type I detection probe P2, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of R1 is 1 μM, and the concentration of Dz2 is 1 μM. Apply an annealing program to ensure the correct binding of R2 and Dz2 to obtain the type I double-stranded nucleic acid detection probe P2.

[0070] The annealing program is set as follows: 90 °C, 2 min; 80 °C, 2 min; 70 °C, 2 min; 60 °C, 2 min; 50 °C, 2 min; 40 °C, 2 min; 37 °C, 2 min; 4 °C, hold.

[0071] 2.2 Preparation of double-stranded targets The double-stranded targets used are DNA double-stranded targets composed of T1 and T2, or DNA-RNA complex targets composed of T1 and T3. The preparation method is as follows: Synthesize and modify single-stranded nucleic acids according to Table 1 to obtain T1, T2, and T3.

[0072] The reaction buffer environment is 1 × λ exonuclease buffer. At room temperature, add T1 and T2 to synthesize the DNA double-stranded target, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of T1 in the system is 500 nM, and the concentration of T2 is 500 nM. Apply an annealing program to ensure the correct binding of T1 and T2 to obtain the DNA double-stranded target.

[0073] The reaction buffer environment is 1 × λ exonuclease buffer. At room temperature, add T1 and T3 to synthesize the DNA-RNA complex target, and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of T1 in the system is 500 nM, and the concentration of T2 (T3) is 500 nM. Apply an annealing program to ensure the correct binding of T1 and T3 to obtain the DNA-RNA complex target.

[0074] The annealing program is set as follows: 90 °C, 2 min; 80 °C, 2 min; 70 °C, 2 min; 60 °C, 2 min; 50 °C, 2 min; 40 °C, 2 min; 37 °C, 2 min; 4 °C, hold.

[0075] 2.3 Establishment of type I double-stranded nucleic acid detection probe and signal amplification system Using the DNA double-stranded target system and DNA-RNA complex target system prepared in 2.2 above as the targets to be measured in this example, dilute the double-stranded targets prepared in 2.2 above into a concentration gradient (specific concentrations are 5 pM, 10 pM, 15 pM, 50 pM, 100 pM) as the samples to be measured, and set a blank control (blank).

[0076] Take 2 μL of type I detection probe P1 with a concentration of 1 μM to make its concentration in the detection system 100 nM, add 2 μL of the sample to be measured, add λ exonuclease to make its concentration in the system 125 U / mL, and add the artificially synthesized specific substrate S1 to make its concentration in the detection system 500 nM. If the total volume of the system is less than 20 μL, supplement it with ddH2O. Place it in a real-time fluorescence detector for detection at 37 °C, set the detection time interval to 5 seconds per cycle, the number of cycles to 1440 times, and the detection duration to 120 minutes, to obtain the fluorescence intensity-time trajectory as shown in Figure 1 (F).

[0077] Take 2 μL of type I detection probe P2 with a concentration of 1 μM to make its concentration in the detection system 100 nM, add 2 μL of the sample to be measured, add λ exonuclease to make its concentration in the system 125 U / mL, and add the artificially synthesized specific substrate S2 to make its concentration in the detection system 500 nM. If the total volume of the system is less than 20 μL, supplement it with ddH2O. Place it in a real-time fluorescence detector for detection at 37 °C, set the detection time interval to 10 seconds per cycle, the number of cycles to 720 times, and the detection duration to 120 minutes, to obtain the fluorescence intensity-time trajectory as shown in Figure 1 (G).

[0078] The fluorescence value-time trajectory of the reaction process is captured and recorded by a real-time fluorescence instrument and output as a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0079] Figure 1 The experimental results in (F) and (G) show that when there is no double-stranded target in the sample to be measured, the fluorescence value remains at a low level with no obvious change; when there is a double-stranded target in the sample, the fluorescence value increases significantly over time. The higher the concentration of the double-stranded target in the sample to be measured, the greater the fluorescence value, showing a positive correlation between the concentration and the fluorescence signal. This indicates that this detection method has good sensitivity in identifying and quantifying double-stranded targets.

[0080] Example 2 1. Design of type II double-stranded nucleic acid detection probe and signal amplification system Based on the type I detection probe in Example 1, this example designs a type II double-stranded nucleic acid detection probe.

[0081] The type II double-stranded nucleic acid detection probe consists of one recognition strand R and n short Dz single strands, where n is a natural number from 2 to 10. In the type II detection probe, the molar ratio of the recognition strand R to Dz is 1:n, and in this example, n is taken as 2.

[0082] The modifying group is any one of a phosphate group, a carboxyl group, and an amino group. In this example, carboxylation modification is adopted.

[0083] After the type II double-stranded DNA probe in this example recognizes and accesses the double-stranded target, the recognition strand R is digested by λ exonuclease (λ exonuclease) and releases twice the amount of Dz. The Dz enters the cycle of the enzyme cleavage specific substrate S to generate and amplify the fluorescence signal.

[0084] This example specifically designs a type II double-stranded nucleic acid detection probe named P3: Detection probe P3: Used to detect the double-stranded target composed of T1 and T2 in Table 1, consisting of 1 recognition strand R3 (see Table 2) and two short single strands Dz1 (see Table 1). The short single strand Dz1 can cleave the enzyme cleavage specific substrate S1 (see Table 1), and the DNA enzyme used is the 10-23 DNA enzyme shown in (B) of Figure 1 After the detection probe P3 recognizes the double-stranded target, the recognition strand R3 is digested by λ exonuclease (λ exonuclease) and releases Dz1 that is twice that of the recognition strand R2. The Dz1 enters the cycle of the enzyme cleavage specific substrate S to generate and amplify the fluorescence signal.

[0085] The sequence of R3 is shown as SEQ ID No:11 in the sequence listing, with a length of 99 nucleotides. Its first position is modified with a carboxylic acid group. R3 is composed of a target binding domain (positions 1-45), two consecutive Dz binding domains (positions 46-72, 73-99) connected in the 5'-3' direction; wherein the target binding domain is reverse complementary to positions 21-65 of one strand T1 in the double-stranded target, and the two Dz binding domains are both reverse complementary to Dz1.

[0086] The sequences of the recognition strand R3, Dz1, the reporter molecule S1, and the double-stranded target -T (composed of T1 and T2) involved in this example are shown in Table 2.

[0087]

[0088] Note: COOH in Table 2 indicates carboxylation modification.

[0089] Figure 2(A) is a schematic diagram of the reaction of type II detection probe recognizing and processing double-stranded nucleic acid: the target binding domain of the recognition chain R recognizes T1 in the double-stranded target and binds to it to form a double-stranded DNA with a 5'-COOH end, and an enzymatic digestion reaction occurs under the action of λ nuclease, and the double-stranded DNA with a 5'-COOH end is digested, releasing 2 times the amount of Dz (10-23 DNA enzyme in this embodiment) of the recognition chain R.

[0090] The released Dz (10-23 DNA enzyme in this example) has deoxyribozyme activity in the presence of magnesium ions, and can cleave the substrate (specific substrate S, the substrate of 10-23 DNA enzyme is S1) to generate a fluorescent signal, and circulate to amplify the fluorescent signal.

[0091] 2. Preparation of type II double-stranded nucleic acid detection probe and signal amplification system 2.1 Preparation of type II double-stranded nucleic acid detection probe According to Table 2, single-stranded DNA was artificially synthesized and modified to obtain R3, and according to Table 1, single-stranded Dz1 was artificially synthesized and modified.

[0092] The reaction buffer environment was 1 × λ exonuclease buffer. At room temperature, R3 and Dz1 were added to synthesize the type I detection probe, and the remaining volume was supplemented with ddH2O to make the total volume of the system equal to 20 μL, the concentration of R2 was 1 μM, and the concentration of Dz was 2 μM. The annealing procedure was applied to ensure that R3 and Dz1 were correctly combined to obtain the type II double-stranded nucleic acid detection probe P3.

[0093] The annealing program was set as follows: 90 ℃, 2 min; 80 ℃, 2 min; 70 ℃, 2 min; 60 ℃, 2 min; 50 ℃, 2 min; 40 ℃, 2 min; 37 ℃, 2 min; 4 ℃, hold.

[0094] 2.2 Preparation of double-stranded targets The double-stranded target used is a double-stranded DNA target composed of T1 and T2. The preparation method is shown in 2.2 of Example 1.

[0095] 2.3 Establishment of type II double-stranded nucleic acid detection probe and signal amplification system The double-stranded target prepared in 2.2 above was used as the target to be tested in this example. The double-stranded target prepared in 2.2 above was diluted into a concentration gradient (specific concentrations were 500 fM, 1 pM, 10 pM, 50 pM, 100 pM, 200 pM, 500 pM, 1 nM) as the sample to be tested, and a blank control was set.

[0096] Take 2 μL of the type II double-stranded nucleic acid detection probe P3 with a concentration of 1 μM, so that its concentration in the detection system is 100 nM, add 2 μL of the sample to be tested, add λ exonuclease so that its concentration in the system is 125 U / mL, and add the artificially synthesized specific substrate S1 so that its concentration in the detection system is 500 nM. If the total volume of the system is less than 20 μL, supplement it with ddH2O. Place it in a real-time fluorescence detector at 37 °C for detection, set the detection time interval to one cycle every 10 seconds, the number of cycles is 1440 times, and the detection duration is 240 minutes to obtain the fluorescence intensity-time trajectory.

[0097] The fluorescence value-time trajectory of the reaction process is captured and recorded by a real-time fluorescence instrument and output as a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0098] Figure 2 The experimental results in (B) show that when there is no double-stranded target in the sample to be tested, the fluorescence value remains stable and there is no obvious upward change; when there is a double-stranded target in the sample, the fluorescence value increases significantly with time, showing the activity of the reaction. In addition, the higher the concentration of the double-stranded target in the sample to be tested, the higher the corresponding fluorescence value, further proving the positive correlation between the concentration and the fluorescence signal. This indicates that the detection technology has good sensitivity and specificity in identifying and quantifying double-stranded targets.

[0099] Example 3 1. Design of type I double-stranded nucleic acid detection probes modified with different groups In this example, based on the type I detection probe P1 in Example 1, type I double-stranded nucleic acid detection probes modified with different groups were designed. In this example, four type I detection probes named P1, P4, P5, and P6 were specifically designed according to the recognition strand R of different modification groups: The detection probe P1 is the detection probe P1 in Example 1 above.

[0100] The compositions of the detection probes P4, P5, and P6 are the same as that of the detection probe P1, and the difference lies in the modification groups of the recognition strand R. The 5'-end of R1 is carboxylated, the 5'-end of R4 is carboxyphosphorylated, the 5'-end of R5 is aminated, and the 5'-end of R6 is mercapto-modified.

[0101]

[0102] Note: COOH in Table 3 represents carboxylation modification (5'-carboxyl modification); P represents phosphorylation modification (5'-phosphorylation modification); NH2 represents amination modification (5'-amino modification), and HS represents mercapto-modification (5'-mercapto modification).

[0103] Figure 3 (A) of the figure is a schematic diagram of the reaction of the detection probe P recognizing and processing a double-stranded DNA target: As Figure 3 shown in (B) of the figure, the ends of the recognition strands R of the three probes are respectively modified with a carboxylic acid group, a phosphate group, an amino group, and a thiol group. In the presence of λ exonuclease, the target-binding domain of the recognition strand R recognizes T1 in the double-stranded target and binds to it to form a double-stranded DNA with a functional group modification at the 5'-end. An enzymatic digestion reaction occurs under the action of λ exonuclease, and the double-stranded DNA with a functional group modification at the 5'-end is digested, releasing Dz.

[0104] The released Dz1 has DNA enzyme activity in the presence of magnesium ions, can cleave the substrate S1 to generate a fluorescence signal, and the fluorescence signal is amplified through cycling.

[0105] 2. Preparation of Type I double-stranded nucleic acid detection probe and signal amplification system 2.1 Preparation of Type I detection probe Prepare Type I detection probes P (P1, P4, P5, P6): Synthesize and modify single-stranded DNA according to Table 3 to obtain R (R1, R4, R5, R6), and synthesize and modify single-stranded Dz1 according to Table 1.

[0106] The reaction buffer environment is 1 × λ exonuclease buffer. Under normal temperature conditions, add R (R1, R4, R5, R6) and Dz1 to synthesize Type I detection probe P (P1, P4, P5, P6), and the remaining volume is supplemented with ddH2O to make the total volume of the system equal to 20 μL. The concentration of R (R1, R4, R5, R6) is 1 μM, and the concentration of Dz1 is 1 μM. Apply an annealing program to ensure the correct binding of R (R1, R4, R5, R6) and Dz1 to obtain Type I double-stranded nucleic acid detection probe P1.

[0107] The annealing program is set as follows: 90 °C, 2 min; 80 °C, 2 min; 70 °C, 2 min; 60 °C, 2 min; 50 °C, 2 min; 40 °C, 2 min; 37 °C, 2 min; 4 °C, hold.

[0108] 2.2 Preparation of double-stranded target The double-stranded target used is a DNA double-stranded target composed of T1 and T2. The preparation method is shown in 2.2 of Example 1.

[0109] 2.3 Establishment of Type I double-stranded nucleic acid detection probe and signal amplification system Using the double-stranded target prepared in 2.2 above as the target to be measured in this example, dilute the double-stranded target prepared in 2.2 above to 5 nM as the sample to be measured, and set a blank control (blank).

[0110] The detection systems of the four kinds of probes modified with different functional groups were constructed as follows: Take 1 μL of the detection probe (P1, P4, P5 or P6) with a concentration of 1 μM, so that its concentration in the detection system is 50 nM, add 2 μL of the sample to be measured, and add λ exonuclease so that its concentration in the system is 125 U / mL. If the total volume of the system is less than 20 μL, supplement it with ddH2O. Place it in a real-time fluorescence detector at 37 °C for detection, set the detection time interval to 10 seconds per cycle, the number of cycles is 600 times, and the detection duration is 100 minutes, to obtain the fluorescence intensity-time trajectory as shown in Figure 3 (C) shown.

[0111] The fluorescence value-time trajectory of the reaction process was captured and recorded by a real-time fluorescence instrument, and output as a fluorescence intensity-time trajectory CSV (Comma-Separated Values, CSV) file.

[0112] When there is no double-stranded target in the sample to be measured, the fluorescence value does not change significantly; when there is a double-stranded target in the sample to be measured, the fluorescence values in the treatments with the four different functionalized group-modified probes all increase significantly with time, and the fluorescence value of the carboxylated modification is the highest.

[0113] Example 4 Detect the non-small cell lung cancer-related gene EGFR-L858R using a type I double-stranded nucleic acid detection probe.

[0114] This example is based on a type I double-stranded nucleic acid detection probe for detection, and the specific steps are as follows: 1. The specific detection probe used in this example is the type I double-stranded nucleic acid detection probe P7, which is composed of Dz1 (see Table 1) and R7 in a molar ratio of 1:1 for the non-small cell lung cancer-related gene EGFR-L858R. The specific sequence of R7 is shown in Table 4:

[0115] Note: COOH in Table 4 indicates carboxylation modification.

[0116] The target gene is EGFR-L858R. One strand of the double-stranded target on it has a length of 45 nucleotides, and the sequence is the same as the 1st to 45th positions of R7. The sequence of the other strand is reverse complementary to the 1st to 45th positions of R7.

[0117] The structure of probe P7 is as shown in Figure 1As shown in (C), it is composed of two DNA single strands, R7 and Dz1, and the molar ratio of R7 to Dz1 is 1:1.

[0118] The sequence of R7 is as shown in SEQ ID No:15 in the sequence listing, with a length of 72 nucleotides. Its first position is modified with a carboxylic acid group. R7 is formed by connecting a target binding domain (positions 1-45) and a Dz binding domain (positions 46-72) in the 5'-3' direction; wherein the target binding domain is reverse complementary to one strand of the double-stranded target of the target gene EGFR-L858R, and the Dz binding domain is reverse complementary to the Dz1.

[0119] According to Table 4, synthesize the required type I double-stranded nucleic acid detection probe P7.

[0120] 2. Add the test sample, exonuclease (λ exonuclease), and reporter molecule S1 to the probe system containing the above type I double-stranded nucleic acid detection probe P7 to form a detection system, and record the fluorescence intensity. 2.1 Preparation of type I double-stranded DNA specific detection probe Prepare the probe (20 μL): The reaction buffer environment is 1 × λ exonuclease buffer. Add the type I double-stranded nucleic acid detection probe P7 so that the concentration of each single-stranded DNA in the type I double-stranded nucleic acid detection probe P7 in the system is 1 μM. Make the total volume of the system equal to 20 μL, and the remaining volume is supplemented with ddH2O.

[0121] Apply the annealing program to ensure the correct binding of the oligonucleotide chains that make up the probe. The annealing program is set as follows: 90°C, 2 min; 85°C, 1 min; 80°C, 1 min; 75°C, 1 min; 70°C, 1 min; 65°C, 1 min; 60°C, 1 min; 55°C, 1 min; 50°C, 1 min; 45°C, 1 min; 40°C, 1 min; 37°C, 2 min; 16°C, 1 min; 4°C, hold.

[0122] The test samples used are as follows: Take tumor tissue samples from non-small cell lung cancer patients (from Tianjin Chest Hospital, 20 people) and normal tissue samples from the lungs of healthy people (from Tianjin Chest Hospital, 10 people) respectively, and use a nucleic acid extraction kit (Yeasen, MolPure ® Blood / Cell / Tissue / Bacteria DNA fast Kit) to extract tissue DNA. The genomic DNA obtained is the test sample from patients and the test sample from healthy people, and a blank control is set.

[0123] 2.2 Detection 20 μL detection system: Take 2 μL of the above-mentioned type I double-stranded nucleic acid detection probe P7 with a concentration of 1 μM, so that its concentration in the system is 100 nM, add 10 μL of the sample to be tested, add λ exonuclease so that its concentration in the system is 125 U / mL, and add the reporter molecule S1 so that its concentration in the system is 500 nM. If the total volume of the system is less than 20 μL, make up with ddH2O. Place it in a real-time fluorescence detector for detection at 37 °C, set the detection time interval to 10 seconds per cycle, with 900 cycles, and the detection duration is about 150 minutes to obtain the fluorescence intensity-time trajectory. When the target nucleic acid sequence does not exist in the detection target, the fluorescence value does not show an obvious upward change; if the fluorescence value increases significantly, it is considered that the target nucleic acid sequence exists in the detection target.

[0124] 2.3 Analyze the fluorescence intensity Analyze the fluorescence intensity recorded in the detection to determine whether there is a double-stranded DNA double-stranded target in the sample to be tested: Figure 4 The results show that if there is a double-stranded target in the sample to be tested, the fluorescence value increases significantly; if there is no double-stranded target in the sample to be tested, the fluorescence value does not show a significant upward change.

[0125] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A composition for detecting double-stranded nucleic acid, characterized in that, It includes a double-stranded nucleic acid detection probe, λ exonuclease, and a reporter molecule; The double-stranded nucleic acid detection probe is used to identify whether the target double-stranded nucleic acid named double-stranded target is contained in the sample to be tested; the double-stranded nucleic acid detection probe includes a single-stranded DNA named recognition strand R, and n single-stranded DNAs named Dz that are connected to the recognition strand R through base complementary pairing; the recognition strand R contains a target binding domain and n Dz binding domains that are adjacent to each other in sequence from the 5'-end to the 3'-end; the n Dz are combined with the recognition strand R through the n Dz binding domains to form a linear DNA complex; the target binding domain can bind to one strand of the double-stranded target; a functional group is modified at the 5'-end of the recognition strand R to achieve the binding of the double-stranded nucleic acid detection probe to λ exonuclease; the Dz is a deoxyribozyme that can cleave the reporter molecule, and the 5'-end of the Dz is modified to prevent the Dz from being degraded by λ exonuclease; The reporter molecule is a specific substrate of the Dz, and the reporter molecule is a compound labeled with a fluorescent group and a corresponding quenching group.

2. The composition according to claim 1, wherein: The deoxyribozyme is selected from any one of 10-23 DNAzyme, 8-17 DNAzyme, 17E DNAzyme, Mg5 DNAzyme, 39E DNAzyme, EtNa DNAzyme, Ce13d DNAzyme.

3. The composition according to claim 2, characterized in that: The core catalytic domain sequence of the 10-23 DNAzyme is shown as positions 2-16 of SEQ ID No:1 in the sequence listing, where the letter M represents A or C, the letter H represents A, C or T, the letter N represents A, C, G or T, and the letter D represents A, G or T; the core catalytic domain of the 8-17 DNAzyme is any one of 5'-NNNNAGYNNNNNCGNN-3', 5'-NNNNAGYNNNNCGNN-3', 5'-NNNNAGYNNNNNCGN-3', 5'-NNNNAGYNNNNCGN-3', where the letter N represents A, C, G or T, and the letter Y represents T or C.

4. The composition according to any one of claims 1-3, characterized in that: The n is a natural number from 1 to 10.

5. The composition according to any one of claims 1-3, characterized in that: The functional group is any one of a phosphate group, a carboxyl group, an amino group, and a mercapto group.

6. The composition according to any one of claims 1-3, characterized in that: The quenching group is BHQ1; the fluorescent group is FAM.

7. The composition according to any one of claims 1-3, characterized in that: The composition is any set of P1-P7: P1, a double-stranded formed by a single-stranded DNA with the sequence of SEQ ID No:8 and a single-stranded DNA with the sequence of SEQ ID No:9 or a single-stranded RNA with the sequence of SEQ ID No:10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by binding a single-stranded DNA with the sequence of SEQ ID No:2 and a single-stranded DNA with the sequence of SEQ ID No:4 through base complementary pairing, and the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No:6; P2. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 3 and a single-stranded DNA with the sequence of SEQ ID No: 5; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:

7. P3. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 11 and two single-stranded DNAs with the sequence of SEQ ID No: 4; the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:

6. P4. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 12 and a single-stranded DNA with the sequence of SEQ ID No: 4, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:

6. P5. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 13 and a single-stranded DNA with the sequence of SEQ ID No: 4, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:

6. P6. Using the double-strand composed of the single-stranded DNA with the sequence of SEQ ID No: 8 and the single-stranded DNA with the sequence of SEQ ID No: 9 or the single-stranded RNA with the sequence of SEQ ID No: 10 as the double-stranded target, the double-stranded nucleic acid detection probe is formed by the base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 14 and a single-stranded DNA with the sequence of SEQ ID No: 4, and the corresponding specific substrate is the DNA-RNA chimera with the sequence of SEQ ID No:

6. Using as the double-stranded target a double-stranded DNA composed of a single-stranded DNA with the 1st to 45th positions of SEQ ID No: 15 and a single-stranded DNA with a sequence reverse complementary to the 1st to 45th positions of SEQ ID No: 15, the double-stranded nucleic acid detection probe is formed by base complementary pairing of a single-stranded DNA with the sequence of SEQ ID No: 15 and a single-stranded DNA with the sequence of SEQ ID No: 4, and the corresponding specific substrate is a DNA-RNA chimera with the sequence of SEQ ID No:

6.

8. The double-stranded nucleic acid detection probe according to any one of claims 1-7.

9. A method for detecting whether a sample to be tested contains a target double-stranded nucleic acid, characterized in that, The method includes reacting the composition according to any one of claims 1-7 with a sample to be tested, detecting the fluorescence signal of the reaction system, and determining whether there is target double-stranded nucleic acid in the sample to be tested based on the fluorescence signal.

10. Application, characterized in that, The application is any one of Y1-Y3: Y1. The application of the composition according to any one of claims 1-7 in the preparation of a nucleic acid detection reagent; Y2. The application of the double-stranded nucleic acid detection probe according to claim 8 in the preparation of a nucleic acid detection reagent; Y3. The application of the method according to claim 9 in the preparation of a nucleic acid detection reagent.

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