阅读说明：本技术 基于crispr技术进行目标核酸检测的方法和系统 (Method and system for detecting target nucleic acid based on CRISPR technology ) 是由 梁亚峰 段志强 孙洁 于 2020-06-05 设计创作，主要内容包括：本发明提供了一种基于CRISPR技术进行目标核酸检测的方法和系统,具体地,涉及一种基于CRISPR技术检测目标核酸中是否存在待检测的特征序列的方法、系统和试剂盒,所述的检测方法包括向含有目标核酸的反应体系中加入核酸外切酶、gRNA、Cas蛋白和单链寡核苷酸。该方法可以有效缩短检测时间且具有更高的灵敏度。(The invention provides a method and a system for detecting target nucleic acid based on a CRISPR technology, and particularly relates to a method, a system and a kit for detecting whether a characteristic sequence to be detected exists in the target nucleic acid based on the CRISPR technology. The method can effectively shorten the detection time and has higher sensitivity.)

1. A method for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, comprising:

(1) providing a target nucleic acid, an exonuclease, a gRNA, a Cas protein, and a single-stranded oligonucleotide;

(2) the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least part of a single stranded nucleic acid;

(3) the gRNA can target a characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein excites the activity of trans (trans) single-stranded DNA cleavage after recognizing the characteristic sequence to be detected;

(4) the Cas protein cleaves the single-stranded oligonucleotide by a trans (trans) single-stranded DNA cleaving activity, the single-stranded oligonucleotide exhibiting a detectable difference after cleavage by the Cas protein compared to the single-stranded oligonucleotide before cleavage by the Cas protein;

(5) testing whether the detectable difference of step (4) is detectable; if the detectable difference in step (4) can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; or, if the detectable difference in step (4) is not detected, it indicates that the target nucleic acid does not contain the signature sequence to be detected;

the Cas protein is a protein having trans cleavage activity, preferably, Cas12a and/or Cas12 b.

2. The method of claim 1, wherein the exonuclease comprises 5 '→ 3' exonuclease or 3 '→ 5' exonuclease; preferably, the exonuclease is 5 '→ 3' exonuclease; more preferably, the exonuclease is selected from one or more of T5 exonuclease, T7 exonuclease, lambda exonuclease and exonuclease VIII.

3. The method of any one of claims 1-2,

the 5 'end and the 3' end of the single-stranded oligonucleotide are respectively provided with different reporter groups, when the single-stranded oligonucleotide is cut, a detectable reporter signal can be shown, and whether the target nucleic acid contains a characteristic sequence to be detected is reflected by the existence of the reporter signal;

or, the 5 'end and the 3' end of the single-stranded oligonucleotide are respectively provided with different labeling molecules, and the results of the single-stranded oligonucleotide before and after cleavage by the Cas protein are detected by a lateral flow test strip, for example, a colloidal gold test strip detection method, so as to reflect whether the target nucleic acid contains the characteristic sequence to be detected.

4. A system for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, the system comprising: exonuclease, gRNA, Cas protein, and single-stranded oligonucleotide;

wherein the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least part of a single stranded nucleic acid;

the gRNA can target a characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein excites the activity of trans (trans) single-stranded DNA cleavage after recognizing the characteristic sequence to be detected;

the Cas protein cleaves the single-stranded oligonucleotide by a trans (trans) single-stranded DNA cleaving activity, the single-stranded oligonucleotide exhibiting a detectable difference after cleavage by the Cas protein compared to the single-stranded oligonucleotide before cleavage by the Cas protein;

if the detectable difference can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; or, if the detectable difference is not detectable, the target nucleic acid does not contain the signature sequence to be detected; the Cas protein is a protein having trans cleavage activity, preferably, Cas12a and/or Cas12 b.

5. A system for detecting the presence or absence of a characteristic sequence to be detected in a target nucleic acid, the system comprising a detection device, a reaction system and a detection agent,

the reaction system comprises exonuclease, gRNA, Cas protein and single-stranded oligonucleotide;

the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least a portion of a single stranded nucleic acid; the gRNA can target a characteristic sequence to be detected, the Cas protein identifies the characteristic sequence to be detected under the action of the gRNA, the Cas protein has trans (trans) single-stranded DNA cleavage activity, and the Cas protein activates the trans (trans) single-stranded DNA cleavage activity after identifying the characteristic sequence to be detected so as to cleave the single-stranded oligonucleotide;

one end of the single-stranded oligonucleotide is connected with a first marker molecule, and the other end of the single-stranded oligonucleotide is connected with a second marker molecule;

the detection agent is linked to a first binding molecule capable of binding to a first label molecule;

the detection device is provided with a first detection line provided with a first substance that can capture a first binding molecule and a second detection line provided with a second substance that can capture a second labeling molecule;

the Cas protein is preferably Cas12a and/or Cas12 b.

6. A method for detecting the presence of a signature sequence to be detected in a target nucleic acid using the system of claim 5,

obtaining target nucleic acid, reacting the target nucleic acid with the reaction system for a period of time, and then contacting the detection agent with the reaction system;

judging whether the characteristic sequence to be detected exists in the target nucleic acid according to the following results:

if the target nucleic acid does not have the characteristic sequence to be detected, the single-stranded oligonucleotide is not cleaved, and after the detection agent is contacted with the reaction system, more signal is captured by the second substance and accumulated at the second detection line, and less signal appears in the first detection line or no signal appears in the first detection line;

alternatively, if the sequence to be detected is present in the target nucleic acid, the single-stranded oligonucleotide will be cleaved and, upon contact of the detection agent with the reaction system, more of the signal will be captured by the first substance and accumulate at the first detection line, with less signal present at the second detection line or no signal present at the second detection line.

7. A kit for detecting the presence of a signature sequence to be detected in a target nucleic acid based on CRISPR technology, comprising the system of any one of claims 4 to 5; preferably, the kit further comprises primers for amplifying the target nucleic acid.

8. Use of the system according to any one of claims 5 to 6 or the kit according to claim 7 for diagnosing or detecting the presence of a characteristic sequence to be detected in a sample to be tested.

9. The method according to any one of claims 1 to 3 or the use according to claim 8, wherein the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence associated with a disease, a specific mutation site or a SNP site; more preferably, the virus is a plant virus or an animal virus; more preferably, the virus is a coronavirus.

10. The method of any one of claims 1 to 3 or the use of claim 8, wherein the target nucleic acid is derived from a virus, a bacterium, a microorganism, soil, a water source, a human.

Technical Field

The invention relates to the field of nucleic acid detection, in particular to a method and a system for detecting target nucleic acid based on CRISPR technology.

Background

The method for specifically detecting Nucleic acid molecules (Nucleic acid detection) has important application values, such as pathogen detection, genetic disease detection and the like. In the aspect of pathogen detection, each pathogenic microorganism has a unique characteristic nucleic acid molecule sequence, so that nucleic acid molecule detection for a specific species, also called Nucleic Acid Diagnostics (NADs), can be developed, and is important in the fields of food safety, detection of environmental microbial contamination, infection of human pathogenic bacteria, and the like. Another aspect is the detection of Single Nucleotide Polymorphisms (SNPs) in humans or other species. Understanding the relationship between genetic variation and biological functions at the genomic level provides a new perspective for modern molecular biology, and SNPs are closely related to biological functions, evolution, diseases and the like, so the development of detection and analysis techniques of SNPs is particularly important.

The detection of specific nucleic acid molecules established today usually requires two steps, the first step being the amplification of the nucleic acid of interest and the second step being the detection of the nucleic acid of interest. The existing detection technologies include restriction endonuclease methods, Southern, Northern, dot blot, fluorescent PCR detection technologies, LAMP loop-mediated isothermal amplification technologies, recombinase polymerase amplification technologies (RPA) and the like. After 2012, CRISPR gene editing technology arose, a new nucleic acid diagnosis technology (SHERLOCK technology) of targeted RNA with Cas13 as a core was developed by the zhanfeng team based on RPA technology, a diagnosis technology (DETECTR technology) with Cas12 enzyme as a core was developed by the Doudna team, and a new nucleic acid detection technology (HOLMES technology) based on Cas12 was also developed by the royal doctor of the institute of physiology and ecology of plants in the shanghai of the chinese academy of sciences. Nucleic acid detection techniques developed based on CRISPR technology are playing an increasingly important role.

Despite the numerous existing nucleic acid detection technologies, how to perform faster, easier, cheaper, and more accurate detection is still an important direction for improving the detection technology. Therefore, the development of novel detection systems and detection methods is still of great significance in the field of nucleic acid detection.

Disclosure of Invention

The invention provides a method, a system and a kit for detecting target nucleic acid based on CRISPR technology.

In one aspect, the present invention provides a method for detecting the presence or absence of a characteristic sequence to be detected in a target nucleic acid based on CRISPR technology, the method comprising:

(1) providing a target nucleic acid, an exonuclease, a gRNA, a Cas protein, and a single-stranded oligonucleotide;

(2) the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least a portion of a single stranded nucleic acid;

(3) the gRNA can target a characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein excites the activity of trans (trans) single-stranded DNA cleavage after recognizing the characteristic sequence to be detected;

(4) the Cas protein cleaves the single-stranded oligonucleotide by a trans (trans) single-stranded DNA cleaving activity, the single-stranded oligonucleotide exhibiting a detectable difference after cleavage by the Cas protein compared to the single-stranded oligonucleotide before cleavage by the Cas protein;

(5) testing whether the detectable difference of step (4) is detectable; if the detectable difference in step (4) can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable difference in step (4) is not detected, it is indicative that the target nucleic acid does not contain the signature sequence to be detected.

In the present invention, the exonuclease is used to digest the target nucleic acid to form a single-stranded nucleic acid, and as shown in FIG. 1, the double-stranded target nucleic acid is cleaved into a form containing a part of the single-stranded nucleic acid by the exonuclease.

In one embodiment, the target nucleic acid comprises DNA, RNA, preferably double-stranded nucleic acid.

In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is the product of PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM amplification.

In one embodiment, the method further comprises the step of obtaining the target nucleic acid from the sample.

In one embodiment, the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence associated with a disease, a specific mutation site or an SNP site; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. If the target nucleic acid has the characteristic sequence to be detected, the sample from which the target nucleic acid is derived can be reflected to be a certain virus, a certain bacterium, or infected with a certain virus, a certain bacterium, or a certain disease, or to have a specific mutation site or SNP site.

In one embodiment, the Cas protein is a protein having double-stranded and/or single-stranded cleavage activity.

In one embodiment, the Cas protein is a protein having Cis and trans cleavage activity.

In one embodiment, the Cas protein is selected from the group consisting of a type V CRISPR/Cas effector protein comprising: cas12, Cas13, Cas14 family proteins, or mutants thereof.

In one embodiment, the Cas protein mutant comprises amino acid substitutions, deletions or substitutions, and the mutant retains at least its trans cleavage activity. Preferably, the mutant has Cis and trans cleavage activity.

In one embodiment, the Cas protein is preferably a Cas12 family, including but not limited to Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12 h; cas12a, Cas12b are preferred.

The Cas12a, also called Cpf1, for example, Chinese patent application CN107488710A discloses that Cas12a can be used for detecting nucleic acid by cleaving a DNA probe with trans cleavage activity.

Further, the Cas12a is selected from a protein consisting of the following sequences:

(1) a protein shown as SEQ ID No. 1;

(2) derived proteins which are formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues in the amino acid sequence shown in SEQ ID No.1 or active fragments thereof and have basically the same functions.

In one embodiment, the Cas12a protein further includes proteins having 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, sequence identity to the above sequences and having trans activity.

The Cas12b, also known as C2C1, for example, chinese patent application CN109689875A discloses C2C1 protein; chinese patent application CN110551800A discloses that Cas12b can be used for detecting nucleic acid by cleaving a DNA probe with trans cleavage activity. In the present invention, the Cas12b is preferably a protein consisting of the following sequence:

(1) a protein shown as SEQ ID No. 4;

(2) derived proteins which are formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues in the amino acid sequence shown in SEQ ID No.4 or active fragments thereof and have basically the same functions.

In one embodiment, the Cas12b protein further includes proteins having 50%, preferably 55%, preferably 60%, preferably 65%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95%, sequence identity to the above sequences and having trans activity.

In one embodiment, the gRNA comprises a sequence targeting the signature sequence to be detected (a guide sequence) and a sequence recognizing the Cas protein (a direct repeat or a portion thereof).

In one embodiment, the targeting sequence comprises 10-25 bp; preferably, 12-23 bp; preferably, 15-23 bp; preferably, 16-18 bp.

In one embodiment, the gRNA has at least a 50% match to the signature sequence to be detected, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

In one embodiment, when the signature sequence contains one or more signature sites (e.g., a particular mutation site or SNP), the signature site is a perfect match to the gRNA.

In one embodiment, one or more grnas targeting different sequences may be included in the detection method, targeting different signature sequences.

In one embodiment, the exonuclease is selected from a 5 'end exonuclease or a 3' end exonuclease; preferably, the exonuclease is a 5' end exonuclease; alternatively referred to as, 5 '→ 3' exonuclease or 3 '→ 5' exonuclease, preferably, 5 '→ 3' exonuclease.

In one embodiment, the exonuclease is selected from the group consisting of: t5 exonuclease, T7 exonuclease, lambda exonuclease or exonuclease VIII.

In one embodiment, said identifying said feature sequence to be detected comprises binding and/or cleaving the feature sequence to be detected.

In one embodiment, the steps (4) and (5) may be implemented by: the 5 'end and the 3' end of the single-stranded oligonucleotide are respectively provided with different reporter groups, when the single-stranded oligonucleotide is cut, a detectable reporter signal can be shown, and whether the target nucleic acid contains a characteristic sequence to be detected is reflected by the existence of the reporter signal; if the reporter signal can be detected, the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the reporter signal is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected. For example, two ends of the single-stranded oligonucleotide are respectively provided with a fluorescent group and a quenching group, when the single-stranded oligonucleotide is cut, a detectable fluorescent signal can be shown, and whether the target nucleic acid contains the characteristic sequence to be detected is reflected by the existence of the fluorescent signal; the fluorescent signal can be detected, and the target nucleic acid contains a characteristic sequence to be detected; alternatively, if the fluorescent signal is not detected, it indicates that the target nucleic acid does not contain the signature sequence to be detected.

In one embodiment, the fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC Red 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In one embodiment, the steps (4) and (5) can also be realized by other ways: the 5 'end and the 3' end of the single-stranded oligonucleotide are respectively provided with different marker molecules, and the colloidal gold test results of the single-stranded oligonucleotide before and after being cut by the Cas protein are detected in a colloidal gold detection mode so as to reflect whether the target nucleic acid contains a characteristic sequence to be detected; the single-stranded oligonucleotide shows different color development results on a colloidal gold detection line and a quality control line before and after being cut by the Cas protein.

In one embodiment, the Cas protein and gRNA are used in a molar ratio of (0.8-1.2): 1.

in one embodiment, the Cas protein is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, more preferably, 50 nM.

In one embodiment, the gRNA is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, and more preferably, 50 nM.

In one embodiment, the target nucleic acid is used in a final concentration of 5-100nM, preferably, 10-50 nM.

In one embodiment, the single stranded oligonucleotide is used at a final concentration of 100-.

In one embodiment, the exonuclease is used in a final concentration of 0.1 to 3.0U/. mu.l, preferably, 0.2 to 2.0U/. mu.l, more preferably, 0.5 to 1.0U/. mu.l.

In one embodiment, the single stranded oligonucleotide has 3-100 nucleotides, preferably, 3-30 nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides.

In one embodiment, the single stranded oligonucleotide is a single stranded DNA molecule.

In one embodiment, the method can be used for the quantitative detection of the signature sequence to be detected.

In another aspect, the present invention also provides a system for detecting whether a characteristic sequence to be detected exists in a target nucleic acid based on CRISPR technology, the system comprising: exonuclease, gRNA, Cas protein, and single-stranded oligonucleotide;

wherein the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least part of a single stranded nucleic acid;

the gRNA can target a characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein excites the activity of trans (trans) single-stranded DNA cleavage after recognizing the characteristic sequence to be detected;

the Cas protein cleaves the single-stranded oligonucleotide by a trans (trans) single-stranded DNA cleaving activity, the single-stranded oligonucleotide exhibiting a detectable difference after cleavage by the Cas protein compared to the single-stranded oligonucleotide before cleavage by the Cas protein;

if the detectable difference can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable difference is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected.

In another aspect, the present invention also provides a kit for detecting the presence or absence of a characteristic sequence to be detected in a target nucleic acid based on CRISPR technology, the kit comprising: exonuclease, gRNA, Cas protein, and single-stranded oligonucleotide;

wherein the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least part of a single stranded nucleic acid;

the gRNA can target a characteristic sequence to be detected, the Cas protein recognizes the characteristic sequence to be detected under the action of the gRNA, and the Cas protein excites the activity of trans (trans) single-stranded DNA cleavage after recognizing the characteristic sequence to be detected;

the Cas protein cleaves the single-stranded oligonucleotide by a trans (trans) single-stranded DNA cleaving activity, the single-stranded oligonucleotide exhibiting a detectable difference after cleavage by the Cas protein compared to the single-stranded oligonucleotide before cleavage by the Cas protein;

if the detectable difference can be detected, reflecting that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable difference is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected.

Further, the kit also comprises a primer for amplifying the target nucleic acid.

In another aspect, the invention also provides the use of the system or the kit in diagnosing whether the characteristic sequence to be detected exists in a sample to be detected.

Further, the use comprises obtaining target nucleic acid from a sample to be detected, and further detecting whether the characteristic sequence to be detected exists in the target nucleic acid.

Preferably, the target nucleic acid may be obtained from the sample to be tested by nucleic acid sequencing-based amplification (NASBA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), or Nicking Enzyme Amplification Reaction (NEAR), PCR, Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR), or derivative amplification methods (RAM).

In a preferred embodiment, the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence related to a disease, a specific mutation site or an SNP site; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. If the target nucleic acid has the characteristic sequence to be detected, the sample from which the target nucleic acid is derived can be reflected to be a certain virus, a certain bacterium, or infected with a certain virus, a certain bacterium, or a certain disease, or to have a specific mutation site or SNP site.

In another aspect, the present invention provides a system for detecting the presence of a signature sequence to be detected in a target nucleic acid, the system comprising a detection device, a reaction system, and a detection agent;

the reaction system comprises exonuclease, gRNA, Cas protein and single-stranded oligonucleotide;

wherein the exonuclease is used to digest the target nucleic acid to form a nucleic acid sequence comprising at least part of a single stranded nucleic acid; the gRNA can target a characteristic sequence to be detected, the Cas protein identifies the characteristic sequence to be detected under the action of the gRNA, the Cas protein has trans (trans) single-stranded DNA cleavage activity, and the Cas protein activates the trans (trans) single-stranded DNA cleavage activity after identifying the characteristic sequence to be detected so as to cleave the single-stranded oligonucleotide;

one end of the single-stranded oligonucleotide is connected with a first marker molecule, and the other end of the single-stranded oligonucleotide is connected with a second marker molecule;

the detection agent is linked with a first binding molecule, and the first labeling molecule can be combined with the first binding molecule;

the detection means (preferably, a test strip) is provided with a first detection line provided with a first substance that can capture a first binding molecule and a second detection line provided with a second substance that can capture a second labeling molecule.

In a preferred embodiment, the detection means is a test strip, preferably a lateral flow test strip.

In one embodiment, the first detection line is downstream of the second detection line in a direction of flow.

Further, the detection agent is preferably disposed on the detection means, and more preferably, the detection agent is disposed upstream of the first detection line and the second detection line in the direction of flow.

In another embodiment, the first detection line and the second detection line are also referred to as a quality control line and a detection line.

In one embodiment, at least a portion of the detection agent or all of the detection agent is contacted with the reaction system after the reaction system has reacted for a period of time.

The detection agent is a colloidal metal, which may comprise water-insoluble metal particles or metal compounds dispersed in a liquid, hydrosol or metal sol, with preferred metals including gold, silver, aluminum, ruthenium, zinc, iron, nickel and calcium, and other suitable metals including their various oxidation states: lithium, sodium, magnesium, potassium, scandium, titanium, vanadium, chromium, manganese, cobalt, copper, gallium, strontium, niobium, molybdenum, palladium, indium, tin, tungsten, rhenium, platinum, and gadolinium; in a preferred embodiment, the colloidal metal is colloidal gold.

The detection agent is nano-scale particle, preferably 2nm-100nm, more preferably 10nm-80nm, more preferably 15nm, 20nm, 30 nm.

After the detection agent is contacted with the reaction system, the detection agent will present different capture signals in the first detection line and the second detection line according to the reaction result of the reaction system.

If the target nucleic acid does not have the characteristic sequence to be detected, the single-stranded oligonucleotide is not cleaved, and after the detection agent is contacted with the reaction system, more signal is captured by the second substance and accumulated at the second detection line, and less signal is present at the first detection line or no signal is present at the first detection line.

If the sequence to be detected is present in the target nucleic acid, the single-stranded oligonucleotide will be cleaved and, upon contact of the detection agent with the reaction system, more of the signal will be captured by the first substance and accumulate at the first detection line, with less signal present at the second detection line or no signal present at the second detection line.

Thus, according to the comparison of the signal intensity of the first detection line and the second detection line, whether the target nucleic acid has the characteristic sequence to be detected can be judged.

In a preferred embodiment, the first labeling molecule is fluorescein, for example Fluorescein Isothiocyanate (FITC) or carboxyfluorescein (FAM), or Digoxin (DIG), 5-carboxytetramethylrhodamine (TAMRA). The second marker molecule is Biotin (Biotin); preferably, the first labeling molecule is disposed at the 5 'end of the single stranded oligonucleotide and the second labeling molecule is disposed at the 3' end of the single stranded oligonucleotide.

Preferably, the first binding molecule is a first antibody capable of binding to a first labeling molecule, and the first substance is a second antibody capable of binding to the first binding molecule. In a preferred embodiment, the first antibody is an anti-FITC antibody, an anti-FAM antibody, an anti-digoxin antibody, an anti-5-carboxytetramethylrhodamine antibody.

Preferably, the second substance is avidin, preferably streptavidin, which can bind to biotin.

In another aspect, the present invention also provides a kit for detecting the presence of a signature sequence to be detected in a target nucleic acid, comprising the above system.

Further, the kit also comprises a primer for amplifying the target nucleic acid.

In another aspect, the invention also provides the use of the system or the kit in diagnosing whether the characteristic sequence to be detected exists in a sample to be detected.

Further, the use comprises obtaining target nucleic acid from a sample to be detected, and further detecting whether the characteristic sequence to be detected exists in the target nucleic acid.

Preferably, the target nucleic acid may be obtained from the sample to be tested by nucleic acid sequencing-based amplification (NASBA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), or Nicking Enzyme Amplification Reaction (NEAR), PCR, Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR), or derivative amplification methods (RAM).

In a preferred embodiment, the characteristic sequence to be detected is a virus-specific sequence, a bacteria-specific sequence, a characteristic sequence related to a disease, a specific mutation site or an SNP site; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. If the target nucleic acid has the characteristic sequence to be detected, the sample from which the target nucleic acid is derived can be reflected to be a certain virus, a certain bacterium, or infected with a certain virus, a certain bacterium, or a certain disease, or to have a specific mutation site or SNP site.

In another aspect, the present invention also provides the use of the system described above for detecting the presence of a signature sequence to be detected in a target nucleic acid.

In another aspect, the invention also provides the use of the system in diagnosing or detecting the presence of the characteristic sequence to be detected in a sample to be detected.

Further, the use comprises obtaining target nucleic acid from a sample to be detected, and further detecting whether the characteristic sequence to be detected exists in the target nucleic acid.

In one embodiment, the Cas protein and gRNA are used in a molar ratio of (0.8-1.2): 1.

in one embodiment, the Cas protein is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, more preferably, 50 nM.

In one embodiment, the gRNA is used in a final concentration of 20-200nM, preferably, 30-100nM, more preferably, 40-80nM, and more preferably, 50 nM.

In one embodiment, the target nucleic acid is used in a final concentration of 5-100nM, preferably, 10-50 nM.

In one embodiment, the single stranded oligonucleotide is used at a final concentration of 100-.

In one embodiment, the exonuclease is used in a final concentration of 0.1 to 3.0U/. mu.l, preferably, 0.2 to 2.0U/. mu.l, more preferably, 0.5 to 1.0U/. mu.l.

General definition:

unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The term "amino acid" refers to a carboxylic acid containing an amino group. Each protein in an organism is composed of 20 basic amino acids.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, whether double-stranded or single-stranded.

The term "oligonucleotide" refers to a sequence of 3 to 100 nucleotides, preferably 3 to 30 nucleotides, preferably 4 to 20 nucleotides, more preferably 5 to 15 nucleotides.

The term "homology" or "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When two sequences to be compared are substituted by the same base or amino acid monomer at a position

When a subunit is occupied (e.g., a position in each of two DNA molecules is occupied by adenine,

or a position in each of the two polypeptides is occupied by a lysine), then the respective molecule is at that position

Are identical. Between the two sequences. Typically, this is done when the two sequences are aligned to yield maximum identity

And (6) comparing. Such an alignment can be determined by using, for example, the identity of the amino acid sequences by conventional methods, as taught by, for example, Smith and Waterman,1981, adv.Appl.Math.2:482Pearson & Lipman,1988, Proc.Natl.Acad.Sci.USA 85:2444, Thompson et al, 1994, Nucleic Acids Res 22:467380, etc., by computerized operational algorithms (GAP, BESTFIT, FASTA, and TFASTA, Genetics Computer Group in the Wisconsin Genetics software package). The BLAST algorithm, available from the national center for Biotechnology information (NCBI www.ncbi.nlm.nih.gov /), can also be used, determined using default parameters.

As used herein, the "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats) derived from the immune system of a microorganism.

As used herein, "biotin", also known as vitamin H, is a small molecule vitamin with a molecular weight of 244 Da. "avidin", also called avidin, is a basic glycoprotein having 4 binding sites with extremely high affinity to biotin, and streptavidin is a commonly used avidin. The very strong affinity of biotin to avidin can be used to amplify or enhance the detection signal in the detection system. For example, biotin is easily bonded to a protein (such as an antibody) by a covalent bond, and an avidin molecule bonded to an enzyme reacts with a biotin molecule bonded to a specific antibody, so that not only is a multi-stage amplification effect achieved, but also color is developed due to the catalytic effect of the enzyme when the enzyme meets a corresponding substrate, and the purpose of detecting an unknown antigen (or antibody) molecule is achieved.

Characteristic sequence

As used herein, the terms "signature sequence" or "signature sequence to be detected" are used interchangeably and refer to a nucleic acid sequence that characterizes an organism-specific or certain characteristic feature that hybridizes to a gRNA guide sequence to promote formation of a CRISPR complex. The signature sequence is a DNA polynucleotide, which can comprise a portion complementary to the gRNA guide sequence in an amount that is the same as or slightly less than the portion complementary to the gRNA guide sequence. In certain embodiments the organisms include animals, plants and microorganisms. The microorganism includes bacteria, fungi, yeast, protozoa, parasites or viruses. For example, the signature sequence may be a nucleic acid sequence which characterises the virus (including a DNA sequence formed by reverse transcription if the virus is an RNA sequence); for example, the signature sequence may be a sequence containing a specific mutation site, such as a tumor-inducing gene mutation site in an animal cell, or some gene mutation site that alters a plant trait in a plant (e.g., a specific mutation site that confers herbicide resistance to an ALS protein).

In certain embodiments, the virus comprises a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus, a retrovirus, or a combination thereof, or the viral infection is caused by a virus of the family Coronaviridae (Coronaviridae), Picornaviridae (Picornaviridae), Caliciviridae (Caliciviridae), Flaviviridae (Flaviviridae), Togaviridae (Togaviridae), Filoviridae (Filoviridae), Paramyxoviridae (Paramyxoviridae), Pneumoviridae (Pneumoviridae), Rhabdoviridae (Rhabdoviridae), Arenaviridae (Arenaviridae), Bunyaviridae (Bunyaviridae), Orthomyxoviridae (Orthomyxoviridae), or Delta viruses, or the viral infection is caused by a coronavirus (Corona virus), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus a), Rhinovirus (Rhinovirus), Rhinovirus (Rhinovirus) or a), Rhinovirus (Rhinovirus) infection by a), Rhinovirus (Rhinovirus) or a), Hepatitis C virus (Hepatitis Cvirus), Dengue virus (Dengue River virus), Zikavirus (Zikavirus), Rubella virus (Rubella virus), Ross River virus (Ross River virus), Sindbis virus (Sindbis virus), Chikungunya virus (Chikungunya virus), Borna disease virus (Borna disease virus), Ebola virus (Ebola virus), novel coronavirus (2019-nCoV), Marburg virus (Marburg virus), measles virus (Measles virus), Mumps virus (Mumps virus), Nipah virus (Nipah virus), Hendra virus (Hendra virus), Newcastle disease virus (Newcastle disease virus), human respiratory syncytial virus (Humanrespiratory syncytial virus), Rabies virus (rabis virus), Lassa virus (Lassa virus), Hantavirus (Hantavirus), Crimean-Congo hemorrhagic fever virus (Crimean-conmorrharge virus), Influenza (inflenza), or Hepatitis d virus (hepatis dvis).

In certain exemplary embodiments, the virus may be a plant virus selected from the group consisting of: tobacco Mosaic Virus (TMV), Tomato Spotted Wilt Virus (TSWV), Cucumber Mosaic Virus (CMV), Potato Virus Y (PVY), RT virus cauliflower mosaic virus (CaMV), plum blossom pox virus (PPV), Brome Mosaic Virus (BMV), Potato Virus X (PVX), Citrus Tristeza Virus (CTV), Barley Yellow Dwarf Virus (BYDV), potato leafroll virus (PLRV), tomato clumping trick virus (TBSV), rice corm virus (RTSV), Rice Yellow Mottle Virus (RYMV), rice grey white virus (RHBV), maize raleigh phenanthroline virus (MRFV), Maize Dwarf Mosaic Virus (MDMV), sugarcane mosaic virus (SCMV), sweet potato feather mottle virus (SPMV), sweet potato sedimentary vein nematode virus (SPV), grape flabellum virus (GFLV), Grape Virus A (GVA), Grape Virus B (GVB), grape spotted virus (GFkV), Grape leaf curl virus-related viruses-1, -2, and-3, (GLRaV-1, -2, and-3), arabis mosaic virus (ArMV), or larch numb-locus-related virus (RSPaV).

In certain embodiments, examples of bacteria include, but are not limited to, one or more (or a combination of) the following: actinobacillus (Actinobacillus), Actinomycetes (Actinomycetes), Actinomycetes (Actinomyces), Aeromonas (Aeromonas) such as Aeromonas hydrophylla, Aeromonas campestris and Aeromonas campestris, Anaplasia angularis, Anaplasia marcescens Alcaligenes Xyloxifragans, Acetobacter baumii, Actinomyces Actinomycetes, Escherichia coli, Fusobacterium nuclearum, Gardnerella vagenalis, Gemelalla Morblillum, Haemophilus (Haemophilus) species (e.g.Haemophilus influezae, Haemophilus ducreyi, Haemophilus saegypticus, Haemophilus parahaemophilus parainfluenzae, Haemophilus Haemophilus and Haemophilus parahaemolyticus, Helicobacter (e.g.Helicobacter pararhizophilus, Helicobacter cinalis and Helicobacter fennellae), Kingella kingii, Klebsiella (Klebsiella) species, Lactobacillus (Lactobacillus) species, Listeria monocytogenes, Leptomonas, Lactobacillus (Lactobacillus) species, Corynebacterium, Lactobacillus species, Mycobacterium species, such as strains, strains such as strains of Bacillus species of Bacillus, strains of Bacillus (Lactobacillus species such as strains of Bacillus, strains of Bacillus, strains of Bacillus, strains of Bacillus, strains of strains, Pasteurella multocida, Pityrosporum orbiculare (Malassezia furur), Providence sp (Providence), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia sp, Salmonella (Salmonella) species (Salmonella enterica, Salmonella typhi, Salmonella serotype spathui, Salmonella enterica, Salmonella serotype serovar, Salmonella enterica, Salmonella choleraesuis, Salmonella typhi, Salmonella typhii, Shigella resistant (Shigella) species (Shigella, Shigella resistant (Shigella), Shigella resistant (Shigella resistant) species (Shigella resistant), such as Shigella resistant Streptococcus pneumoniae, Shigella resistant (Shigella resistant), Shigella resistant Streptococcus pneumoniae, Shigella resistant (Shigella) species (Shigella resistant), Shigella resistant Streptococcus pneumoniae, Shigella resistant Streptococcus (Shigella) species (Shigella resistant), Shigella resistant) species (Shigella resistant) such as Shigella resistant Streptococcus pneumoniae, Shigella resistant Streptococcus (Shigella resistant) S4, Shigella resistant (Shigella resistant) species (Shigella resistant), Streptococcus, Shigella resistant) species (Shigella resistant) such as Shigella resistant to Streptococcus, Streptococcus (Shigella resistant to Shi, Tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, Omptoxin-resistant serotype 14 Streptococcus pneumoniae, rifampin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Yersinia species (Yersinia) such as Yersinia enterocolitica, Yersinia pestis, and Yersinia pseudoticus, and Xanthomonas maphila, and the like.

Target nucleic acid

As used herein, the "target nucleic acid" refers to a polynucleotide molecule extracted from a biological sample (a sample to be tested). The biological sample is any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to single-celled organisms such as bacteria, yeasts, protozoa and amoebae and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or superficially healthy human subjects or human patients affected by a condition or disease to be diagnosed or investigated, e.g. infection by a pathogenic microorganism such as a pathogenic bacterium or virus). For example, the biological sample may be a biological fluid obtained from, for example, blood, plasma, serum, urine, feces, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, exudate (e.g., obtained from an abscess or any other site of infection or inflammation), or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of a skin or mucosal surface. The sample may also be a sample obtained from any organ or tissue (including biopsies or autopsy specimens, e.g., tumor biopsies) or may comprise cells (primary cells or cultured cells) or culture medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cytocentrifuge preparations, cytological smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin-embedded tissue sections).

In other embodiments, the biological sample may be a plant cell, callus, tissue or organ (e.g., root, stem, leaf, flower, seed, fruit), and the like.

In the present invention, the target nucleic acid also includes a DNA molecule formed by reverse transcription of RNA, and further, the target nucleic acid may be amplified by a technique known in the art, such as isothermal amplification techniques, such as nucleic acid sequencing-based amplification (NASBA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), or nickase amplification (NEAR), and non-isothermal amplification techniques. In certain exemplary embodiments, non-isothermal amplification methods may be used, including, but not limited to, PCR, Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR), or derivative amplification methods (RAM).

Further, the detection method of the present invention further comprises a step of amplifying the target nucleic acid; the detection system further comprises a reagent for amplifying the target nucleic acid. The reagents for amplification include one or more of the following: DNA polymerase, strand displacing enzyme, helicase, recombinase, single-strand binding protein, and the like.

Cas protein

As used herein, "Cas protein" refers to a CRISPR-associated protein, preferably from type V CRISPR/Cas protein, which upon binding to a signature sequence (target sequence) to be detected (i.e., forming a ternary complex of Cas protein-gRNA-target sequence) can induce its trans activity, i.e., random cleavage of non-targeted single-stranded nucleotides (i.e., single-stranded oligonucleotides, preferably single-stranded dna (ssdna), as described herein). When the Cas protein is combined with the characteristic sequence, the protein can induce the trans activity by cutting or not cutting the characteristic sequence; preferably, it induces its trans activity by cleaving the signature sequence; more preferably, it induces its trans activity by cleaving the single-stranded signature sequence. The Cas protein recognizes the characteristic sequence by recognizing PAM (proto-spaacedjacent motif) adjacent to the characteristic sequence.

The Cas protein is a protein at least having trans cleavage activity, and preferably, the Cas protein is a protein having Cis and trans cleavage activity. The Cis activity refers to the activity that the Cas protein can recognize a PAM site and specifically cut a target sequence under the action of the gRNA.

The Cas protein provided by the invention comprises V-type CRISPR/CAS effector proteins, including protein families such as Cas12, Cas13 and Cas 14. Preferably, e.g. Cas12 proteins, e.g. Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12 h; preferably, the Cas protein is Cas12a, Cas12 b.

In embodiments, a Cas protein, as referred to herein, such as Cas12, also encompasses a functional variant of Cas or a homolog or ortholog thereof. As used herein, a "functional variant" of a protein refers to a variant of such a protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Also included in functional variants are fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be artificial. Advantageous embodiments may relate to engineered or non-naturally occurring V-type DNA targeting effector proteins.

In one embodiment, one or more nucleic acid molecules encoding a Cas protein, such as Cas12, or orthologs or homologs thereof, may be codon optimized for expression in a eukaryotic cell. Eukaryotes can be as described herein. One or more nucleic acid molecules may be engineered or non-naturally occurring.

In one embodiment, the Cas12 protein or ortholog or homolog thereof may comprise one or more mutations (and thus the nucleic acid molecule encoding it may have one or more mutations.

In one embodiment, the Cas protein may be from: cilium, listeria, corynebacterium, satt's, legionella, treponema, Proteus, eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivola, Flavobacterium, Sphaerochaeta, Azospirillum, gluconacetobacter, Neisseria, Rochelia, Parvibaculum, Staphylococcus, Nitratifroctor, Mycoplasma, Campylobacter, and Muspirillum.

The Cas protein can be obtained by recombinant expression vector technology, namely, a nucleic acid molecule encoding the protein is constructed on a proper vector and then is transformed into a host cell, so that the encoding nucleic acid molecule is expressed in the cell, and the corresponding protein is obtained. The protein can be secreted by cells, or the protein can be obtained by breaking cells through a conventional extraction technology. The encoding nucleic acid molecule may or may not be integrated into the genome of the host cell for expression. The vector may further comprise regulatory elements which facilitate sequence integration, or self-replication. The vector may be, for example, of the plasmid, virus, cosmid, phage, etc. type, which are well known to those skilled in the art, and preferably, the expression vector of the present invention is a plasmid. The vector further comprises one or more regulatory elements selected from the group consisting of promoters, enhancers, ribosome binding sites for translation initiation, terminators, polyadenylation sequences, and selectable marker genes.

The host cell may be a prokaryotic cell, such as E.coli, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell.

gRNA

As used herein, the "gRNA" is also referred to as guide RNA or guide RNA and has a meaning commonly understood by those skilled in the art. In general, the guide RNA may comprise, or consist essentially of, a direct repeat and a guide sequence (guidessequence), also referred to as a spacer (spacer) in the context of an endogenous CRISPR system. grnas may include crRNA and tracrRNA or only crRNA depending on Cas protein on which they depend in different CRISPR systems. The crRNA and tracrRNA may be artificially engineered to fuse to form single guide RNA (sgRNA). In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence (the signature sequence described in the present invention) to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence, typically having a sequence length of 12-25 nt. The direct repeat sequence can fold to form a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The targeting sequence need not be 100% complementary to the signature sequence (target sequence). The targeting sequence is not complementary to the single stranded oligonucleotide.

In certain embodiments, the degree of complementarity (degree of match) between a targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, when optimally aligned. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, the Smith-Waterman algorithm in matlab (Smith-Waterman), Bowtie, Geneius, Biopython, and SeqMan.

The gRNA of the invention can be natural, and can also be artificially modified or designed and synthesized.

Exonuclease

The exonuclease or exonuclease refers to that the exonuclease can digest one nucleotide chain in double-stranded DNA in a specific direction, i.e. hydrolyze a single nucleotide to form a single-stranded nucleic acid (also called a sticky end), and comprises a 5 'end exonuclease and a 3' exonuclease. The 5 'terminal exonuclease means that it digests the nucleotide chain in the 5' → 3 'direction, and the 3' exonuclease means that it digests the nucleotide chain in the 3 '→ 5' direction. The exonuclease in the invention is preferably DNA exonuclease. Such exonucleases include, but are not limited to: t5 exonuclease, T7 exonuclease, lambda exonuclease or exonuclease VIII and functional variants thereof.

The exonuclease of the invention is preferably an enzyme that does not substantially digest single-stranded oligonucleotides.

Single-stranded oligonucleotides

The single-stranded oligonucleotide of the present invention refers to a sequence containing 3 to 100 nucleotides, preferably, 3 to 30 nucleotides, preferably, 4 to 20 nucleotides, and more preferably, 5 to 15 nucleotides. Preferably a single stranded oligo-DNA molecule.

In one embodiment, the single stranded oligonucleotide may comprise A, T, C, G four mononucleotides.

The single-stranded oligonucleotide is used in a detection method or system to report whether a characteristic sequence is contained. The oligonucleotide includes different reporter groups or marker molecules at both ends, which do not exhibit a reporter signal when in an initial state (i.e., non-cleaved state) and which exhibit a detectable signal when the oligonucleotide is cleaved, i.e., exhibits a detectable difference after cleavage from before cleavage. In the present invention, if a detectable difference can be detected, it is reflected that the target nucleic acid contains the characteristic sequence to be detected; alternatively, if the detectable difference is not detectable, it is indicative that the target nucleic acid does not contain the signature sequence to be detected.

In one embodiment, the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In one embodiment, the single stranded oligonucleotide has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded oligonucleotide is matched with a flow strip to detect a characteristic sequence (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody that binds to a first molecule (i.e. a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (control line), and an antibody that binds to a second molecule (i.e. a second molecular antibody, such as avidin) at the second line (test line). As the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g. color). As more reporters are cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (side) flow test or a (side) flow immunochromatographic assay. In certain aspects, the molecules in the oligonucleotide chain may be substituted for each other, or the position of the molecules may be changed, and the modified forms are also included in the present invention as long as the reporting principle is the same as or similar to that of the present invention.

The detection method can be used for quantitative detection of the characteristic sequence to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of a fluorescent group, or the width of a color development strip.

The invention provides a system, a composition and a combination for detecting whether a characteristic sequence to be detected exists in target nucleic acid based on a CRISPR technology, wherein the system, the composition and the combination comprise: exonuclease, gRNA, Cas protein, and single-stranded oligonucleotide; in one embodiment, the test system, composition, or combination further comprises a buffer.

The invention also provides a kit containing the system, the composition and the combined device.

The system, composition, combination and kit of the invention further comprise reagents and primers for nucleic acid amplification.

Drawings

FIG. 1 is a schematic diagram of the technical scheme of the invention.

Figure 2. effect of T5 exonuclease on Cas12a detection system sensitivity. Wherein, 1 is blank control, 2 is an experiment group which adds T5 exonuclease but does not add target nucleic acid, 3 is an experiment group which adds target nucleic acid but does not add T5 exonuclease, and 4 is an experiment group which adds target nucleic acid and T5 exonuclease simultaneously.

Figure 3. effect of different exonucleases on the sensitivity of Cas12b detection system. Wherein, 1 is blank control, 2 is an experiment group which adds T5 exonuclease but does not add target nucleic acid, 3 is an experiment group which adds T7 exonuclease but does not add target nucleic acid, 4 is an experiment group which adds target nucleic acid but does not add exonuclease, 5 is an experiment group which simultaneously adds target nucleic acid and T5 exonuclease, and 6 is an experiment group which simultaneously adds target nucleic acid and T7 exonuclease.

FIG. 4. effect of T5 exonuclease on sensitivity of Cas12a test strip detection system.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

The technical scheme of the invention is based on the following principle, as shown in figure 1, double-stranded target nucleic acid is obtained in a sample to be detected by an amplification method, the target nucleic acid contains a characteristic sequence, and during actual operation, a proper primer can be designed according to the characteristic sequence to amplify the target nucleic acid by taking the sample to be detected as a template; double-stranded target nucleic acid forms single-stranded nucleic acid containing a signature sequence under the action of an exonuclease, e.g., 5 '→ 3' exonuclease; guiding the recognition and binding of the Cas protein on the characteristic sequence by using a gRNA which can be matched with the characteristic sequence; subsequently, the Cas protein activates trans (trans) single-stranded DNA cleavage activity, which can cleave single-stranded oligonucleotides in the system; in this embodiment, the two ends of the single-stranded oligonucleotide are respectively provided with a fluorescent group and a quenching group, and if the single-stranded oligonucleotide is cut, fluorescence is excited; in other embodiments, the single-stranded oligonucleotide may be further provided with a label at both ends thereof, the label being detectable by colloidal gold.