Nucleic acid detection method based on sulfur-modified nucleic acid and sulfur-modified nucleic acid recognition protein

Abstract

The present application provides a nucleic acid detection method based on sulfur-modified nucleic acid and sulfur-modified nucleic acid recognition protein and application. Specifically, the present application provides a nucleic acid detection effector which is used in combination with sulfur-modified guide nucleic acid to realize nucleic acid detection function, the effector comprises a targeting element and a reporter element, wherein the targeting element and the reporter element form a fusion protein, the targeting element comprises a sulfur-modified nucleic acid recognition protein, and the reporter element is a restriction endonuclease or a fluorescent double-molecular complementation reporter system. The present application first combines sulfur-modified recognition protein with nucleic acid detection, and constructs a system for detecting by matching and combining sulfur-modified guide nucleic acid and target nucleic acid and combining sulfur-modified recognition protein. The present application has the advantages of non-invasiveness, specificity, high throughput and the like, can be completed within 1h, and the sensitivity reaches 1000 copies or less.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a system and method for detecting target nucleic acids, and more particularly to a nucleic acid detection method based on sulfur-modified nucleic acids and sulfur-modified nucleic acid recognition proteins and its application. Background Technology

[0002] Nucleic acid testing is suitable for pathogen detection, cancer screening, single nucleotide polymorphism (SNP) analysis, and bacterial resistance gene screening, and can be widely applied in molecular medicine diagnostics, environmental microbiology monitoring, and food safety. In recent years, safety issues caused by pathogenic microorganisms have become increasingly widespread and serious, posing a significant threat to human health and property. Accurate and rapid detection of pathogenic microorganisms within a short timeframe is crucial for food safety monitoring and early diagnosis.

[0003] Currently, traditional nucleic acid detection technologies based on polymerase chain reaction (PCR) remain important methods, including qPCR and DNA sequencing. DNA sequencing offers high accuracy but suffers from drawbacks such as high cost and long processing time. qPCR is not only a rapid and highly sensitive detection method but also allows for quantitative analysis; however, it still suffers from low accuracy. Furthermore, both qPCR and DNA sequencing technologies are dependent on specialized laboratory equipment, limiting their widespread use in primary care laboratories.

[0004] In recent years, nucleic acid isothermal amplification technology has shown promising applications in detection. Nucleic acid sequence-based amplification (NASBA) is a technique for amplifying RNA targets at 41°C and can be combined with electrochemiluminescence immunoassay for detection. Rolling circle amplification (RCA) uses single-stranded circular oligonucleotides as templates for linear synthesis, ultimately generating a linear single-stranded DNA or RNA containing a repetitive target sequence. Loop-mediated isothermal amplification (LAMP) uses 4-6 primers at 65°C to rapidly synthesize the target sequence using strand displacement DNA polymerase. Adding reverse transcriptase or using a polymerase with reverse transcription activity simultaneously can amplify RNA, i.e., RT-LAMP. Recombinase polymerase amplification (RPA) mimics the in vivo replication mechanism, using recombinase, single-strand binding proteins, and DNA polymerase at 37°C to achieve exponential amplification of the target sequence. The reaction does not require an annealing process and offers high sensitivity and specificity. The nicking endonuclease amplification reaction (NEAR) technology utilizes nicking enzymes to recognize specific short sequences of viral nucleic acids and synthesize single-stranded target sequences at 56°C.

[0005] Recently, nucleic acid detection technologies that combine isothermal amplification with clustered regularly interspaced short palindromic repeats (CRISPR / Cas) and related proteins have opened up new avenues for establishing rapid and effective detection methods. In 2017, Zhang Feng's team published a novel nucleic acid detection system, SHERLOCK, in *Science*. In this system, researchers utilized the property of Cas13a to cleave other RNAs after binding to a specific target RNA, and designed an RNA fluorescence reporter system using RPA. This technology can rapidly detect pathogens such as Zika virus under field (non-laboratory) conditions, but the instability of RNA increases the operational difficulty. In 2018, Doudna's team utilized the activity of Cas12a protein to non-specifically cleave single-stranded DNA after cleaving target dsDNA, and combined it with RPA to develop the DETECTR technology. However, the CRISPR system suffers from off-target problems, leading to false positives; furthermore, this system is limited by PAM sequences when selecting target DNA regions.

[0006] Therefore, there is an urgent need in this field to develop a nucleic acid detection technology that is highly specific, highly sensitive, has no sequence limitations, does not require complex instruments, and can be used in a variety of scenarios. Summary of the Invention

[0007] The purpose of this invention is to provide a nucleic acid detection method based on sulfur-modified nucleic acids and sulfur-modified nucleic acid recognition proteins, and its application.

[0008] In a first aspect, the present invention provides a nucleic acid detection effector comprising a targeting element and a reporter element, wherein the targeting element and the reporter element form a fusion protein, and wherein the targeting element comprises a sulfur-modified nucleic acid recognition protein.

[0009] In another preferred embodiment, the nucleic acid detection effector, guided by a sulfur-modified guide nucleic acid, can bind to a predetermined region of the nucleic acid to be detected and generate a reporting signal by contacting a molecular beacon through a reporting element.

[0010] In another preferred embodiment, the sulfur-modified guide nucleic acid and the nucleic acid to be detected form a double-stranded complementary structure within a predetermined region.

[0011] In another preferred embodiment, the sulfur-modified guide nucleic acid is a single-stranded phosphorus-thioyl modified nucleic acid.

[0012] In another preferred embodiment, the sulfur-modified guide nucleic acid comprises phosphorus-thioylated modified gDNA or gRNA.

[0013] In another preferred embodiment, the sequence length of the gDNA or gRNA is greater than 10 nt and less than 100 nt, and contains at least one sulfur-modified nucleotide.

[0014] In another preferred embodiment, the sequence length of the gDNA or gRNA is 10-30 nt.

[0015] In another preferred embodiment, the sulfur-modified guide nucleic acid may also have other chemical modifications in addition to sulfur modification, the chemical modifications being selected from the group consisting of: biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, locked nucleic acid (LNA) or combinations thereof.

[0016] In another preferred embodiment, the nucleic acid detection effector is a single-stranded nucleic acid detection effector.

[0017] In another preferred embodiment, the single-stranded nucleic acid is single-stranded DNA, single-stranded RNA, or a combination thereof.

[0018] In another preferred embodiment, the sulfur-modified nucleic acid recognition protein is a sulfur-binding domain protein (SBD) that specifically recognizes sulfur-modified nucleic acids.

[0019] In another preferred embodiment, the sulfur-modified nucleic acid refers to an OH (O or O) group in the phosphate group of the nucleic acid. - ) by SH (or S - The ) replaced by or a =0 was replaced by =S.

[0020] In another preferred embodiment, the sulfur-modified nucleic acid refers to the nucleic acid... Modified as Or its corresponding isomer.

[0021] In another preferred embodiment, the sulfur-modified nucleotide may be a single nucleotide.

[0022] In another preferred embodiment, the sulfur-modified nucleotide can be N consecutive nucleotides (1 ≤ N ≤ 10).

[0023] In another preferred embodiment, the sulfur-modified nucleotide is preferably C or G.

[0024] In another preferred embodiment, the sulfur-binding domain protein (SBD) binds to DNA / DNA double strands or DNA / RNA hybrid double strands.

[0025] In another preferred embodiment, the sulfur-binding domain protein (SBD) includes: SBDgsu, SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu, or an SBD homolog.

[0026] In another preferred embodiment, the sulfur-binding domain protein (SBD) is derived from species selected from the group consisting of GsuMcrA, MMoMcrA, ScoMcrA, SprMcrA, HgaMcrA, and EcoMcrA; preferably, it is derived from GsuMcrA.

[0027] In another preferred embodiment, the SBD homologous protein may bind to a sulfur-modified oligonucleotide and has one or more of the following conditions:

[0028] C1) A protein domain that has sulfur-modified oligonucleotide (including nucleotides such as DNA or RNA) binding activity;

[0029] C2) has an amino acid sequence identity of ≥20% with any one of the proteins SBDgsu, SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, and SBDtcu (preferably ≥50%, more preferably ≥70%, most preferably ≥80% or ≥90%).

[0030] C3) contains PLW motifs or ALW motifs (e.g., P86-L90-W96 in SBDgsu; P75-L79-W85 in SBDmmo; P79-L83-W89 in SBDspr; A73-L77-W83 in SBDtcu).

[0031] In another preferred embodiment, the reporting element includes a monomolecular reporting element and a bimolecular complementary reporting element.

[0032] In another preferred embodiment, the single-molecule reporter element is a restriction endonuclease.

[0033] In another preferred embodiment, the restriction endonuclease specifically recognizes a specific DNA sequence; preferably, the restriction endonuclease is BclI, which specifically recognizes the DNA sequence 5'-TGATCA-3' / 5'-TGATCA-3'.

[0034] In another preferred embodiment, the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system.

[0035] In another preferred embodiment, the fluorescent bimolecular complementary reporter system includes (but is not limited to): Fluc-N and Fluc-C, LgBiT and SmBiT, LacA and LacB.

[0036] In another preferred embodiment, the reporting element is selected from the group consisting of restriction endonucleases, fluorescent bimolecular complementary reporter systems, or combinations thereof.

[0037] In another preferred embodiment, the molecular beacon is selected from the group consisting of double-stranded DNA, luciferase, or combinations thereof.

[0038] In another preferred embodiment, the double-stranded DNA contains a sequence that can be specifically recognized by restriction endonucleases.

[0039] In another preferred embodiment, the double-stranded DNA contains a sequence that can be specifically recognized by the BclI enzyme, namely the DNA sequence 5'-TGATCA-3' / 5'-TGATCA-3'.

[0040] In another preferred embodiment, the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0041] In another preferred embodiment, the fluorescent group includes (but is not limited to): fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), tetrachlorofluorescein (TET), and hexachlorofluorescein (HEX); preferably, the fluorescent group is FAM.

[0042] In another preferred embodiment, the quenching group includes (but is not limited to): BHQ-1, BHQ-2, 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL); preferably, the quenching group is BHQ-1.

[0043] In another preferred embodiment, the targeting element and / or the reporter element are each independently a corresponding element of the nucleic acid detection effector prepared in the embodiments.

[0044] In another preferred embodiment, the targeting element and the reporter element are directly linked by chemical bonds (such as peptide bonds) or connected by a connector.

[0045] In another preferred embodiment, the joint includes a flexible joint and a non-flexible joint.

[0046] In another preferred embodiment, the connector is selected from the group (GGGGS)n, where n is a positive integer from 1 to 4, and preferably n is a positive integer from 2 to 3.

[0047] In another preferred embodiment, the fusion protein contains one or more SBDs.

[0048] In another preferred embodiment, the plurality of SBDs may be the same or different.

[0049] In another preferred embodiment, the plurality of SBDs may be located on the same side (e.g., left or right) of the reporting element, or on both sides of the reporting element.

[0050] In another preferred embodiment, the structure of the fusion protein is shown in formula I, I', or I”:

[0051] ALB(I)

[0052] BLA(I')

[0053] ALBLA(I”)

[0054] In the formula,

[0055] A represents the target element SBD;

[0056] B is the reporting element;

[0057] L can be either absent or linked peptides independently;

[0058] Each "-" represents a chemical bond independently.

[0059] In another preferred embodiment, the chemical bond includes a peptide bond or a covalent bond.

[0060] In another preferred embodiment, the sequence of the fusion protein is shown in SEQ ID No:6, SEQ ID No:32 or SEQ ID No:33.

[0061] A second aspect of the present invention provides a method for nucleic acid detection, comprising the steps of:

[0062] (i) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector as described in the first aspect of the invention forms a "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex with the nucleic acid to be detected; and

[0063] (ii) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal.

[0064] In another preferred embodiment, the nucleic acid detection effector includes a targeting element SBD and a reporter element.

[0065] In another preferred embodiment, the SBD is selected from the group consisting of SBD proteins or combinations thereof that share ≥20% amino acid sequence identity with any one of SBDgsu, SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, and SBDtcu; preferably, the SBD is SBDgsu.

[0066] In another preferred embodiment, the reporting element includes a monomolecular reporting element and a bimolecular complementary reporting element.

[0067] In another preferred embodiment, the single-molecule reporter element is a restriction endonuclease; preferably, the restriction endonuclease is BclI.

[0068] In another preferred embodiment, the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system.

[0069] In another preferred embodiment, the sulfur-modified guide nucleic acid is a sulfur-modified single-stranded gDNA or gRNA.

[0070] In another preferred embodiment, the sulfur-modified guide nucleic acid may also have other chemical modifications in addition to sulfur modification, including (but not limited to) biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, and locked nucleic acid (LNA); preferably, the sulfur-modified guide nucleic acid is also biotin-modified.

[0071] In another preferred embodiment, the nucleic acid to be detected is selected from the group consisting of single-stranded DNA, single-stranded RNA, or combinations thereof.

[0072] In another preferred embodiment, the nucleic acid to be detected is enriched in large quantities by isothermal amplification.

[0073] In another preferred embodiment, the isothermal amplification includes the following methods: nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and nicking endonuclease amplification reaction (NEAR).

[0074] In another preferred embodiment, the molecular beacon is selected from the group consisting of double-stranded DNA, luciferase, or combinations thereof.

[0075] In another preferred embodiment, the molecular beacon is double-stranded DNA.

[0076] In another preferred embodiment, the double-stranded DNA contains a sequence that can be specifically recognized by restriction endonucleases.

[0077] In another preferred embodiment, the double-stranded DNA contains a sequence that can be specifically recognized by the BclI enzyme, namely the DNA sequence 5'-TGATCA-3' / 5'-TGATCA-3'.

[0078] In another preferred embodiment, the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0079] In another preferred embodiment, the fluorescent group includes (but is not limited to) fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), tetrachlorofluorescein (TET), and hexachlorofluorescein (HEX); preferably, the fluorescent group is FAM.

[0080] In another preferred embodiment, the quenching group includes (but is not limited to) BHQ-1, BHQ-2, and 4-(4-oxaneaminophenylazo)benzoic acid (DABCYL); preferably, the quenching group is BHQ-1.

[0081] In another preferred embodiment, the method includes the following steps:

[0082] (1) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector and the nucleic acid to be detected form a complex of “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected”;

[0083] (2) Using magnetic beads, the “nucleic acid detection effector-sulfur-modified guide nucleic acid-detectable nucleic acid” complex is separated from the detectable nucleic acid that has not formed a complex; and

[0084] (3) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal;

[0085] The nucleic acid detection effector contains a targeting element SBD and a reporter element, wherein the reporter element is a single-molecule reporter element, and the single-molecule reporter element is a restriction endonuclease;

[0086] The sulfur-modified guide nucleic acid, in addition to sulfur modification, also has other chemical modifications, including (but not limited to) biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, and locked nucleic acid (LNA); preferably, the sulfur-modified guide nucleic acid also has biotin modification.

[0087] The molecular beacon is a double-stranded DNA containing a sequence that can be specifically recognized by restriction endonucleases. Furthermore, the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0088] In another preferred embodiment, the method includes the following steps:

[0089] (1) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector and the nucleic acid to be detected form a complex of “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected”;

[0090] (2) Optionally, magnetic beads are used to separate the “nucleic acid detection effector-sulfur-modified guide nucleic acid-detectable nucleic acid” complex from the detectable nucleic acid that has not formed a complex; and

[0091] (3) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal;

[0092] The nucleic acid detection effector includes a targeting element (SBD) and a reporter element, wherein the reporter element is a bimolecular complementary reporter element, and the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system;

[0093] In addition to sulfur modification, the sulfur-modified guide nucleic acid may optionally also have other chemical modifications, including (but not limited to) biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, and locked nucleic acid (LNA).

[0094] The molecular beacon is luciferase.

[0095] In another preferred embodiment, the magnetic beads are labeled magnetic beads, and the magnetic beads bind to sulfur-modified guide nucleic acids with other chemical modifications through the label, thereby separating the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex from the nucleic acid to be detected that has not formed a complex;

[0096] The labeling includes (but is not limited to) streptavidin labeling, thiol labeling, carboxyl labeling, polystyrene coating labeling, alkynyl labeling, azide labeling, epoxy labeling, and hydroxyl labeling; preferably, the magnetic beads are streptavidin-labeled magnetic beads.

[0097] A third aspect of the present invention provides a reaction system for nucleic acid detection, the reaction system comprising:

[0098] (a) The nucleic acid detection effector as described in the first aspect of the present invention;

[0099] (b) A sulfur-modified guide nucleic acid, wherein the sulfur-modified guide nucleic acid guides the nucleic acid detection effector to specifically bind to a predetermined region of the nucleic acid to be detected; and

[0100] (c) A molecular beacon that generates a reporting signal upon contact with the nucleic acid detection effector.

[0101] In another preferred embodiment, the detection system further includes: (d) magnetic beads, which are used to separate the nucleic acid to be detected from the nucleic acid that has not formed a complex, namely, the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex.

[0102] In another preferred embodiment, the detection system further includes: (e) a buffer solution.

[0103] In another preferred embodiment, the nucleic acid to be detected is single-stranded DNA, single-stranded RNA, or a combination thereof.

[0104] In another preferred embodiment, the nucleic acid to be detected is enriched in large quantities by isothermal amplification.

[0105] In another preferred embodiment, the nucleic acid to be detected includes nucleic acids derived from the group consisting of: plants, animals, insects, microorganisms, viruses, environmental samples, clinical samples, or combinations thereof.

[0106] In another preferred embodiment, the nucleic acid to be detected is either artificially synthesized or naturally occurring DNA.

[0107] In another preferred embodiment, the nucleic acid to be detected includes wild-type or mutant DNA.

[0108] In another preferred embodiment, the concentration of the nucleic acid to be detected in the reaction system is 1 × 10⁻⁶. 3 Up to 1×10 8 Copies / µL, preferably 1×10 4 Up to 1×10 7 Copy / microliter.

[0109] In another preferred embodiment, the concentration of the sulfur-modified guide nucleic acid in the detection system is 0.1-10 nM, more preferably 1-10 nM, and even more preferably 1-5 nM.

[0110] In another preferred embodiment, the sulfur-modified guide nucleic acid is sulfur-modified gDNA or gRNA.

[0111] In another preferred embodiment, the sulfur-modified guide nucleic acid may have other chemical modifications in addition to sulfur modification, the chemical modifications being selected from the group consisting of: biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, locked nucleic acid (LNA) or combinations thereof.

[0112] In another preferred embodiment, the length of the gDNA or gRNA is 10-100 nt, more preferably 10-30 nt.

[0113] In another preferred embodiment, the concentration of the molecular beacon in the reaction system is 160 to 2560 nM, more preferably 320 to 1280 nM, and even more preferably 640 nM.

[0114] In another preferred embodiment, the molecular beacon is double-stranded DNA or luciferase.

[0115] In another preferred embodiment, the amount of magnetic beads used is 20 to 400 μg; preferably, the amount used is 100 μg.

[0116] In another preferred embodiment, the magnetic beads are labeled magnetic beads, and the magnetic beads bind to sulfur-modified guide nucleic acids with other chemical modifications through the label, thereby separating the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex from the nucleic acid to be detected that has not formed a complex;

[0117] The labeling includes (but is not limited to) streptavidin labeling, thiol labeling, carboxyl labeling, polystyrene coating labeling, alkynyl labeling, azide labeling, epoxy labeling, and hydroxyl labeling; preferably, the magnetic beads are streptavidin-labeled magnetic beads.

[0118] In another preferred embodiment, the detection system includes:

[0119] (a1) A nucleic acid detection effector; the nucleic acid detection effector comprises a targeting element SBD and a single-molecule reporter element restriction endonuclease;

[0120] (b1) Sulfur-modified guide nucleic acids;

[0121] (c1) A molecular beacon, wherein the molecular beacon is a double-stranded DNA containing a sequence specifically recognized by restriction endonucleases, and wherein the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end; and

[0122] (d1) Magnetic bead.

[0123] In another preferred embodiment, the detection system includes:

[0124] (a2) Nucleic acid detection effector; the nucleic acid detection effector includes a targeting element SBD and a bimolecular complementary reporter element, wherein the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system;

[0125] (b2) Sulfur-modified guide nucleic acids; and

[0126] (c2) Molecular beacon, wherein the molecular beacon is luciferase.

[0127] In a fourth aspect, the present invention provides a nucleic acid that encodes a nucleic acid detection effector as described in the first aspect of the present invention.

[0128] In another preferred embodiment, the nucleic acid is a linear sequence.

[0129] In another preferred embodiment, the nucleic acid has a 5'-3' (5' to 3') formula II or II' structure:

[0130] P1-X1-L1-X2(II)

[0131] P1-X2-L1-X1(II');

[0132] In the formula, P1 is the first promoter sequence;

[0133] X1 is the coding sequence for a sulfur-binding domain protein (SBD);

[0134] L1 is the encoded sequence without any OR connections;

[0135] X2 is the encoding sequence of the reporting element;

[0136] Furthermore, each "-" is an independent chemical bond.

[0137] In another preferred embodiment, the nucleic acid construct contains one or more SBD coding sequences.

[0138] In another preferred embodiment, the plurality of SBD encoded sequences may be the same or different.

[0139] In another preferred embodiment, the plurality of SBD encoded sequences may be located on the same side (e.g., left or right) of the report element encoded sequence, or on both sides of the report element encoded sequence.

[0140] In a fifth aspect, the present invention provides a carrier containing nucleic acid as described in the fourth aspect of the present invention.

[0141] In another preferred embodiment, the vector includes an expression vector.

[0142] In another preferred embodiment, the vector includes a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0143] In a sixth aspect, the present invention provides a genetically engineered cell containing a vector as described in the fifth aspect of the present invention, or whose genome integrates nucleic acids as described in the fourth aspect of the present invention.

[0144] In another preferred embodiment, the cells are transformed or transfected by the vector or nucleic acid.

[0145] In another preferred embodiment, the cells are further transfected with sulfur-modified gDNA or gRNA or their expression vector.

[0146] In another preferred embodiment, the cells include eukaryotic cells and prokaryotic cells.

[0147] In another preferred embodiment, the cells include bacteria, yeast, mammalian cells, and plant cells.

[0148] A seventh aspect of the present invention provides a kit comprising:

[0149] (C1) A first container, and a nucleic acid detection effector of the first aspect of the present invention, or its coding sequence, or a vector containing the coding sequence, located in the first container;

[0150] (C2) A second container, and a sulfur-modified guide nucleic acid located within the second container; and

[0151] (C3) The third container, and the molecular beacon located in the third container;

[0152] The nucleic acid detection effector, guided by sulfur-modified guide nucleic acid, can bind to a predetermined nucleic acid region to form a "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex. After the complex comes into contact with the molecular beacon, it generates a detectable reporting signal.

[0153] In another preferred embodiment, the sulfur-modified guide nucleic acid includes sulfur-modified gDNA, gRNA, or a combination thereof.

[0154] In another preferred embodiment, the sulfur-modified guide nucleic acid may also have other chemical modifications in addition to sulfur modification, the chemical modifications being selected from the group consisting of: biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, locked nucleic acid (LNA) or combinations thereof.

[0155] In another preferred embodiment, the molecular beacon comprises double-stranded DNA or luciferase.

[0156] In another preferred embodiment, the kit further comprises: (C4) a fourth container, and magnetic beads located in the fourth container, the magnetic beads being labeled magnetic beads, and the magnetic beads binding to sulfur-modified guide nucleic acids with other chemical modifications via the label, thereby separating the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex from the nucleic acid to be detected that has not formed a complex.

[0157] In another preferred embodiment, the nucleic acid detection effector comprises an SBD and a restriction endonuclease; and the molecular beacon is a double-stranded DNA containing a sequence that can be specifically recognized by the restriction endonuclease, and the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0158] In another preferred embodiment, the nucleic acid detection effector comprises an SBD and a fluorescent bimolecular complementary reporter system; while the molecular beacon is a luciferase.

[0159] An eighth aspect of the present invention provides the use of a nucleic acid detection effector as described in the first aspect of the present invention for preparing reagents or kits for nucleic acid detection.

[0160] In another preferred embodiment, the nucleic acid is single-stranded DNA, single-stranded RNA, or a combination thereof.

[0161] In another preferred embodiment, the nucleic acid includes nucleic acids to be detected derived from a group selected from: plants, animals, insects, microorganisms, viruses, environmental samples, clinical samples, or combinations thereof.

[0162] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0163] Figure 1 The amino acid sequence alignment of SBD homologous proteins is shown; the triangular annotations indicate PLW or ALW motifs.

[0164] Figure 2 The chemical structure of sulfur-modified DNA is shown.

[0165] Figure 3 The binding activity of the sulfur-modified DNA-binding domain to sulfur-modified nucleic acids was demonstrated.

[0166] Figure 4 The SBDgsu-BclI cleavage activity was verified.

[0167] Figure 5 A schematic diagram of the process for nucleic acid detection using the SBDgsu-BclI fusion protein is shown.

[0168] Figure 6 The single-stranded DNA in the detection system using the SBDgsu-BclI fusion protein is shown.

[0169] Figure 7 This demonstrates the use of NEAR amplification technology coupled with the SBDgsu-BclI detection system for the detection of single-stranded DNA.

[0170] Figure 8 This demonstrates the use of NEAR amplification technology coupled with the SBDgsu-BclI detection system for detecting double-stranded DNA.

[0171] Figure 9 This demonstrates the use of NEAR amplification technology coupled with the SBDgsu-BclI detection system for detecting Candida albicans genomic DNA.

[0172] Figure 10 A schematic diagram of the process for nucleic acid detection using the SBDgsu-FlucN and SBDgsu-FlucC fusion proteins is shown.

[0173] Figure 11 The results show the detection of single-stranded DNA in the system using the SBDgsu-FlucN and SBDgsu-FlucC fusion proteins.

[0174] Figure 12 This demonstrates the use of RPA amplification technology coupled with the SBDgsu-BclI detection system for the detection of single-stranded DNA.

[0175] Figure 13 This demonstrates the use of RPA amplification technology coupled with the SBDgsu-BclI detection system for the detection of Candida albicans DNA. Detailed Implementation

[0176] Through extensive and in-depth research, focusing on the recognition domains of sulfur-modified DNA, the inventors have developed for the first time a target nucleic acid detection method based on sulfur-modified nucleic acids and their recognition proteins. They have also provided a detection system and kit containing the nucleic acid detection effectors required to execute the method. Experimental results show that the method of this invention successfully completes detection within one hour, with a sensitivity of up to 1000 copies. Based on this, the present invention was completed.

[0177] the term

[0178] As used herein, the term "sulfur modification" may also be referred to as "phosphothioylation modification," which refers to the modification of the sulfur modification guide nucleic acid of the nucleic acid detection effector described herein; specifically, it refers to the modification that occurs when a non-bridging oxygen atom on the DNA or RNA phosphate backbone is replaced by a sulfur atom.

[0179] As used in this article, the terms “sulfur-modified nucleic acid recognition protein,” “sulfur-binding domain protein,” and “SBD” are used interchangeably and all refer to proteins that can specifically recognize sulfur-modified nucleic acids.

[0180] As used herein, the terms "sulfur-modified nucleic acid" or "sulfur-modified nucleic acid" refer to nucleic acids that are modified with sulfur atoms; wherein the nucleic acid can be RNA or DNA.

[0181] As used herein, the term "gRNA" or "gDNA" refers to a single-stranded nucleic acid sequence that can pair complementaryly with the bases of the nucleic acid to be detected, containing sulfur modifications at one or more sites to guide the SBD to the nucleic acid to be detected. In a preferred embodiment of the invention, the "gRNA" or "gDNA" also has other chemical modifications, such as biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, locked nucleic acid (LNA), etc.

[0182] As used herein, the term "molecular beacon" refers to a molecule that can interact (contact) with the reporter element in the nucleic acid detection effector of the present invention to generate a detectable report signal.

[0183] In a preferred embodiment of the invention, the molecular beacon is a double-stranded DNA, comprising a sequence specifically recognizable by a reporter element (restriction endonuclease) in a nucleic acid detection effector. Further, the 5' and 3' ends of the double-stranded DNA are labeled with a fluorescent group and a quencher group, respectively. When the double-stranded DNA molecule comes into contact with and is specifically recognized by the nucleic acid detection effector, the double-stranded DNA molecule is cleaved, causing the fluorescent and quencher groups originally labeled at the 5' and 3' ends to move away from each other, thereby emitting a detectable fluorescent signal.

[0184] In another preferred embodiment of the invention, the molecular beacon is a luciferase. When the luciferase comes into contact with a nucleic acid detection effector (containing a fluorescent bimolecular complementary reporter system as a reporter element), a detectable fluorescent signal is generated.

[0185] Nucleic acid detection effector

[0186] In one aspect of the invention, a nucleic acid detection effector is provided. As used herein, the terms "nucleic acid detection effector of the present invention" and "fusion protein of the present invention" are used interchangeably and both refer to the nucleic acid detection effector described in the first aspect of the invention, i.e., a fusion protein comprising at least one targeting element and a reporter element, wherein the targeting element comprises a sulfur-modified nucleic acid recognition protein, and the reporter element comprises a single-molecule reporter element and a bimolecular complementary reporter element; wherein, guided by a sulfur-modified guide nucleic acid, the nucleic acid detection effector can bind to a predetermined region of the nucleic acid to be detected and generate a reporting signal by contacting a molecular beacon through the reporter element.

[0187] In a preferred embodiment of the present invention, the nucleic acid detection effector includes an SBD and a single-molecule reporter element. The single-molecule reporter element can function independently and generate a reporter signal after contacting a molecular beacon, without the assistance of other molecules. The single-molecule reporter element can be a restriction endonuclease. In one embodiment of the present invention, a fusion protein SBDgsu-BclI comprising an SBD derived from GsuMcrA, namely SBDgsu, and the restriction endonuclease BclI is constructed as a nucleic acid detection effector.

[0188] In another preferred embodiment of the present invention, the nucleic acid detection effector includes an SBD and a bimolecular complementary reporter element, wherein the bimolecular complementary reporter element is linked to one SBD or simultaneously to at least one SBD protein. The two molecules in the bimolecular complementary reporter element cooperate with each other to generate a reporter signal upon contact with a molecular beacon. In one embodiment of the present invention, a fusion protein comprising an SBD derived from GsuMcrA, namely SBDgsu, and fluorescent complementary bimolecules Fluc-N and Fluc-C is constructed, with SBDgsu-FlucN and SBDgsu-FlucC serving as the nucleic acid detection effector.

[0189] Sulfide-binding domain protein (SBD)

[0190] Sulfur-binding domain protein, abbreviated as SBD. In this invention, a fusion protein was constructed, with the SBD serving as a targeting element to deliver the nucleic acid detection effector of this invention to a predetermined region of the nucleic acid to be detected.

[0191] SBDs can be located at the N-terminus or C-terminus of the fusion protein. Multiple SBD domains can also exist within the fusion protein, such as in the form of SBD-reporter element-SBD, to improve detection efficiency. Furthermore, SBDs and reporter elements can be arranged in various combinations, such as SBD-SBD-reporter element, reporter element-SBD-SBD, reporter element-SBD-reporter element, etc.

[0192] In this invention, representative sulfur-binding domain proteins (SBDs) include: SBDgsu, SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu, or SBD homologs.

[0193] Amino acid sequence alignment results of some representative SBD homologs are shown below. Figure 1 .

[0194] sulfur-modified nucleic acids

[0195] In this invention, the sulfur-modified nucleic acid refers to a nucleic acid containing one OH (O or O) group in its phosphate group. - ) by SH (or S- The ) replaced by or a =0 was replaced by =S.

[0196] In another preferred embodiment, the sulfur-modified nucleic acid refers to the nucleic acid... Modified as Or its corresponding isomer.

[0197] The structure of a representative sulfur-modified nucleic acid is as follows: Figure 2 As shown.

[0198] The binding activity of sulfur-modified nucleic acid binding domains to certain sulfur-modified nucleic acids, such as... Figure 3 As shown.

[0199] The nucleic acid detection method, detection system, and reagent kit of the present invention

[0200] The present invention also provides a method, detection system, and reagent kit for nucleic acid detection using the nucleic acid detection effector of the present invention.

[0201] In general, the nucleic acid detection method of the present invention includes the steps of: (i) in the presence of sulfur-modified guide nucleic acid, causing the nucleic acid detection effector described in the first aspect of the present invention to form a "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex with the nucleic acid to be detected; and (ii) contacting the molecular beacon with the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex and detecting a report signal.

[0202] In a preferred embodiment of the present invention, the method includes the following steps:

[0203] (1) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector and the nucleic acid to be detected form a complex of “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected”;

[0204] (2) Using magnetic beads, the “nucleic acid detection effector-sulfur-modified guide nucleic acid-detectable nucleic acid” complex is separated from the detectable nucleic acid that has not formed a complex; and

[0205] (3) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal;

[0206] The nucleic acid detection effector contains a targeting element SBD and a reporter element, wherein the reporter element is a single-molecule reporter element, and the single-molecule reporter element is a restriction endonuclease;

[0207] The sulfur-modified guide nucleic acid, in addition to sulfur modification, also has other chemical modifications, including (but not limited to) biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, and locked nucleic acid (LNA); preferably, the sulfur-modified guide nucleic acid also has biotin modification.

[0208] The molecular beacon is a double-stranded DNA containing a sequence that can be specifically recognized by restriction endonucleases. Furthermore, the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0209] When using this detection method, a detection system containing the following components or a kit containing the following components should be used:

[0210] (a1) A nucleic acid detection effector; the nucleic acid detection effector comprises a targeting element SBD and a single-molecule reporter element restriction endonuclease;

[0211] (b1) Sulfur-modified guide nucleic acids; and

[0212] (c1) A molecular beacon, wherein the molecular beacon is a double-stranded DNA containing a sequence specifically recognizable by restriction endonucleases, and the double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end; and

[0213] (d1) Magnetic beads, wherein the magnetic beads are labeled magnetic beads, and the magnetic beads bind to sulfur-modified guide nucleic acids with other chemical modifications through the label, thereby separating the "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex from the nucleic acid to be detected that has not formed a complex;

[0214] The labeling includes (but is not limited to) streptavidin labeling, thiol labeling, carboxyl labeling, polystyrene coating labeling, alkynyl labeling, azide labeling, epoxy labeling, and hydroxyl labeling; preferably, the magnetic beads are streptavidin-labeled magnetic beads.

[0215] In another preferred embodiment of the present invention, the method includes the following steps:

[0216] (1) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector and the nucleic acid to be detected form a complex of “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected”;

[0217] (2) Optionally, magnetic beads are used to separate the “nucleic acid detection effector-sulfur-modified guide nucleic acid-detectable nucleic acid” complex from the detectable nucleic acid that has not formed a complex; and

[0218] (3) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal;

[0219] The nucleic acid detection effector includes a targeting element (SBD) and a reporter element, wherein the reporter element is a bimolecular complementary reporter element, and the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system;

[0220] In addition to sulfur modification, the sulfur-modified guide nucleic acid may optionally also have other chemical modifications, including (but not limited to) biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, and locked nucleic acid (LNA).

[0221] The molecular beacon is luciferase.

[0222] When using this detection method, a detection system containing the following components or a kit containing the following components should be used:

[0223] (a2) Nucleic acid detection effector; the nucleic acid detection effector includes a targeting element SBD and a bimolecular complementary reporter element, wherein the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system;

[0224] (b2) Sulfur-modified guide nucleic acids; and

[0225] (c2) Molecular beacon, wherein the molecular beacon is luciferase.

[0226] The main advantages of this invention are:

[0227] (1) Fast: If the testing conditions are ready, it takes only about 1 hour from receiving the sample to receiving the test results.

[0228] (2) High efficiency: This invention has extremely high sensitivity and can detect 1000 copies / µL of target DNA.

[0229] (3) Simple: No special or complicated steps.

[0230] (4) Multi-purpose: This invention can be applied to various scenarios such as detecting pathogenic microorganisms, cancer screening, gene mutation, and bacterial drug resistance gene detection.

[0231] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional procedures, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition) (Science Press Co., Ltd., 2017), or according to the conditions described in the product manufacturer's instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the embodiments are commercially available.

[0232] Example 1

[0233] Purification and activity verification of SBDgsu-BclI fusion protein

[0234] 1.1 Construction of SBDgsu-BclI fusion protein expression vector

[0235] BclI was synthesized into the E. coli expression vector pBAD-Myc-HisA via gene synthesis, and EcoRI and KpnI restriction sites were added. The synthesized sequence is as follows (SEQ ID NO:1):

[0236]

[0237] The underlined parts represent KpnI and EcoRI restriction sites, while the italicized parts represent BclI sequences.

[0238] Using the plasmid described above as a template, fragment 1 was amplified using the following primers:

[0239] BclI-F:

[0240] CTGGAAGAACAAGCCCATCTGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCATGCAGCCGAACCCGAAATTT (SEQ ID NO: 2)

[0241] BclI-R:CAGTGGGTGGTGGTGGTGGTGCTCGAGTCATTTATAATACTGATTCA(SEQ ID NO:3)

[0242] Using a plasmid containing the SBDgsu sequence as a template, fragment 2 was amplified using the following primers:

[0243] SBDgsu-F:AGCAAATGGGTCGCGGATCCGAATTCATGACCAGTCTGACCCCAAG(SEQ ID NO:4)

[0244] SBDgsu-R:

[0245] AAATTTCGGGTTCGGCTGCATGCTGCCGCCGCCGCCGCTGCCGCCGCCGCCCAGATGGGCTTGTTCTTCCAG (SEQ ID NO: 5)

[0246] After PCR, fragments 1 and 2 were recovered using a gel extraction kit (OMEGA). Fragment 3 was then amplified using these two fragments as templates in the same system with primers SBDgsu-F and BclI-R. Following PCR, the PCR product was constructed into the commercial expression vector pET28a using a homologous recombination kit (Novizan), and sequenced for verification.

[0247] The amino acid sequence of the SBDgsu-BclI fusion protein is shown in SEQ ID NO:6:

[0248] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMTSLTPSLKLFSSLSRAPGAVWTEATRRKAPHKPLLLLAVLDLVHRGVITSPFIAVSGDLVELNELFNLYWRRVVPLGLTSSIAFPFSRLSREPFWELVPQPGIAITDAVINNTSSVSYLRKYALGAKLDDGLFRVMQSGEGREALREALLLSCFSADAAALLREQSVINREAFDYSRVLEEQAHL GGGGSGGGGS MQPNPKFINKSSAFWAYAKLLSEQLGYSKDGVVISYSEAQARAKLKKLGINVKEGIFKDVLRYLKYRAELLNKHKDYLMDVEEARKYFQVALKQHQQNNYTCKLPLNKQKNEKKDYAYFTCIINIIAETELRYFANNNGLVYGK DIYFDDNPMNLSYILNFNRELEGIMSRRFDGAFPSTVNPILIWEIKEYYYTTTFGSRIADGVYETQLDGYEIKTIREETNKNIQHIYFIDDYNTWWNMGKSYLCRIIDMLHMGLVDEVIMGKEVFERWPQILRAVLNQYYK(SEQ IDNO:6)

[0249] 1.2 Expression of SBDgsu-BclI fusion protein

[0250] Heterologous expression of the SBDgsu-BclI fusion protein was performed in the E. coli host BL21(DE3). Single clones carrying the expression vector were picked and cultured in LB liquid medium at 37°C and 220 rpm until OD200 was reached. 600 When the concentration was 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.2 mM. The mixture was then cultured at 16°C and 220 rpm for 20 hours to induce protein expression.

[0251] 1.3 Purification of SBDgsu-BclI fusion protein.

[0252] Bacterial lysis: After the culture was completed, the bacterial cells were collected, the supernatant was discarded, and the bacterial cells were resuspended in 50 mL of Ni column Buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 50 mM imidazole). The bacterial cells were lysed using a pre-cooled cell disruptor. The cell lysate was centrifuged at 10,000 rpm for 1 h at 4 °C. The supernatant was collected, filtered through a 0.45 μm filter membrane, and placed on ice.

[0253] Ni column purification: The Ni affinity chromatography column (GE) was equilibrated with Ni column buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 50 mMimidazole). Cell lysis buffer was loaded onto the Ni affinity chromatography column. After washing with Ni column buffer A for 20 column volumes, the column was washed with Ni column buffer B (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 500 mMimidazole) for 2 column volumes, and the lysate was collected.

[0254] Heparin column purification: The Heparin affinity chromatography column (GE) was equilibrated with heparin column buffer A (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT). The protein purified from the Ni column was diluted and loaded onto the column. After washing with heparin column buffer A for 5 column volumes, heparin column buffer A was mixed with buffer B (20 mM Tris-HCl pH 8.0, 1000 mM NaCl, 1 mM DTT). During elution, the concentration of buffer B increased linearly, and the SBDgsu-BclI fusion protein was eluted within the range of approximately 30%-60% B concentration. The entire process was performed on an AKTA purification system (GE).

[0255] Molecular sieve purification: The molecular sieve (GE) was equilibrated with molecular sieve buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl). The protein purified from the heparin column was loaded onto the sample, and the sample was washed with molecular sieve buffer at a constant flow rate of 1 mL / min for 1.1 column volumes. The SBDgsu-BclI fusion protein was washed off in approximately 13-16 min. The entire process was performed on an AKTA purification system (GE).

[0256] Protein quantification and preservation: Collect samples, concentrate them using ultrafiltration tubes, quantify them using the Bradford Protein Assay Kit, and store them in a -80°C freezer.

[0257] 1.4 Validation of the activity of the SBDgsu-BclI fusion protein

[0258] The reaction substrate for detecting cleavage activity was plasmid pET28a, containing only one BclI cleavage site. pET28a was transformed into E. coli JM110, and single colonies were picked from the plates and cultured overnight in LB liquid medium at 37°C and 220 rpm. After culture, plasmid pET28a was extracted. First, 200 ng of pET28a and 2.5, 5, 10, 20, 40, 80, and 160 ng of SBDgsu-BclI fusion protein were mixed in a reaction buffer of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCl, and 0.02% Triton X-100. The mixed sample was incubated at 50°C for 30 minutes, followed by electrophoresis on a 0.8% agarose gel at 120 V for 30 minutes. After electrophoresis, the agarose gel was developed with EB nucleic acid dye.

[0259] like Figure 4 As shown, the SBDgsu-BclI fusion protein can cleave plasmid pET28a.

[0260] Example 2

[0261] Single-stranded DNA in the SBDgsu-BclI fusion protein detection system

[0262] 2.1 Single-stranded DNA preparation

[0263] The single-stranded DNA sequence is shown below:

[0264] T1: AGAGTAAGTAGTTCGCCAGTTAATAGTTTG (SEQ ID NO:7)

[0265] T2:ACTGAGATACCTACAGCGTGAGCTATGAGA(SEQ ID NO:8)

[0266] After dissolving in deionized water, a stock solution with a concentration of approximately 100 μM was prepared. The template was quantified using nanodrop, and the template was diluted with deionized water to create concentration gradients of 50 μM, 5 μM, 500 nM, and 50 nM.

[0267] 2.2 Primer and Signal DNA Design

[0268] The gDNA sequence used for the detection is shown below:

[0269] gDNA-1: B-CAAACTATTAACTGGCGsAACTACTTACTCT (SEQ ID NO:9)

[0270] Where 's' represents sulfur modification and 'B' represents the biotin group. After dissolving in deionized water, a stock solution with a concentration of 100 μM was prepared.

[0271] The signal DNA contains the recognition site for the BclI endonuclease, formed by annealing two primers, and its sequence is as follows:

[0272] Signal DNA-F: FAM-GC TGATCA TG-Q (SEQ ID NO:10)

[0273] Signal DNA-R: CA TGATCA GC (SEQ ID NO:11)

[0274] In this sequence, the underlined sequence represents the BclI recognition sequence, FAM represents the fluorescent group, and Q represents the quencher group. When the fluorescent group and the quencher group are close together, no fluorescent signal is generated. If the signal-DNA is cleaved by SBDgsu-BclI, the fluorescent group and the quencher group move away from each other, and the fluorescent group releases a fluorescent signal, which can be detected.

[0275] 2.3 Detection Methods

[0276] Of the DNA samples described above, T1 can form double-stranded sulfur-modified DNA with gDNA-1 through base complementarity, which is the experimental group T1 / g1 in this embodiment. T2 cannot form double-stranded DNA with gDNA-1, which is the control group T2 / g1 in this embodiment. NaCl to a final concentration of 100 mM and gDNA-1 to 5 μM were added to deionized water, followed by the addition of 5 μM, 500 nM, 50 nM, and 5 nM of T1 or T2, respectively. The mixture was treated at 95°C for 5 minutes, and then slowly cooled to room temperature.

[0277] Take 1 μL of T1 / g1 or T2 / g1 and mix it with 10 μL of streptavidin magnetic beads (10 mg / mL, Purimag) and 2 pmol of SBDgsu-BclI fusion protein in 1 mL of binding buffer for 30 minutes. The binding buffer is: 20 mM Tris-HCl pH 8.0, 1 M NaCl, 1 mM EDTA, and 0.02% Triton X-100.

[0278] The magnetic beads were separated from the liquid using a magnetic rack. 85 μL of reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100) and signal-DNA to a final concentration of 640 nM were added to the beads. The mixture was then transferred to a 96-well plate, and the fluorescence value was measured over 10 minutes at 50 °C. Linear regression was performed on the fluorescence values, and the slope was analyzed for significance.

[0279] A schematic diagram of the above detection process is shown below. Figure 5 As shown.

[0280] 2.4 Test Results

[0281] like Figure 6 As shown, the detection limit of the SBDgsu-BclI-based detection system without amplification is 50 nM template DNA.

[0282] Example 3

[0283] NEAR amplification technology combined with the SBDgsu-BclI fusion protein detection system is used to detect target DNA.

[0284] 3.1 Preparation of target DNA

[0285] The target DNA sequence is shown below:

[0286] Target F1:

[0287] ATCCGCCTCCATCCAGTGAGTCCGGCGTTGCCGGGAAGCTAGAGTAAGTAGAACGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGC (SEQ ID NO: 12)

[0288] Target R1:

[0289] GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGTTCTACTTACTCTAGCTTCCCGGCAACGCCGGACTCACTGGATGGAGGCGGAT (SEQ ID NO: 13)

[0290] After dissolving in deionized water, a stock solution with a concentration of 100 μM was prepared. Target F1 and Target R1, and NaCl, with a final concentration of 10 nM, were added to the deionized water. The solution was treated at 95 °C for 5 minutes, then slowly cooled to room temperature to form a double-stranded DNA template. Single-stranded DNA template Target F1 and double-stranded DNA template were quantified using a nanodropper, and the template was diluted to a concentration of 10 mM with deionized water. 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 Concentration gradient.

[0291] 3.2 Design of sulfur-modified guide DNA (gDNA-2), amplification primers, and signal DNA

[0292] Based on the target sequence, a sulfur-modified gDNA complementary pair was designed, with the following sequence:

[0293] gDNA-2: B-CAAACTATTAACTGGCGsTTCTACTTACTCT (SEQ ID NO: 14)

[0294] Where 's' represents the sulfur modification site and 'B' represents the biotin group. After dissolving in deionized water, a stock solution with a concentration of 100 μM was prepared.

[0295] Based on the target sequence, NEAR amplification primers NF1 and NR1 and insurance primers BF1 and BR1 were designed, with the following sequences:

[0296] NF1: GCTTCTTACTGAGTCCCGGCGTTGCCGGGAAGCT (SEQ ID NO: 15)

[0297] NR1: GCCGTGTATAGAGTCCGTACAAACTATTAACTGG (SEQ ID NO: 16)

[0298] BF1:TCCGCCTCCATCC (SEQ ID NO: 17)

[0299] BR1: GCAATGGCAACA (SEQ ID NO: 18)

[0300] The design and pretreatment of the signal DNA were the same as in Example 2.

[0301] 3.3 Detection Methods

[0302] First, in 25 μL of NEAR amplification mixture (0.75 U Nt.Bst NBI, 1.2 U Bst 3.0 DNA polymerase, 625 μM dNTP, 3 mM MgSO4, 10 mM (NH4)2SO4, 100 mM KCl, 50 mM NaCl, 40 mM Tris-HCl pH 8.4, 0.1% Triton X-100), 800 nM NF1, 100 nM NR1, 100 nM BF1, 100 nM BR1, 5 pmol gDNA-2, and different concentrations of single-stranded DNA template Target F1 or double-stranded DNA template were added. The mixed sample was amplified at 56 °C for 30 minutes, and then slowly cooled to room temperature.

[0303] In 1 mL of binding buffer, mix the sample with 10 μL of streptavidin magnetic beads (10 mg / mL, Purimag) and 2 pmol of SBDgsu-BclI fusion protein for 15 minutes. The binding buffer consisted of 20 mM Tris-HCl pH 8.0, 1 M NaCl, 1 mM EDTA, and 0.02% Triton X-100.

[0304] The magnetic beads were separated from the liquid using a magnetic rack. After washing, 85 μL of reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100) and signal-DNA to a final concentration of 640 nM were added to the magnetic beads. The mixture was then transferred to a 96-well plate, and the fluorescence value was measured over 10 minutes at 50 °C. Linear regression was performed on the fluorescence values, and the slope was analyzed for significance.

[0305] 3.4 Test Results

[0306] like Figure 7 and Figure 8 As shown, when combined with isothermal amplification NEAR, the detection limit for both ssDNA and dsDNA targets using SBDgsu-BclI is 1000 copies / µL.

[0307] Example 4

[0308] NEAR amplification technology combined with the SBDgsu-BclI fusion protein detection system for the detection of Candida albicans genomic DNA

[0309] 4.1 Preparation of Candida albicans genomic DNA

[0310] Genomic DNA was extracted from *Candida albicans* SC5314 using a yeast genomic DNA extraction kit (Tiangen). After extraction, it was detected by electrophoresis on a 0.8% agarose gel at 120V for 30 minutes. After electrophoresis, the agarose gel was developed with EB nucleic acid dye. The successfully detected *Candida albicans* genomic DNA served as the experimental group. *Helicobacter pylori* genomic DNA prepared using a similar method served as the negative control group.

[0311] 4.2 Design of sulfur-modified guide DNA (gDNA-3), amplification primers, and signal DNA

[0312] Based on the DNA sequence of the Candida albicans virulence gene SAP2, complementary pairing gDNA with sulfur modification and corresponding amplification primers were designed, as follows:

[0313] gDNA-3: B-CAGCATCTGGAGsAATTAAGATAAAGTGAAT (SEQ ID NO: 19)

[0314] NF2:AAAAAACAAGGAGTCATTGCTAAGAATGCTT(SEQ ID NO:20)

[0315] NR2: GCCGTGTATAGAGTCCGTAGTGGCAGCATCTGGAG (SEQ ID NO: 21)

[0316] BF2:GTCCCTGTCACTT(SEQ ID NO:22)

[0317] BR2:CCGAAAATTATTTGT(SEQ ID NO:23)

[0318] Where 's' represents the sulfur modification site and 'B' represents the biotin group. After dissolving in deionized water, a stock solution with a concentration of 100 μM was prepared.

[0319] The design and pretreatment of the signal DNA were the same as in Example 2.

[0320] 4.3 Detection Methods

[0321] First, in 25 μL of NEAR amplification mixture (0.75 U Nt.Bst NBI, 1.2 U Bst 3.0 DNA polymerase, 625 μM dNTP, 3 mM MgSO4, 10 mM (NH4)2SO4, 100 mM KCl, 50 mM NaCl, 40 mM Tris-HCl pH 8.4, 0.1% Triton X-100), 800 nM NF2, 100 nM NR2, 100 nM BF2, 100 nM BR2, 5 pmol gDNA-3, and Candida albicans or Helicobacter pylori genomic DNA were added to a final concentration. The mixed sample was amplified at 56 °C for 30 minutes, and then slowly cooled to room temperature.

[0322] In 1 mL of binding buffer, mix the sample with 10 μL of streptavidin magnetic beads (10 mg / mL, Purimag) and 2 pmol of SBDgsu-BclI fusion protein for 15 minutes. The binding buffer consisted of 20 mM Tris-HCl pH 8.0, 1 M NaCl, 1 mM EDTA, and 0.02% Triton X-100.

[0323] The magnetic beads were separated from the liquid using a magnetic rack. After washing, 85 μL of reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NaCl, 0.02% Triton X-100) and signal-DNA to a final concentration of 640 nM were added to the magnetic beads. The mixture was then transferred to a 96-well plate, and the fluorescence value was measured over 10 minutes at 50 °C. Linear regression was performed on the fluorescence values, and the slope was analyzed for significance.

[0324] 4.4 Test Results

[0325] like Figure 9 As shown, the NEAR amplification technology combined with the SBDgsu-BclI detection system can specifically detect Candida albicans genomic DNA.

[0326] Example 5

[0327] Purification of SBDgsu-FlucN and SBDgsu-FlucC fusion proteins

[0328] 5.1 Construction of expression vectors for SBDgsu-FlucN and SBDgsu-FlucC fusion proteins

[0329] Using plasmids containing SBDgsu and Fluc as templates, the following amplification primers were used:

[0330] SBDgsu-FC:

[0331] AAAGGTGGCAAGATCGCGGTGGGCGGCGGCGGCAGCGGAGGAGGAGGAAGTACCAGTCTGACCCCAAGTCTG(SEQ ID No:24)

[0332] SBDgsu-FN:tgtattttcagggcgaattcACCAGTCTGACCCCAAGTCTGA(SEQ ID No:25)

[0333] SBDgsu-RC:acggagctcgaattcggatccTCACAGATGGGCTTGTTCTTCCAG(SEQ ID No:26)

[0334] SBDgsu-RN:

[0335] CTTAATGTTTTTCGCATCTTCACTTCCTCCTCCTCCGCTGCCGCCGCCGCCCAGATGGGCTTGTTCTTCCAG(SEQ ID No:27)

[0336] FlucN-F:

[0337] CTGGAAGAACAAGCCCATCTGGGCGGCGGCGGCAGCGGAGGAGGAGGAAGTGAAGATGCGAAAAACATTAAG(SEQ ID No:28)

[0338] FlucN-R:cctgcaggtcgactctagaggatccACCGTCTTTATCAATCAGCG(SEQ ID No:29)

[0339] FlucC-F:tgccacgaggaagccatatgAGCGGTTACGTGAACAACCC(SEQ ID No:30)

[0340] FlucC-R:

[0341] CAGACTTGGGGTCAGACTGGTACTTCCTCCTCCTCCGCTGCCGCCGCCGCCCACCGCGATCTTGCCACCTTT(SEQ ID No:31)

[0342] In the primers, lowercase letters represent sequences matching the vector, and uppercase letters represent sequences matching the fusion protein. After PCR, the PCR products were constructed into expression vectors pMAL and pET28a using a homologous recombination kit (Novizan), and then sequenced for verification. The amino acid sequences of the SBDgsu-FlucN and SBDgsu-FlucC fusion proteins are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.

[0343] SBDgsu-FlucN amino acid sequence:

[0344]

[0345] SBDgsu-FlucC amino acid sequence:

[0346] MGSSHHHHHHSSGLVPRGSHMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVGGGGSGGGGSTSLT PSLKLFSSLSRAPGAVWTEATRRKAPHKPLLLLAVLDLVHRGVITSPFIAVSGDLVELNELFNLYWRRVVPLGLTSSIAFPFSRLSREPFWELVPQPGIAITDAVINNTSSVSYLRKYALGAKLDDGLFRVMQSGEGREALREALLLSCFSADAAALLREQSVINREAFDYSRVLEEQAHL(SEQID NO:33)

[0347] 5.2 Induction and purification of SBDgsu-FlucN and SBDgsu-FlucC fusion proteins

[0348] First, the SBDgsu-FlucN and SBDgsu-FlucC fusion protein expression vectors were transformed into E. coli BL21(DE3) hosts and cultured in LB liquid medium at 37°C and 220 rpm until OD200. 600 When the concentration of the bacterial cell lysate was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM. The cells were cultured at 16°C and 220 rpm for 20 hours. After the culture was completed, the supernatant of the bacterial cell lysate was collected. The fusion protein was purified sequentially using a nickel ion affinity chromatography column (GE) and a heparin affinity chromatography column (GE). The protein was then quantified using a Bradford protein assay kit and stored at -80°C.

[0349] Example 6

[0350] Single-stranded DNA in the SBDgsu-FlucN and SBDgsu-FlucC fusion protein detection system

[0351] 6.1 Preparation of target single-stranded DNA

[0352] The target single-stranded DNA sequence was selected from a segment of the Candida albicans virulence gene SAP2, as shown below:

[0353] Target F2: CTTATATAAAGTACCTTGAGATGAAGATCCATCACCATAACCAATTTTGA (SEQ IDNO: 34)

[0354] After dissolving in deionized water, a stock solution with a concentration of approximately 100 μM was prepared.

[0355] 6.2 Design of sulfur-modified guide DNAs (gDNA-4 and gDNA-5)

[0356] gDNA-4:ATTGGTTATGsGTGAT (SEQ ID NO: 35)

[0357] gDNA-5: TCAAGsGTACTTTATA (SEQ ID NO: 36)

[0358] Where 's' represents the sulfur modification site. After dissolving in deionized water, a stock solution with a concentration of 100 μM was prepared.

[0359] 6.3 Detection Methods

[0360] 10 pmol of gDNA-4 and gDNA-5 were added to a 25 μL system, followed by different concentrations of template, and annealing was performed. After annealing, SBDgsu-FlucN and SBDgsu-FlucC fusion proteins were added to the system at a final concentration of 100 nM, and the mixture was incubated at room temperature for 10 min. Then, luciferase substrate was added. Real-time fluorescence was detected.

[0361] A schematic diagram of the above detection process is shown below. Figure 10 As shown.

[0362] 6.4 Test Results

[0363] The results are as follows Figure 11 As shown, almost no fluorescence signal is generated without template addition. The fluorescence signal gradually increases with increasing template concentration. In this embodiment, the detection limit is 5 nM.

[0364] Example 7

[0365] RPA amplification technology combined with the SBDgsu-Fluc detection system is used to detect target DNA.

[0366] 7.1 Preparation of target DNA

[0367] The preparation of the target DNA is the same as in Example 6.

[0368] 7.2 Design of sulfur-modified guide DNA and amplification primers

[0369] The sulfur-modified guide DNA design is consistent with that in Example 6.

[0370] Based on the target SAP2 sequence, RPA amplification primers were designed, and the sequences are as follows:

[0371] RF1: ATCAACATCAGCTAAAACTTGATTTTTAAT (SEQ ID NO: 37)

[0372] RR1: GATCCAAGTGGTTCATCAGCTTCACAAGAT (SEQ ID NO: 38)

[0373] 7.3 Detection Method

[0374] First, the target DNA concentration was amplified by asymmetric RPA for 30 min at 37℃ using an RPA kit (Junuode). Then, 10 pmol of gDNA-4 and gDNA-5 were added, followed by annealing. After annealing, SBDgsu-FlucN and SBDgsu-FlucC fusion proteins were added to a final concentration of 100 nM, and the mixture was incubated at room temperature for 10 min. Finally, luciferase substrate was added. Real-time fluorescence was detected.

[0375] 7.4 Test Results

[0376] like Figure 12 As shown, when combined with RPA, the detection limit for target DNA using SBDgsu-FlucN and SBDgsu-FlucC is 1000 copies / µL.

[0377] Example 8

[0378] RPA amplification technology combined with the SBDgsu-Fluc detection system was used to detect Candida albicans genomic DNA.

[0379] 8.1 Preparation of Candida albicans genomic DNA

[0380] The preparation of Candida albicans genomic DNA was the same as in Example 4. Helicobacter pylori genomic DNA prepared using a similar method served as a negative control.

[0381] 8.2 Design of sulfur-modified guide DNA and amplification primers

[0382] The sulfur-modified guide DNA design is consistent with that in Example 6.

[0383] The design of the amplification primers is the same as in Example 7.

[0384] 8.3 Detection Methods

[0385] First, the genomic DNA content of *Candida albicans* was amplified at 37°C for 30 min using an RPA kit (Junuode). Then, 10 pmol of gDNA-4 and gDNA-5 were added, followed by annealing. After annealing, SBDgsu-FlucN and SBDgsu-FlucC fusion proteins were added to a final concentration of 100 nM, and the mixture was incubated at room temperature for 10 min. Finally, luciferase substrate was added. Real-time fluorescence was detected.

[0386] 8.4 Test Results

[0387] like Figure 13 As shown, RPA amplification technology combined with the SBDgsu-Fluc detection system can specifically detect Candida albicans genomic DNA.

[0388] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai Jiao Tong University Hangzhou Bigfly Biotechnology Co., Ltd. <120> Nucleic acid detection methods based on sulfur-modified nucleic acids and sulfur-modified nucleic acid recognition proteins <130> P2022-0414 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 867 <212> DNA <213> Artificial sequence <220> <223> SBDgsu-BclI fusion protein nucleotide sequence <400> 1 ggtaccatgc agccgaaccc gaaatttatt aacaaaagca gcgcgttttg ggcgtatgcg 60 aaactgctga gcgaacagct gggctatagc aaagatggcg tggtgattag ctatagcgaa 120 gcgcaagcgc gcgcgaaact gaaaaaactg ggcattaacg tgaaagaagg cattttaaa 180 gatgtgctgc gctatctgaa atatcgcgcg gaactgctga acaaacacaa agattatctg 240 atggatgtgg aagaagcgcg caaatatttt caagtggcgc tgaaacagca tcagcagaac 300 aactacacct gcaaactgcc gttaaacaag caaaaaaatg aaaaaaaga ttatgcgtat 360 tttacctgca ttattaacat tattgcggaa accgaactgc gctattttgc gaacaataac 420 ggcctggtgt atggcaaaga tatttatttt gatgataacc cgatgaacct gagctatatt 480 ctgaacttta accgcgaact ggaaggcatt atgagccgcc gctttgatgg cgcgtttccg 540 agcaccgtga acccgattct gatttgggaa attaaagaat attactatac cacgaccttt 600 ggcagccgca ttgcggatgg cgtgtatgaa acgcagctgg atggctatga aattaaaacc 660 attcgcgaag aaaccaacaa aaacattcag catatttatt ttattgatga ttataacacc 720 tggtggaaca tgggcaaaag ctatctgtgc cgcattattg atatgctgca tatgggcctg 780 gtggatgaag tgattatggg caaagaagtg tttgaacgct ggccgcagat tctgcgcgcg 840 gtgctgaatc agtattataa agaattc 867 <210> 2 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 2 ctggaagaac aagcccatct gggcggcggc ggcagcggcg gcggcggcag catgcagccg 60 aacccgaaat tt 72 <210> 3 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 3 cagtggtggt ggtggtggtg ctcgagtcat ttataatact gattca 46 <210> 4 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 4 agcaaatggg tcgcggatcc gaattcatga ccagtctgac cccaag 46 <210> 5 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 5 aaatttcggg ttcggctgca tgctgccgcc gccgccgctg ccgccgccgc ccagatgggc 60 ttgttcttcc ag 72 <210> 6 <211> 517 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of SBDgsu-BclI fusion protein <400> 6 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Met Thr Ser Leu Thr Pro Ser Leu Lys Leu Phe Ser 35 40 45 Ser Leu Ser Arg Ala Pro Gly Ala Val Trp Thr Glu Ala Thr Arg Arg 50 55 60 Lys Ala Pro His Lys Pro Leu Leu Leu Leu Ala Val Leu Asp Leu Val 65 70 75 80 His Arg Gly Val Ile Thr Ser Pro Phe Ile Ala Val Ser Gly Asp Leu 85 90 95 Val Glu Leu Asn Glu Leu Phe Asn Leu Tyr Trp Arg Arg Val Val Pro 100 105 110 Leu Gly Leu Thr Ser Ser Ile Ala Phe Pro Phe Ser Arg Leu Ser Arg 115 120 125 Glu Pro Phe Trp Glu Leu Val Pro Gln Pro Gly Ile Ala Ile Thr Asp 130 135 140 Ala Val Ile Asn Asn Thr Ser Ser Val Ser Tyr Leu Arg Lys Tyr Ala 145 150 155 160 Leu Gly Ala Lys Leu Asp Asp Gly Leu Phe Arg Val Met Gln Ser Gly 165 170 175 Glu Gly Arg Glu Ala Leu Arg Glu Ala Leu Leu Leu Ser Cys Phe Ser 180 185 190 Ala Asp Ala Ala Ala Leu Leu Arg Glu Gln Ser Val Ile Asn Arg Glu 195 200 205 Ala Phe Asp Tyr Ser Arg Val Leu Glu Glu Gln Ala His Leu Gly Gly 210 215 220 Gly Gly Ser Gly Gly Gly Gly Ser Met Gln Pro Asn Pro Lys Phe Ile 225 230 235 240 Asn Lys Ser Ser Ala Phe Trp Ala Tyr Ala Lys Leu Leu Ser Glu Gln 245 250 255 Leu Gly Tyr Ser Lys Asp Gly Val Val Ile Ser Tyr Ser Glu Ala Gln 260 265 270 Ala Arg Ala Lys Leu Lys Lys Leu Gly Ile Asn Val Lys Glu Gly Ile 275 280 285 Phe Lys Asp Val Leu Arg Tyr Leu Lys Tyr Arg Ala Glu Leu Asn 290,295,300 Lys His Lys Asp Tyr Leu Met Asp Val Glu Glu Ala Arg Lys Tyr Phe 305 310 315 320 Gln Val Ala Leu Lys Gln His Gln Gln Asn Asn Tyr Thr Cys Lys Leu 325 330 335 Pro Leu Asn Lys Gln Lys Asn Glu Lys Asp Tyr Ala Tyr Phe Thr 340 345 350 Cys Contains Asn Contains Glu Thr Glu Leu Arg Tyr Phe Containing Asn 355 360 365 Asn Asn Gly Leu Val Tyr Gly Lys Asp Ile Tyr Phe Asp Asp Asn Pro 370 375 380 Met Asn To Ser Tyr Ile To Asn Phe Asn Arg Glu To Glu Gly Ile 385 390 395 400 Met Ser Arg Arg Phe Asp Gly Ala Phe Pro Ser Thr Val Asn Pro Ile 405 410 415 Leu Ile Trp Glu Ile Lys Glu Tyr Tyr Tyr Thr Thr Thr Phe Gly Ser 420 425 430 Arg Ile Ala Asp Gly Val Tyr Glu Thr Gln Leu Asp Gly Tyr Glu Ile 435 440 445 Lys Thr Ile Arg Glu Glu Thr Asn Lys Asn Ile Gln His Ile Tyr Phe 450 455 460 Ile Asp Asp Tyr Asn Thr Trp Trp Asn Met Gly Lys Ser Tyr Leu Cys 465 470 475 480 Arg Ile Ile Asp Met Leu His Met Gly Leu Val Asp Glu Val Ile Met 485 490 495 Gly Lys Glu Val Phe Glu Arg Trp Pro Gln Ile Leu Arg Ala Val Leu 500 505 510 Asn Gln Tyr Tyr Lys 515 <210> 7 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Single-stranded DNA sequence T1 <400> 7 agagtaagta gttcgccagt taatagtttg 30 <210> 8 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Single-stranded DNA sequence T2 <400> 8 actgagatac ctacagcgtg agctatgaga 30 <210> 9 <211> 30 <212> DNA <213> Artificial sequence <220> <223> gDNA-1 <400> 9 caaactatta actggcgaac tacttactct 30 <210> 10 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Signal DNA-F <400> 10 gctgatcatg 10 <210> 11 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Signal DNA-R <400> 11 catgatcagc 10 <210> 12 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Target DNA F1 <400> 12 atccgcctcc atccagtgag tccggcgttg ccgggaagct agagtaagta gaacgccagt 60 taatagtttg cgcaacgttg ttgccattgc 90 <210> 13 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Target DNA R1 <400> 13 gcaatggcaa caacgttgcg caaactatta actggcgttc tacttactct agcttcccgg 60 caacgccgga ctcactggat ggaggcggat 90 <210> 14 <211> 30 <212> DNA <213> Artificial sequence <220> <223> gDNA-2 <400> 14 caaactatta actggcgttc tacttactct 30 <210> 15 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 15 gcttcttact gagtccggcg ttgccgggaa gct 33 <210> 16 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 16 gccgtgtata gagtccgtac aaactattaa ctgg 34 <210> 17 <211> 13 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 tccgcctcca tcc 13 <210> 18 <211> 12 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 gcaatggcaa ca 12 <210> 19 <211> 30 <212> DNA <213> Artificial sequence <220> <223> gDNA-3 <400> 19 cagcatctgg agaattaaga taaagtgaat 30 <210> 20 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 20 aaaaaacaag gagtcattgc taagaatgct t 31 <210> twenty one <211> 35 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty one gccgtgtata gagtccgtag tggcagcatc tggag 35 <210> twenty two <211> 13 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty two gtccctgtca ctt 13 <210> twenty three <211> 15 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty three ccgaaaatta tttgt 15 <210> twenty four <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> twenty four aaaggtggca agatcgcggt gggcggcggc ggcagcggag gaggaggaag taccagtctg 60 accccaagtc tg 72 <210> 25 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 25 tgtattttca gggcgaattc accagtctga ccccaagtct ga 42 <210> 26 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 26 acggagctcg aattcggatc ctcacagatg ggcttgttct tccag 45 <210> 27 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 27 cttaatgttt ttcgcatctt cacttcctcc tcctccgctg ccgccgccgc ccagatgggc 60 ttgttcttcc ag 72 <210> 28 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 28 ctggaagaac aagcccatct gggcggcggc ggcagcggag gaggaggaag tgaagatgcg 60 aaaaacatta ag 72 <210> 29 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 29 cctgcaggtc gactctagag gatccaccgt ctttatcaat cagcg 45 <210> 30 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 30 tgccacgagg aagccatatg agcggttacg tgaacaaccc 40 <210> 31 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 31 cagacttggg gtcagactgg tacttcctcc tcctccgctg ccgccgccgc ccaccgcgat 60 cttgccacct tt 72 <210> 32 <211> 1025 <212> PRT <213> Artificial sequence <220> <223> SBDgsu-FlucN amino acid sequence <400> 32 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Leo Gly Ile 370 375 380 Glu Gly Arg Ile Ser Glu Asn Leu Tyr Phe Gln Gly Glu Phe Thr Ser 385 390 395 400 Leu Thr Pro Ser Leu Lys Leu Phe Ser Ser Leu Ser Arg Ala Pro Gly 405 410 415 Ala Val Trp Thr Glu Ala Thr Arg Arg Lys Ala Pro His Lys Pro Leu 420 425 430 Leu Leu Leu Ala Val Leu Asp Leu Val His Arg Gly Val Ile Thr Ser 435 440 445 Pro Phe Ile Ala Val Ser Gly Asp Leu Val Glu Leu Asn Glu Leu Phe 450 455 460 Asn Leu Tyr Trp Arg Arg Val Val Pro Leu Gly Leu Thr Ser Ser Ile 465 470 475 480 Ala Phe Pro Phe Ser Arg Leu Ser Arg Glu Pro Phe Trp Glu Leu Val 485 490 495 Pro Gln Pro Gly Ile Ala Ile Thr Asp Ala Val Ile Asn Asn Thr Ser 500 505 510 Ser Val Ser Tyr Leu Arg Lys Tyr Ala Leu Gly Ala Lys Leu Asp Asp 515 520 525 Gly Leu Phe Arg Val Met Gln Ser Gly Glu Gly Arg Glu Ala Leu Arg 530 535 540 Glu Ala Leu Leu Leu Ser Cys Phe Ser Ala Asp Ala Ala Ala Leu Leu 545 550 555 560 Arg Glu Gln Ser Val Ile Asn Arg Glu Ala Phe Asp Tyr Ser Arg Val 565 570 575 Leu Glu Glu Gln Ala His Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly 580 585 590 Ser Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 595 600 605 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 610 615 620 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 625 630 635 640 Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 645 650 655 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 660 665 670 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 675 680 685 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 690 695 700 Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val 705 710 715 720 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 725 730 735 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 740 745 750 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 755 760 765 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 770 775 780 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 785 790 795 800 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 805 810 815 Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 820 825 830 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 835 840 845 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 850 855 860 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 865 870 875 880 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 885 890 895 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 900 905 910 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 915 920 925 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 930 935 940 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 945 950 955 960 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 965 970 975 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 980 985 990 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 995 1000 1005 Gly Ser Ser Arg Val Asp Leu Gln Ala Lys Leu His His His His 1010 1015 1020 His His 1025 <210> 33 <211> 368 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of SBDgsu-FlucC <400> 33 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ser Gly Tyr Val Asn Asn Pro Glu Ala Thr Asn 20 25 30 Ala Leu Ile Asp Lys Asp Gly Trp Leu His Ser Gly Asp Ile Ala Tyr 35 40 45 Trp Asp Glu Asp Glu His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu 50 55 60 Ile Lys Tyr Lys Gly Tyr Gln Val Ala Pro Ala Glu Leu Glu Ser Ile 65 70 75 80 Leu Leu Gln His Pro Asn Ile Phe Asp Ala Gly Val Ala Gly Leu Pro 85 90 95 Asp Asp Asp Ala Gly Glu Leu Pro Ala Ala Val Val Val Leu Glu His 100 105 110 Gly Lys Thr Met Thr Glu Lys Glu Ile Val Asp Tyr Val Ala Ser Gln 115 120 125 Val Thr Thr Ala Lys Lys Leu Arg Gly Gly Val Val Phe Val Asp Glu 130 135 140 Val Pro Lys Gly Leu Thr Gly Lys Leu Asp Ala Arg Lys Ile Arg Glu 145 150 155 160 Ile Leu Ile Lys Ala Lys Lys Gly Gly Lys Ile Ala Val Gly Gly Gly 165 170 175 Gly Ser Gly Gly Gly Gly Ser Thr Ser Leu Thr Pro Ser Leu Lys Leu 180 185 190 Phe Ser Ser Leu Ser Arg Ala Pro Gly Ala Val Trp Thr Glu Ala Thr 195 200 205 Arg Arg Lys Ala Pro His Lys Pro Leu Leu Leu Leu Ala Val Leu Asp 210 215 220 Leu Val His Arg Gly Val Ile Thr Ser Pro Phe Ile Ala Val Ser Gly 225 230 235 240 Asp Leu Val Glu Leu Asn Glu Leu Phe Asn Leu Tyr Trp Arg Arg Val 245 250 255 Val Pro Leu Gly Leu Thr Ser Ser Ile Ala Phe Pro Phe Ser Arg Leu 260 265 270 Ser Arg Glu Pro Phe Trp Glu Leu Val Pro Gln Pro Gly Ile Ala Ile 275 280 285 Thr Asp Ala Val Ile Asn Asn Thr Ser Ser Val Ser Tyr Leu Arg Lys 290 295 300 Tyr Ala Leu Gly Ala Lys Leu Asp Asp Gly Leu Phe Arg Val Met Gln 305 310 315 320 Ser Gly Glu Gly Arg Glu Ala Leu Arg Glu Ala Leu Leu Leu Ser Cys 325 330 335 Phe Ser Ala Asp Ala Ala Ala Leu Leu Arg Glu Gln Ser Val Ile Asn 340 345 350 Arg Glu Ala Phe Asp Tyr Ser Arg Val Leu Glu Glu Gln Ala His Leu 355 360 365 <210> 34 <211> 50 <212> DNA <213> artificial sequence <220> <223> target single-stranded DNA sequence <400> 34 cttatataaa gtaccttgag atgaagatcc atcaccataa ccaattttga 50 <210> 35 <211> 15 <212> DNA <213> Artificial sequence <220> <223> gDNA-4 <400> 35 attggttatg gtgat 15 <210> 36 <211> 15 <212> DNA <213> Artificial sequence <220> <223> gDNA-5 <400> 36 tcaaggtact ttata 15 <210> 37 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 37 atcaacatca gctaaaactt gatttttaat 30 <210> 38 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 38 gatccaagtg gttcatcagc ttcacaagat 30

Claims

1. A nucleic acid detection effector, characterized in that, The nucleic acid detection effector includes a targeting element and a reporter element, wherein the targeting element and the reporter element form a fusion protein, and wherein the targeting element contains a sulfur-modified nucleic acid recognition protein, which is a sulfur-binding domain protein (SBD) that specifically recognizes sulfur-modified nucleic acids. The sulfur-modified nucleic acid refers to nucleic acids containing sulfur-modified nucleic acids. Modified as The reporter element includes a single-molecule reporter element and a bimolecular complementary reporter element. The single-molecule reporter element is a restriction endonuclease, and the bimolecular complementary reporter element is a fluorescent bimolecular complementary reporter system. The nucleic acid detection effector, guided by sulfur-modified guide nucleic acid, can bind to a predetermined region of the nucleic acid to be detected and generate a reporting signal by contacting a molecular beacon through a reporter element. The sulfur-modified guide nucleic acid comprises phosphorus-thioylated modified gDNA or gRNA, wherein the gDNA or gRNA is a single-stranded nucleic acid sequence that can be complementary to the bases of the nucleic acid to be detected, and contains sulfur modification at one or more sites to guide the SBD to the nucleic acid to be detected; the sequence length of the gDNA or gRNA is greater than 10 nt and less than 100 nt, and contains at least one sulfur-modified nucleotide.

2. The nucleic acid detection effector as described in claim 1, characterized in that, The sequence length of the gDNA or gRNA is 10-30 nt.

3. The nucleic acid detection effector as described in claim 1, characterized in that, The sulfur-modified nucleotide is C or G.

4. The nucleic acid detection effector as described in claim 1, characterized in that, In addition to sulfur modification, the sulfur-modified guide nucleic acid also has other chemical modifications, which are selected from the group consisting of: biotin modification, amino modification, fluorination modification, methylation modification, methoxy modification, locked nucleic acid (LNA) or combinations thereof.

5. The nucleic acid detection effector as described in claim 1, characterized in that, The nucleic acid detection effector is a single-stranded nucleic acid detection effector.

6. The nucleic acid detection effector as described in claim 1, characterized in that, The sulfur-binding domain proteins (SBDs) include: SBDgsu, SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu, or SBD homologs.

7. The nucleic acid detection effector as described in claim 1, characterized in that, The bimolecular complementary reporter elements include: Fluc-N and Fluc-C, LgBiT and SmBiT, LacA and LacB.

8. The nucleic acid detection effector as described in claim 1, characterized in that, The molecular beacon is selected from the group consisting of double-stranded DNA, luciferase, or combinations thereof.

9. The nucleic acid detection effector as described in claim 8, characterized in that, The double-stranded DNA contains a sequence that can be specifically recognized by restriction endonucleases.

10. The nucleic acid detection effector as described in claim 8, characterized in that, The double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

11. The nucleic acid detection effector as described in claim 1, characterized in that, The fusion protein contains one or more SBDs.

12. The nucleic acid detection effector as described in claim 1, characterized in that, The structure of the fusion protein is shown in formula I or I': ALB(I) BLA(I') In the formula, A is the target element; B is the reporting element; L can be either absent or linked peptides independently; Each "-" represents a chemical bond independently.

13. The nucleic acid detection effector as described in claim 1, characterized in that, The sequence of the fusion protein is shown in SEQ ID No:

6.

14. The nucleic acid detection effector as described in claim 1, characterized in that, The sequences of the fusion protein are shown in SEQ ID No: 32 and SEQ ID No:

33.

15. A method for nucleic acid detection, characterized in that, Includes the following steps: (i) In the presence of sulfur-modified guide nucleic acid, the nucleic acid detection effector as described in claim 1 forms a "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex with the nucleic acid to be detected; and (ii) Contact the molecular beacon with the “nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected” complex and detect the report signal.

16. The method as described in claim 15, characterized in that, The sulfur-modified guide nucleic acid is a sulfur-modified single-stranded gDNA or gRNA.

17. The method as described in claim 15, characterized in that, The nucleic acid to be detected is selected from the following group: single-stranded DNA, single-stranded RNA, or combinations thereof.

18. The method as described in claim 15, characterized in that, The nucleic acid to be tested is enriched in large quantities through isothermal amplification.

19. The method as described in claim 15, characterized in that, The molecular beacon is selected from the group consisting of double-stranded DNA, luciferase, or combinations thereof.

20. The method as described in claim 19, characterized in that, The double-stranded DNA contains a sequence that can be specifically recognized by restriction endonucleases.

21. The method as described in claim 19, characterized in that, The double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

22. The method of claim 15, further comprising: The "nucleic acid detection effector-sulfur-modified guide nucleic acid-nucleic acid to be detected" complex was separated from the nucleic acid to be detected that did not form a complex using magnetic beads.

23. A reaction system for nucleic acid detection, characterized in that, The reaction system includes: (a) The nucleic acid detection effector as described in claim 1; (b) A sulfur-modified guide nucleic acid, wherein the sulfur-modified guide nucleic acid guides the nucleic acid detection effector to specifically bind to a predetermined region of the nucleic acid to be detected; and (c) A molecular beacon that generates a reporting signal upon contact with the nucleic acid detection effector.

24. The reaction system as described in claim 23, characterized in that, The sulfur-modified guide nucleic acid is a sulfur-modified single-stranded gDNA or gRNA.

25. The reaction system as described in claim 23, characterized in that, The nucleic acid to be detected is selected from the following group: single-stranded DNA, single-stranded RNA, or combinations thereof.

26. The reaction system as described in claim 23, characterized in that, The nucleic acid to be tested is enriched in large quantities through isothermal amplification.

27. The reaction system as described in claim 23, characterized in that, The molecular beacon is selected from the group consisting of double-stranded DNA, luciferase, or combinations thereof.

28. The reaction system as described in claim 27, characterized in that, The double-stranded DNA contains a sequence that can be specifically recognized by restriction endonucleases.

29. The reaction system as described in claim 27, characterized in that, The double-stranded DNA is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

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