阅读说明：本技术 一种基于CRISPR/Cas9的DNA检测方法及其应用 (DNA detection method based on CRISPR/Cas9 and application thereof ) 是由 王进科 徐新慧 于 2020-04-26 设计创作，主要内容包括：本发明公开了一种基于CRISPR/Cas9的DNA检测方法及其应用,该方法通过将待检DNA分子与一对dCas9-sgRNA室温孵育,形成dCas9-sgRNA-DNA-dCas9-sgRNA复合物,再利用sgRNA上的捕获序列将复合物捕获到固相基质表面并捕获信号报告分子,实现靶DNA分子的检测。本发明的方法可在不经传统核酸检测中进行核酸扩增、核酸杂交等复杂、耗时环节的情况下,快速、简单地实现低至飞摩级DNA分子的检测。本发明成功避免了目前核酸检测和分型领域中核酸杂交和扩增等关键瓶颈问题,实现了可视化和超灵敏的DNA快速检测,在核酸检测领域具有极其广泛的应用价值。(The invention discloses a DNA detection method based on CRISPR/Cas9 and application thereof, the method comprises the steps of incubating a DNA molecule to be detected and a pair of dCas9-sgRNA at room temperature to form a dCas9-sgRNA-DNA-dCas9-sgRNA compound, capturing the compound on the surface of a solid phase matrix by using a capture sequence on the sgRNA, capturing a signal reporter molecule, and realizing the detection of the target DNA molecule. The method can quickly and simply realize the detection of the DNA molecules with low femtomolar level without complicated and time-consuming links such as nucleic acid amplification, nucleic acid hybridization and the like in the traditional nucleic acid detection. The invention successfully avoids the key bottleneck problems of nucleic acid hybridization, amplification and the like in the field of nucleic acid detection and typing at present, realizes visualized and ultrasensitive DNA rapid detection, and has extremely wide application value in the field of nucleic acid detection.)

1. A DNA detection method based on CRISPR/Cas9, which is characterized by comprising the following steps:

(1) incubating the DNA molecule to be detected with a pair of dCas9-sgRNA to form a dCas9-sgRNA-DNA-dCas9-sgRNA compound;

(2) capturing a dCas9-sgRNA-DNA-dCas9-sgRNA complex to the surface of a solid phase substrate by using a capture sequence on the sgRNA of one dCas 9-sgRNA;

(3) the signal reporter was captured using a capture sequence on the sgRNA of another dCas 9-sgRNA.

2. The DNA detection method according to claim 1, wherein the pair of dCas9-sgRNA in step (1) refers to two dCas9-sgRNA complexes, i.e., dCas9-sgRNA and dCas 9-sgRNA; wherein the sgRNA and the sgRNA have different 5 'end target DNA binding sequences and 3' end capture sequences respectively.

3. The DNA detection method according to claim 2, wherein the sequence of the capture sequence at the 3' end of the sgRNA is (SEQ ID No.1) 5'-CGGAA CCTTA CGAAT ACCAG ATGC-3'; the sequence of the capture sequence at the 3' end of the sgRNA is (SEQ ID NO.2) 5'-TACTT CATGT TACAG ACGAC TCCCA C-3' or (SEQ ID NO.3) 5'-ATCTA GTGGA ACCTC AAACA TACC-3'.

4. The DNA detection method according to claim 2, wherein the target DNA binding sequences at the 5' ends of the sgRNA and the sgRNA are 20bp long sequences, and dCas9-sgRNA complex is guided to bind to the target DNA by hybridization with the target DNA to form dCas9-sgRNA-DNA-dCas9-sgRNA complex; the sgRNA and sgRNA, when used to detect papillomavirus DNA molecules, have target DNA binding sequences at the 5' ends selected from one pair of sequences SEQ ID NO. 4-33; when detecting the DNA of the Escherichia coli T7 RNA polymerase, the 5' end target DNA binding sequences of sgRNA and sgRNA are respectively SEQ ID NO. 34-35; when detecting mutant TERT promoter DNA, the target DNA binding sequences of 5' ends of sgRNA and sgRNAb are respectively SEQ ID NO. 36-37.

5. The DNA detection method according to any one of claims 1 to 4, wherein the step (2) of capturing dCas9-sgRNA-DNA-dCas9-sgRNA complex onto the surface of the solid phase substrate using the capture sequence on the sgRNA means that dCas9-sgRNA-DNA-dCas9-sgRNA complex can be captured onto the surface of the solid phase substrate by means of the capture sequence at the 3' end of the sgRNA.

6. The DNA detection method according to claim 5, wherein the solid phase matrix of step (2) preferably comprises various solid phase matrices including microspheres, microwell plates, glass plates or nanoparticles; the surface of the solid phase matrix is fixed with capture oligonucleotide; wherein the sequence of the capture oligonucleotide is base complementary to the 3' capture sequence of the sgRNAa; wherein the capture oligonucleotide has the sequence (SEQ ID NO.38)5'-GCATC TGGTATTCGT AAGGT TCCG-3'.

7. The DNA detection method according to any one of claims 1 to 4, wherein the capturing of the signal reporter by the capture sequence on the sgRNA in step (3) is performed by capturing the signal reporter to dCas9-sgRNA-DNA-dCas9-sgRNA complex by the capture sequence at the 3' end of the sgRNA.

8. The DNA detection method according to claim 7, wherein the signal reporter molecule of step (3) is a hybrid chain reaction hairpin 1; wherein the hybrid chain reaction consists of two DNA molecules of hairpin 1 and hairpin 2; wherein the 3' capture sequence (SEQ ID No.39) 5'-TACTT CATGT TACAG ACGAC TCCCAC-3' of sgRNAb can open hairpin 1 by hybridization; the opened hairpin 1 can be crossed with the hairpin 2, and the hairpin 2 is opened; the opened hairpin 2 can be hybridized with the hairpin 1 to open the hairpin 1; so circulating, and forming a continuously prolonged DNA chain; the 3' end of the hairpin 1 has the sequence of (SEQ ID NO.40) 5'-G TGGGAGTCGT CTGTA ACATG AAGTA-3'.

9. The DNA detection method according to claim 7, wherein the signal reporter molecule in step (3) is a biotin-labeled oligonucleotide having a sequence complementary to a base of the 3' -capture sequence of the sgRNA; wherein the sequence of the biotin-labeled oligonucleotide is (SEQ ID NO.41)5'-TTTTT TGGTA TGTTT GAGGTTCCAC TAGAT-3'.

10. The method for detecting DNA according to claim 9, wherein the biotin-labeled oligonucleotide is one in which a biotin molecule is bonded to an enzyme-labeled streptavidin molecule.

11. The DNA detection method of claim 10, wherein the enzyme is horseradish peroxidase, which can generate a pigment molecule by catalyzing a substrate for reporting a detection signal by detecting a light absorbance value; the substrate is TMB.

12. Use of the CRISPR/Cas 9-based DNA detection method of claim 1 in the preparation of detection reagents for detecting various DNA molecules.

13. Use of the CRISPR/Cas 9-based DNA detection method of claim 1 in the preparation of a detection reagent for detecting papillomavirus DNA molecules.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a DNA detection method based on CRISPR/Cas9 and application thereof.

Background

DNA detection and genotyping have long been important for basic research, various detection and diagnostic applications. Therefore, DNA detection and genotyping techniques have been receiving much attention, thereby promoting the development of such techniques. In conclusion, there are mainly three types of DNA detection and genotyping techniques that are widely used. The first is a variety of techniques based on Polymerase Chain Reaction (PCR). PCR is the most commonly used technique for DNA detection and genotyping. PCR-based DNA detection and genotyping relies mainly on the design of specific primers and multiplex PCR amplification. PCR detection can be achieved by conventional PCR (tpcr), quantitative PCR (qpcr), and recently developed digital PCR. Q-PCR is highly popular in almost all research, detection and diagnostic laboratories because of its obvious advantages, such as real-time detection and high sensitivity. More accurate digital PCR has now been developed with great potential and advantages as a clinical testing tool. However, PCR techniques are limited to multiplex amplification and highly specific primers when used to distinguish between highly related genotypes. In addition to PCR, various DNA hybridization techniques such as DNA microarray are widely used for detecting and typing DNA. However, due to its expensive equipment, complicated detection procedures and inevitable nonspecific hybridization, the DNA microarray technology cannot become a conventional DNA detection and genotyping tool like PCR. DNA sequencing is another effective DNA detection and genotyping technique. Particularly with the advent of Next Generation Sequencing (NGS) technology, more and more DNA sequencing tools are available for NGS platforms such as illumina novaseq. However, they are still not as useful for routine research, detection and diagnosis as PCR due to the need for expensive equipment and chemicals. In addition, in recent years, various isothermal amplification techniques for nucleic acids have been developed for nucleic acid detection, such as Rolling Circle Amplification (RCA), Recombinase Polymerase Amplification (RPA), Multiple Displacement Amplification (MDA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-dependent amplification (NASBA), helicase-dependent amplification (HDA), Nicking Enzyme Amplification Reaction (NEAR), etc., but these techniques all rely on a diverse amplification procedure for a target nucleic acid to detect the nucleic acid.

Ishino et al first discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in the genome of escherichia coli (e.coli) in 1987 and was defined by Jansen et al as CRISPRs in 2002. Currently, known CRISPR systems comprise three different types (types I, II and III). Type I and III systems consist of multiple Cas proteins, whereas type II systems require only one Cas protein Cas 9. Cas9 is associated with CRISPR-associated rna (crrna) and trans-activated crrna (tracrrna). The Cas9 nuclease can be activated by TracrRNA, which is complementary to the 20 nucleotide sequence of the target DNA. The latter therefore determines the specificity of the CRISPR-Cas9 system. The crRNA-guided Cas9 nuclease can bind to target DNA adjacent to a Primitive Adjacent Motif (PAM) and cleave the target DNA three bases upstream of the PAM sequence (NGG). The integration of tracrRNA and crRNA into one single guide rna (sgrna) greatly simplifies the application of type II CRISPR systems. Cas9 was guided by the sgRNA to cleave the target DNA. Currently, the CRISPR-Cas9 system is widely used by many researchers in the field of genome editing due to its simplicity and high efficiency. In addition, dCas9(dead Cas9) is modified from Cas9, the nuclease activity is lost, but a gene transcription Activation Domain (AD) or a gene transcription Inhibition Domain (ID) is reserved, and dCas9(dead Cas9) is widely applied to endogenous gene expression regulation as a novel artificial transcription factor.

Although Cas9/sgRNA has been widely used for gene editing and regulation, it has not been fully explored for use in the field of nucleic acid detection. By virtue of high specificity of DNA recognition and cutting ability (capable of distinguishing single base), Cas9/sgRNA and other CRISPR-associated nucleases (such as Cpf1 and the like) have great potential in DNA detection and typing. More recently, the CRISPR-Cas9 system has been used to detect Zika virus and to be able to type both us and african Zika viruses. In view of the high specificity of the tools of CRISPR, CRISPR-Cas9 can achieve single base resolution in differentiating viral strains, and can perform typing detection on orthologous bacteria and viruses at the single base level. Recently the CRISPR system (Cas13 a/C2C2 of type III) has been applied to the detection of Zika virus and has an ultra high sensitivity (amount of virus particles as low as 2 aM). These studies indicate that CRISPR systems have great potential and advantages for the development of nucleic acid detection techniques. However, in the currently reported Cas 9-based nucleic acid detection methods, they achieve the purpose of RNA typing by first reverse transcribing single-stranded DNA with RNA to be detected, then generating double-stranded DNA, and then cleaving the double-stranded DNA with Cas9/sgRNA system. Thus, the Cas9/sgRNA system has not been fully developed for detection and typing of nucleic acids at present.

Based on the sequence-specific cleavage function of the CRISPR system on nucleic acid molecules, the application of the CRISPR system in the field of nucleic acid detection is being developed gradually. In addition to the Cas9 enzyme, applications of other Cas proteins have also demonstrated application value in the field of nucleic acid detection in CRISPR systems. For example, Cas13a (also called C2C2) of the type III CRISPR system has recently been applied to the detection of Zika virus and has an ultra-high sensitivity (the amount of virus particles is as low as 2aM) (this method is named Sherlock) (Science 2017; 356: 438-. However, Sherlock technology can only be used for detecting RNA because it relies on cas13a enzyme which can only cut RNA; if the DNA needs to be detected, the recombinase amplification (RPA) technology is used for carrying out isothermal amplification on the DNA, a T7 promoter sequence is introduced at the tail end of an amplification product through a primer during amplification, then in-vitro transcription is carried out to generate RNA, then the RNA is subjected to Cas13a specific cleavage, and further the non-specific cleavage activity of the Cas13a on the single-stranded RNA is activated, so that the purpose of detecting the DNA is achieved. The detection technology depends on recombinase amplification and in vitro transcription, and although the detection technology is favorable for improving the sensitivity of detection, the detection process is complicated and the cost is high. Furthermore, based on the property that Cas12a (also referred to as Cpf1) specifically cleaves target double-stranded DNA (dsDNA) followed by non-specific single-stranded DNA (ssDNA), a new technique for detecting target DNA molecules (this method is named Detectr) was developed (Science 2018; 360: 436-439). The Detectr technology can detect amole-grade molecules and has high sensitivity, but like Sherlock, the Detectr also depends on a nucleic acid isothermal amplification process, and is high in cost and time-consuming. Another nucleic acid detection technique similar to Detectr based on Cas12a was named HOLMES (Cell Research 2018; 28: 491-493; Celldiscovery 2018; 4: 20). A similar principle is also proposed for Cas12 b-based HOLMESv2 (HOLMES2.0) technology (ACS Synth. biol.2019; 8:2228-2237) that exploits the ssDNA lateral cleavage activity of Cas12 b. The HOLMESv2 technique also relies on an isothermal nucleic acid amplification technique, LAMP or asymmetric pcr (asymmetric pcr) amplification technique. These studies show that the CRISPR system has great potential and advantages when used for developing nucleic acid detection technology, but at present, there are also various nucleic acid amplification technologies that depend on pre-amplification such as reverse transcription, in vitro transcription, PCR amplification, RPA amplification, LAMP amplification, asymmetric PCR amplification and the like of DNA or RNA, so that the CRISPR system can be used for specific cleavage of various Cas proteins (Cas13a, Cas12a, Cas12b) and grnas and non-specific cleavage (signal amplification) detection of fluorescent reporter molecules.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a DNA detection method based on CRISPR/Cas9, which is called CADD for short, namely CRISPR/Cas9 Assisted DNA detection (CRISPR/Cas9-Assisted DNADIECE). The method can complete DNA detection through three steps, and the DNA detection method of CRISPR/Cas9 can realize the detection of DNA molecules with low to femtomolar (fM) level without complicated procedures such as enzyme reaction, amplification, high-temperature hybridization and the like; in addition, the method realizes efficient DNA molecular typing without traditional procedures such as traditional nucleic acid specific amplification (such as PCR, RPA and the like), high-temperature hybridization (such as chip hybridization), sequencing and the like.

The invention also provides applications of the DNA detection method based on CRISPR/Cas9, including applications in various DNA molecule detection, particularly applications in detection of papilloma virus (HPV).

The technical scheme is as follows: in order to achieve the above object, the DNA detection method based on CRISPR/Cas9 according to the present invention comprises the following steps:

(1) incubating the DNA molecule to be detected with a pair of dCas9-sgRNA at room temperature to form a dCas9-sgRNA-DNA-dCas9-sgRNA compound;

(2) capturing a dCas9-sgRNA-DNA complex to the surface of a solid phase substrate by using a capture sequence on the sgRNA of one dCas 9-sgRNA;

(3) the signal reporter was captured using a capture sequence on the sgRNA of another dCas 9-sgRNA.

Wherein the pair of dCas9-sgRNA in step (1) refers to two dCas9-sgRNA complexes, namely dCas9-sgRNA a and dCas9-sgRNA b; wherein the sgRNA and the sgRNA have different 5 'end target DNA binding sequences and 3' end capture sequences respectively.

Wherein sgRNA means single-guide RNA (single-guide RNA); it differs from the conventional sgRNA in that its 3' end contains a capture sequence, which is a capture sgRNA.

Among them, dCas9 is inactivated Cas9 (inactivated Cas9), and this Cas9 protein loses activity of cutting DNA, but can bind to sgRNA and target-bind to DNA.

Preferably, the preferred sequence of the 3' capture sequence of the sgrna is (SEQ ID No.1) 5'-CGGAACCTTA CGAAT ACCAG ATGC-3'; the preferred sequence of the capture sequence at the 3' end of the sgRNA is (SEQ ID NO.2) 5'-TACTT CATGT TACAG ACGAC TCCCA C-3' or (SEQ ID NO.3) 5'-ATCTA GTGGA ACCTCAAACA TACC-3'.

Wherein, the target DNA binding sequences of the 5' ends of the sgRNAi and the sgRNAb are both sequences with the length of 20 bp; wherein, when detecting papillomavirus DNA molecules, the preferred sequence of the target DNA binding sequence of the 5' ends of the sgRNA a and the sgRNA b is the sequence described in the description sequence list 1.

Wherein the 5 'end target DNA binding sequences of the sgRNA and the sgRNA are both 20bp long sequences and have the functions of guiding dCas9-sgRNA complex to be bound with target DNA through hybridization with the target DNA to form dCas9-sgRNA-DNA-dCas9-sgRNA complex, and the 5' end target DNA binding sequences of the sgRNA and the sgRNA are shown in SEQ ID NO.4-37 respectively.

Sequence Listing 1, sgRNA sequences targeting 15 hrHPV DNAs, T7 RNA polymerase DNA and TERT promoter (SEQ ID NO.4-37 in order)

Wherein, the method of the invention can detect any target DNA, when detecting papillomavirus DNA molecules, the preferred sequences of the target DNA binding sequences of 5' ends of sgRNA and sgRNA are a pair of sequences SEQ ID NO.4-33 in the sequence table 1 of the specification; when detecting the DNA of the Escherichia coli T7 RNA polymerase, the optimal sequences of the 5' end target DNA binding sequences of the sgRNA and the sgRNA are respectively the sequences SEQ ID NO.34-35 in the sequence table 1; when detecting mutant TERT promoter DNA, the preferable sequences of the target DNA binding sequences of 5' ends of sgRNA and sgRNAb are respectively SEQ ID NO.36-37 of the sequence table 1.

Wherein, the step (2) of capturing the dCas9-sgRNA-DNA complex on the surface of the solid phase matrix by using the capture sequence on the sgRNA means that the dCas9-sgRNA-DNA-dCas9-sgRNA complex can be captured on the surface of a solid phase matrix by using the capture sequence at the 3' end of the sgRNA.

Preferably, the solid phase matrix in step (2) includes various solid phase matrices, such as various microspheres (beads) (e.g., magnetic microspheres, encoded microspheres, polymer microspheres, etc.), microwell plates, glass sheets, nanoparticles (e.g., nanogold), and the like; the surface of the solid phase matrix is fixed with capture oligonucleotide; wherein the sequence of the capture oligonucleotide is base complementary to the 3' capture sequence of the sgRNAa; preferably, the capture oligonucleotide has the sequence (SEQ ID NO.38)5'-GCATC TGGTA TTCGT AAGGTTCCG-3'.

Wherein the capturing of the signal reporter molecule by the capture sequence on the sgRNA in step (3) means that the signal reporter molecule is captured onto the dCas9-sgRNA-DNA-dCas9-sgRNA complex by the capture sequence at the 3' end of the sgRNA.

Preferably, the signal reporter molecule in the step (3) is hairpin 1 of hybrid chain reaction; wherein the Hybridization Chain Reaction (HCR) consists of two DNA molecules of Hairpin 1(Hairpin 1) and Hairpin 2(Hairpin 2); wherein the 3' capture sequence (SEQ ID No.39) 5'-TACTT CATGT TACAG ACGACTCCCA C-3' of sgRNAb can open hairpin 1 by hybridization; the opened hairpin 1 can be crossed with the hairpin 2, and the hairpin 2 is opened; the opened hairpin 2 can be hybridized with the hairpin 1 to open the hairpin 1; so circulating, and forming a continuously prolonged DNA chain; preferably, the 3' end sequence of hairpin 1 is (SEQ ID NO.40) 5'-G TGGGA GTCGT CTGTA ACATG AAGTA-3'.

Wherein, hairpin 1 or hairpin 2 or both of the hybrid chain reaction are marked by fluorescent molecules and used for reporting detection signals; wherein, hairpin 1 or hairpin 2 of the hybrid chain reaction can also be double-labeled with a quenching group and a fluorescent molecule to prepare a Molecular Beacon (MB).

Preferably, the signaling reporter molecule in step (3) is a biotin-labeled oligonucleotide, wherein the sequence of the biotin-labeled oligonucleotide is base-complementary to the 3' -capture sequence of the sgrna; preferably, the biotin-labeled oligonucleotide has the sequence (SEQ ID NO.41)5'-TTTTT TGGTA TGTTT GAGGT TCCAC TAGAT-3'.

Wherein, the biotin molecule of the biotin-labeled oligonucleotide can be combined with an enzyme-labeled streptavidin (streptavidin) molecule, wherein the enzyme is preferably Horse Radish Peroxidase (HRP) which can generate a pigment molecule by catalyzing a substrate and is used for reporting a detection signal by detecting a light absorption value; preferably, the substrate is 3,3',5,5' -Tetramethylbenzidine (TMB).

Preferably, the signaling reporter molecule can also be a nanoparticle-labeled oligonucleotide; the nanoparticles include nanogold (AuNPs), Quantum Dots (QDs), and the like; wherein the sequence of the nanoparticle label-derived oligonucleotide is base complementary to the 3' capture sequence of the sgrna; preferably, the sequence of the oligonucleotide in which the nanoparticle is labeled is (SEQ ID NO.41)5'-TTTTT TGGTA TGTTT GAGGT TCCAC TAGAT-3'.

Preferably, the 3' capture sequence of the sgRNA can also be designed into other signal reporting structures, such as rnango structure, MS2 structure, rolling circle primer, Cas13a-sgRNA target, and the like.

By using the technical principle of the invention, the dCas9 can be replaced by other Cas proteins to realize the detection of DNA or RNA, and the detection of RNA can be realized by replacing dCas9 by Cas13a or dCas13 a.

The DNA detection method based on CRISPR/Cas9 is applied to preparation of detection reagents for detecting various DNA molecules. The DNA detection method of the invention can be applied to qualitative and quantitative detection of various DNA molecules.

The DNA detection method based on CRISPR/Cas9 is applied to the preparation of a detection reagent for detecting papilloma virus DNA molecules, and can be applied to the qualitative and quantitative detection of papilloma virus (HPV) DNA molecules; wherein the 5' end target DNA binding sequence of the sgRNA for detecting 15 high-risk papilloma viruses is shown as a sequence SEQ ID NO.4-33 in a sequence table 1.

Wherein the 15 high-risk papillomaviruses refer to HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68 and HPV 73.

The method can be demonstrated by taking any DNA as a detection material and also taking HPV DNA as a material. In addition, the DNA detection method based on CRISPR/Cas9 provided by the invention designs and proves a set of sgRNAs targeting hrHPV, and basically develops a new technical method for detecting clinical samples of ready-to-use HPV.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention develops a novel DNA detection method based on CRSIPR/Cas 9. The method can simply, quickly and super sensitively carry out specific detection and typing on the target DNA molecules. The invention utilizes the specific binding property of the Cas9-sgRNA compound to the target DNA, successfully avoids the key bottleneck problems of DNA hybridization, amplification and the like in the field of DNA detection and typing at present, and realizes the visual, high-specificity and high-sensitivity rapid DNA detection. The novel DNA detection method based on CRSIPR/Cas9 provided by the invention is completely different from the principles of various nucleic acid detection methods (such as Sherlock, Decctector and HOLMES) based on CRISPR systems reported at present in detection principle, and has the most remarkable advantages of not depending on pre-amplification (in vitro transcription, RPA, LAMP and the like) of nucleic acid samples to be detected and not depending on enzyme reactions (such as DNA polymerase reaction, Cas protein nucleic acid cleavage reaction and the like). The novel DNA detection method provided by the invention has wide application value in the field of nucleic acid detection.

The novel DNA detection method based on CRSIPR/Cas9 provided by the invention can realize the report of detection signals by a very flexible signal reporting mode. The invention demonstrates CADD methods of three signal reporting modes by examples, which are Beads-HCR CADD, Beads-ELISA CADD and DNA-Bind-ELISA CADD respectively. In the three signal reporting modes, the signal output modes of CADD detection are respectively carried out by using microsphere hybrid chain reaction (Beads-HCR), microsphere enzyme-linked immunosorbent assay (Beads-ELISA) and DNA binding ELISA (DNA-Bind-ELISA). In Beads-HCR, using fluorescence labeled HCR hairpin molecule report detection signal; in the Beads-ELISA and in the DNA-Bind-ELISA, color reaction was observed by streptavidin-conjugated HRP catalyzed TMB to report the detection signal. Among them, ELISA is an abbreviation of the English enzymelinkedimunosorbent assay.

In conclusion, the method can rapidly and simply realize the detection of the DNA molecules with low to femtomolar level without complex and time-consuming links such as nucleic acid amplification, nucleic acid high-temperature hybridization and the like in the traditional nucleic acid detection. The invention utilizes the specificity recognition and combination characteristics of the Cas9-sgRNA compound on DNA molecules, successfully avoids the key bottleneck problems of nucleic acid hybridization, amplification and the like in the field of nucleic acid detection and typing at present, realizes visualized and ultrasensitive DNA rapid detection, and has extremely wide application value in the field of nucleic acid detection. The DNA detection method based on CRISPR/Cas9 can be applied to the preparation of detection reagents for detecting various DNA molecules and can also be applied to the preparation of detection reagents for detecting papilloma virus DNA molecules.

Drawings

FIG. 1 is a schematic diagram of the principle of the Beads-HCR CADD, A: the principle of the Beads-HCR CADD is shown schematically. dCas9, dCas9 protein; sgRNA, guide RNA; beads, microspheres; capture; hairpin, Hairpin; target DNA, Target DNA; mix, mixing; bind, Bind; HCR, Hybridization Chain Reaction (Hybridization Chain Reaction); FAM, fluorescein; beads @ FAM, microspheres attached to FAM molecules. B: liquid phase detection of HCR reaction. Initiator, Initiator; initiator oligo, Initiator oligonucleotide. C: adding sgRNA, sgRNA (initiator) and initiator oligo into HCR reaction solution containing Hairpin 1 and Hairpin 2, and detecting reaction products by agarose gel electrophoresis; a reaction without the initiator oligo (no initiator oligo) was used as a negative control. The right side of the electrophoresis chart is the secondary structure of the Hairpin 1 and the Hairpin 2, and the lower side is the sequence and the fluorescence modification of the Hairpin 1 and the Hairpin 2.

FIG. 2 shows the detection of T7 polymerase DNA using the Beads-HCR CADD. A and B: different concentrations of T7 polymerase DNA were detected using the Beads-HCR CADD. A: beads fluorescence images; b: and (5) measuring and analyzing the results of the Beads fluorescence. A linear relationship between DNA concentration and fluorescence signal exists between 100pM and 10 fM. C-D: genomic DNA (gDNA) of both BL21 and DH 5. alpha. bacteria was detected at different concentrations using the Beads-HCR CADD. C: beads fluorescence images; d: and (5) quantitatively analyzing results of the Beads fluorescence. Each field shows bright field and fluorescent images.

FIG. 3 is the detection of TERT promoter DNA using Beads-HCR CADD. A: beads fluorescence images; b: and (5) quantitatively analyzing results of the Beads fluorescence.

FIG. 4 shows the detection of HPV16 DNA using the Beads-HCR CADD. A and B: different concentrations of HPV16 DNA were detected using the Beads-HCR CADD. The sgRNA used targets HPV 16. A: beads fluorescence images; b: and (5) quantitatively analyzing results of the Beads fluorescence. A linear relationship between DNA concentration and fluorescence signal exists between 100pM and 10 fM. C and D: detection of HPV16 and HPV18 DNA (pMD-HPV16 and pMD-HPV18) was performed using the Beads-HCR CADD. C: beads fluorescence images; d: and (5) quantitatively analyzing results of the Beads fluorescence. The sgRNA used targets the sgRNA of HPV16 and HPV18 (sgRNA16 and sgRNA 18). sgrnact (sgrnacocktail) is an equimolar mixture of sgrnas targeting 15 hrHPV.

FIG. 5 shows the detection of DNA of cervical cancer cell line by Beads-HCR CADD. A: beads fluorescence images; b: and (5) quantitatively analyzing results of the Beads fluorescence. Extracting genome DNA (gDNA) of three cervical cancer cell strains, and detecting three gDNA samples by sgRNA targeting HPV16 and HPV18 respectively.

FIG. 6 shows detection of HPV DNA using Beads-HCR CADD. A and B: detection of HPV16 and HPV18 DNA was performed using the Beads-HCR CADD. The sgRNA used was sgRNA and sgRNA that targeted 15 hrHPV. A: various sgrnas detect Beads fluorescence images of pMD-HPV 16; b: the result of the Beads fluorescence quantitative analysis of pMD-HPV18 was detected for various sgRNAs. C-D: the Beads-HCR CADD was used to detect pMD-HPV16 and pMD-HPV 18. C: beads fluorescence images; d: and (5) performing quantitative analysis on the Beads fluorescence.

FIG. 7 shows HPV DNA detection using Beads-HCR CADD. The DNA of 15 hrHPV were detected using the Beads-HCR CADD. The sgRNA used was sgRNA and sgRNA that targeted 15 hrHPV. A: beads fluorescence images; only the fluorescence images of each DNA detected by its corresponding sgRNA and sgRNAct are shown. B-C: and (3) detecting the result of the quantitative analysis of the fluorescence of the Beads of various HPV DNAs by various sgRNAs. B: quantitative analysis of Beads fluorescence images of each HPV DNA detected by its corresponding sgRNA (left) and sgRNAct (right). C: quantitative analysis of Beads fluorescence images detected by various sgrnas for each HPV DNA. D: results of quantitative analysis of Beads fluorescence images (mean fluorescence intensity heatmap) detected by various sgrnas for each HPV DNA. Both panels C and D show that each HPV DNA was detected by its corresponding sgRNA and sgRNA only, while no fluorescence signal was detected by the other sgrnas.

FIG. 8 is a first HPV clinical sample tested with the Beads-HCR CADD. Beads-HCR CADD was used to test 15 hrHPV infections in 31 clinical samples. The sgRNA used was sgRNA and sgRNA that targeted 15 hrHPV. A: beads fluorescence images; only fluorescence images of sample 36 detected by various sgrnas are shown (right side is an enlarged view of the sgRNAct detected sample). B: results of quantitative analysis of Beads fluorescence images (mean fluorescence intensity heatmap) for detection of HPV infection in 31 clinical samples by various sgrnas. C: comparison of the results of PCR and CADD detection of HPV infection in 31 clinical samples. PCR detection is finished by a general hospital in the east war zone; hrHPV: high risk HPV; PCR-rd: HPV45 in samples 2 and 37 and HPV59 in sample 11 were again detected by specific PCR amplification.

FIG. 9 shows the re-detection of HPV45 in samples 2 and 37 and HPV59 infection in sample 11 by specific PCR amplification. A pair of HPV45 and HPV59 specific primers were designed to amplify different DNA samples (templates), respectively.

FIG. 10 shows the detection of HPV16 DNA using Beads-ELISA CADD. A: the principle of the Beads-ELISA CADD is shown schematically. B: different concentrations of HPV16 DNA were detected with Beads-ELISA CADD. The sgRNA used targets HPV16(sgRNA 16). The pictures show imaging of a TMB developed microplate. Each column is four repeats. C: quantitative analysis of TMB color development absorbance. A linear relationship between DNA concentration and absorbance exists between 100pM and 1 fM. D: absorption spectra measured with a microplate.

FIG. 11 is detection of HPV DNA using Beads-ELISA CADD. DNA of 15 hrHPV were detected using Beads-ELISA CADD. The sgRNA used was sgRNA and sgRNA that targeted 15 hrHPV. A: Beads-ELISA microplate image; images of Beads-ELISA microplates with each HPV DNA detected by a variety of sgRNAs are shown. B: results of quantitative analysis of absorbance of Beads-ELISA microplates in A (heat map). C: DNA samples formed from a mixture of two different HPV DNAs were detected with different sgrnas. The images of the Beads-ELISA microplates are shown. D: results of quantitative analysis of absorbance in Beads-ELISA microplates in C (heat map). E: HPV clinical samples were detected with Beads-ELISA CADD (test specificity). The right-hand table shows the DNA samples (text) in each well and the sgRNAs used (color: dark grey for sgRNA, light grey for sgRNA sp, sgRNA sp indicating the specific sgRNA, i.e.the sgRNA for detection of a certain HPV, of the same type as the DNA samples in the same column).

FIG. 12 shows the detection of HPV16 DNA using DNA-Bind-ELISA CADD. A: schematic representation of DNA-Bind-ELISA CADD. B: different concentrations of HPV16 DNA were detected using DNA-Bind-ELISA CADD. The sgRNA used targets HPV16(sgRNA 16). The pictures show imaging of a TMB developed microplate. Each column is three replicates. C: quantitative analysis of TMB color development absorbance. A linear relationship between DNA concentration and absorbance exists between 100pM and 1 fM.

FIG. 13 is a second HPV clinical sample tested with DNA-Bind-ELISA CADD. DNA-Bind-ELISACADD was used to detect infection of 15 hrHPV in 33 clinical samples. The sgRNA used was sgRNA and sgRNA that targeted 15 hrHPV. A: imaging a DNA-Bind-ELISA microporous plate; pMD-HPVct is a mixture containing 15 plasmids (pMD-HPV 16-pMD-HPV 73) and is used as a positive control; empty plasmid pMD served as negative control. B: DNA-Bind-ELISA absorbance quantitative analysis results (heat maps) of HPV infection in 33 clinical samples were detected by each sgRNA. C: comparison of the results of PCR and CADD detection of HPV infection in 33 clinical samples. PCR detection is finished by a general hospital in the east war zone; hrHPV: high risk HPV; recaDD: the 5 samples were again tested for HPV infection using DNA-Bind-ELISA CADD. D and E: the 5 samples were again tested for HPV infection using DNA-Bind-ELISACADD. D: imaging the DNA-Bind-ELISA micropore plate; e: DNA-Bind-ELISA Absorbance quantification assay results (heat map).

Detailed Description

The invention is further illustrated by the following figures and examples.