CN113969308A - Nucleic acid detection method based on gene editing and flow analysis technology - Google Patents

Nucleic acid detection method based on gene editing and flow analysis technology Download PDF

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CN113969308A
CN113969308A CN202111265031.6A CN202111265031A CN113969308A CN 113969308 A CN113969308 A CN 113969308A CN 202111265031 A CN202111265031 A CN 202111265031A CN 113969308 A CN113969308 A CN 113969308A
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nucleic acid
magnetic beads
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韩坤
鞠婷
翟星帏
吴雪兰
李靖雯
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Suzhou Institute of Biomedical Engineering and Technology of CAS
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Abstract

The invention discloses a nucleic acid detection method based on gene editing and flow analysis technology, which comprises the following steps: mixing a sample to be detected, CRISPR-Cas12a protein, crRNA and an initiator, and incubating; adding magnetic beads for reaction, separating and taking out the magnetic beads after reaction, mixing the magnetic beads with the hairpin nucleic acid modified with the marker after washing, and incubating; and finally, separating and taking out the reacted magnetic beads, detecting the fluorescence intensity through flow cytometry after washing, and then calculating the concentration of the nucleic acid target to be detected in the sample to be detected. The method for rapidly detecting the DNA is constructed by combining CRISPR-Cas12a, Hybrid Chain Reaction (HCR) and flow cytometry, can efficiently identify the target in a short time, has the advantages of high specificity, high sensitivity and the like, has the detection limit as low as 2pM, and is expected to become a powerful detection means for biomedical research and clinical diagnosis.

Description

Nucleic acid detection method based on gene editing and flow analysis technology
Technical Field
The invention relates to the field of biomedical sensors, in particular to a nucleic acid detection method based on gene editing and flow analysis technology.
Background
With the introduction of the concept of precise medicine, precise medicine has been gradually accepted by people, and tumors are one of the first solution tasks of precise medicine. In recent years, liquid biopsy is rapidly becoming an important minimally invasive means of standard tumor biopsy. Among them, circulating tumor DNA (ctDNA) has become one of tumor markers and is of great interest. ctDNA is not only a diagnostic marker, but is also critical for the assessment of clinical therapeutic efficacy and tumor metastasis. The development of a new ctDNA detection method has important significance in clinical application.
The CRISPR-Cas system is an adaptive immune system for bacteria to resist invasion of foreign nucleic acids, and because of convenient use and simple construction, CRISPR is considered as a revolutionary technology in the field of genetic research. The CRISPR-Cas12a (Cpf1) protein is an RNA-guided enzyme, unlike the CRISPR/Cas9 protein, Cas12a endonuclease requires only crRNA (CRISPR-derived RNAs) and does not require tracrRNA (trans-activating crRNA) as their guide. The Cas12a protein can specifically cleave double-stranded dna (dsdna) or single-stranded dna (ssdna) by specific crRNA. In addition, Cas12a protein also produces single-stranded dnase activity when bound to dsDNA or ssDNA complementary to crRNA. Thus, non-specific ssDNA can be degraded by the Cas12 a/CrRNA/target DNA ternary complex, and this cleavage activity is referred to as trans-cleavage.
Dai et al reported an electrochemical biosensor (E-CRISPR) based on CRISPR-Cas12a (cpf1) that investigated the trans-cleavage effect of Cas12a on non-specific ssDNA by the cis-cleavage method. A nonspecific ssDNA with Methylene Blue (MB) as an electrochemical labeling signal was designed, and the other end was tethered to the sensor surface via a thiol moiety to obtain an electrical signal. In the presence of the target, Cas12a trans-cleavage activity is activated, separating MB-ssDNA from the electrode surface, thereby reducing transmission of MB signals and reducing electrochemical signals; in the absence of target, Cas12a trans-cleavage activity was silenced, retaining MB-ssDNA on the surface (y.dai, r.a.somoza, l.wang, j.f.welter, y.li, a.i.caplan and c.c.liu, angelw Chem Int Ed Engl 2019). The scheme performs signal transduction by combining CRISPR-Cas12a (cpf1) with electrochemistry, but does not realize signal amplification, so that the method has the defects of detection sensitivity and precision.
Therefore, a more reliable solution is now needed.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a nucleic acid detection method based on gene editing and flow analysis technology, aiming at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a nucleic acid detection method based on gene editing and flow analysis technology comprises a CRISPR-Cas protein enzyme digestion system and a Hybrid Chain Reaction (HCR) system; the method mainly comprises the following steps: mixing a sample to be detected, CRISPR-Cas protein, crRNA and an initiator, and incubating to obtain an enzyme digestion solution A, wherein the CRISPR-Cas is Cas12, Cas13, Cas9 or Cas14 protein; then adopting the processing mode of (1) or (2):
(1) adding magnetic beads into the enzyme digestion solution A for reaction, separating and taking out the magnetic beads after reaction, mixing the magnetic beads with probe hairpin nucleic acid (DNA chain or RNA chain) after washing, and incubating; finally, separating and taking out the magnetic beads after reaction;
(2) adding probe hairpin nucleic acid (DNA chain or RNA chain) into the enzyme digestion solution A, mixing and incubating; then adding magnetic beads, and finally separating and taking out the magnetic beads after reaction;
and (3) detecting fluorescence by using the washed and separated magnetic bead solution through flow cytometry, and analyzing the type and the concentration of the nucleic acid target to be detected in the sample to be detected. Under the condition of no nucleic acid target, the CRISPR-Cas protein trans-cleavage activity is silenced, an initiator is reserved, the initiator is combined with a magnetic bead, and an HCR reaction is triggered through the initiator, so that hairpin nucleic acid modified with a label is combined to the surface of the magnetic bead, and the fluorescence of the magnetic bead is enhanced;
under the condition of a nucleic acid target, a Cas-crRNA binary complex formed by the CRISPR-Cas protein and the crRNA is specifically combined with the nucleic acid target, so that the trans-cleavage activity of the CRISPR-Cas protein is activated, an initiator is cracked, an HCR reaction cannot be triggered, hairpin nucleic acid modified with a marker cannot be combined to the surface of a magnetic bead, and the fluorescence of the magnetic bead cannot be enhanced; therefore, the concentration of the nucleic acid target can be calculated by establishing the relation between the concentration of the nucleic acid target to be detected and the fluorescence intensity of the magnetic beads after reaction and detecting the fluorescence intensity result of the magnetic beads after reaction by using flow cytometry, and the detection of the nucleic acid target is realized.
The method can efficiently identify the target in a short time, and has the advantages of high specificity, high sensitivity and the like.
Wherein the initiator is a nucleic acid chain, namely: a DNA strand or an RNA strand; the combination mode of the magnetic beads and the magnetic beads comprises: covalent binding, electrostatic adsorption, antigen-antibody binding, aptamer-ligand binding, enzyme-substrate binding.
Preferably, the combination of the initiator and the magnetic beads is as follows: biotin is bound to streptavidin, or antigen is bound to antibody, or sulfhydryl is bound to gold, or amino is condensed with carboxyl.
Preferably, the hairpin nucleic acid comprises hairpin H1 and hairpin H2, and the labels are modified on the hairpin H1 and/or hairpin H2.
Preferably, the label is a quantum dot or a fluorophore.
Preferably, wherein the sample to be detected, the CRISPR-Cas protein, the crRNA and the initiator are mixed, an RNase inhibitor is added to the reaction system.
Preferably, the incubation temperature is from-10 ℃ to 100 ℃. More preferably, the incubation temperature is 32 to 40 ℃.
Preferably, the incubation time is 0 to 24 hours, and more preferably, the incubation time is 0.5 to 2.5 hours.
Preferably, the sequence of the crRNA is complementary to a partial sequence of the nucleic acid target.
In a Hybrid Chain Reaction (HCR) system, the initiator and hairpin loop H1/H2 design principle is as follows: h1 includes four portions a, b, c and b, wherein b and b have 18 pairs of bases complementary to form a double strand as the stem of H1, c is the loop of the hairpin structure, and a is a single-stranded sticky end extending from the stem of the hairpin structure; h2 includes four portions c, b, a, and b. Its design is similar to H1, and a, b, c are respectively complementary to a, b, c. When no priming strand (a × b) exists, although partial strands in H1 and H2 can be complementary, because H1 and H2 form stem loops respectively, the two can exist stably in solution and cannot be mutually hybridized to open each other; when priming strand a b is added, a b is first complementary to the sticky end ab of the stem of H1, opening the stem-loop structure of H1, exposing the c b strand, opening the hairpin structure of H2, then exposing a b complementary to H1, and can continue to open H1, repeating in sequence. After the strand-directed hybridization reaction was initiated, the hairpins H1 and H2 were successively opened by H1 and H2, and a long-chain nicked double-stranded DNA polymer was formed by the alternate hybridization of H1 and H2 (see FIG. 7).
In a more preferred embodiment, the sequence of the crRNA is:
5'-UAAUUUCUACUAAGUGUAGAUGCAUGAGCUGCAUGAUGAGCU G-3' are provided. In the CRISPR/Cas12a system, the direct repeat sequence forming the stem-loop structure is "UAAUUUCUACUAAGUGUAGAU" and the spacer sequence is complementary to the detection sequence. The spacer sequence of crRNA is complementary to the Target sequence;
the sequence of the nucleic acid target is:
5’-CTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCG-3’;
the initiator has the sequence:
5'-AGTCTAGGATTCGGCGTGGGTTAATTTTTT-3', the 3' end of the initiator is marked with biotin;
the sequence of the hairpin DNA H1 is as follows:
5'-TTAACCCACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG-3', the 5 ' end of the hairpin DNA H1 is modified with a marker FAM;
the sequence of the hairpin DNA H2 is as follows:
5’-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG-3’。
the invention has the beneficial effects that: the method for rapidly detecting the DNA is constructed by combining CRISPR-Cas12a, HCR (hybrid chain reaction) and flow cytometry, can efficiently identify the target in a short time, has the advantages of high specificity, high sensitivity and the like, has the detection limit as low as 2pM, and is expected to become a powerful detection means for biomedical research and clinical diagnosis.
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FIG. 1 is a schematic diagram of a nucleic acid detection method based on gene editing and flow analysis techniques according to the present invention;
fig. 2 shows the results of verification of CRISPR-Cas trans-cleavage activity in example 2;
FIG. 3 shows the result of verifying the feasibility of the HCR (hybrid chain reaction) system in example 3;
FIG. 4 shows the results of the validation of the feasibility of the reaction system in example 4;
FIG. 5 shows the results of evaluating the detection sensitivity of the reaction system in example 5;
FIG. 6 shows the results of evaluation of selectivity and specificity of detection in the reaction system of example 6;
FIG. 7 shows the sequence design principle of the HCR system of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The nucleic acid detection method based on gene editing and flow analysis technology of the embodiment comprises a CRISPR-Cas protein enzyme digestion system and a Hybrid Chain Reaction (HCR) system; the method mainly comprises the following steps: mixing a sample to be detected, CRISPR-Cas protein, crRNA and an initiator, and incubating to obtain an enzyme digestion solution A, wherein the CRISPR-Cas is Cas12, Cas13, Cas9 or Cas14 protein; then adopting the processing mode of (1) or (2):
(1) adding magnetic beads into the enzyme digestion solution A for reaction, separating and taking out the magnetic beads after reaction, mixing the magnetic beads with the probe hairpin DNA after washing, and incubating; finally, separating and taking out the magnetic beads after reaction;
(2) adding probe hairpin DNA into the enzyme digestion solution A, mixing and incubating; then adding magnetic beads, and finally separating and taking out the magnetic beads after reaction;
and (3) detecting fluorescence by using the washed and separated magnetic bead solution through flow cytometry, and analyzing the type and the concentration of the nucleic acid target to be detected in the sample to be detected.
Under the condition of no nucleic acid target, the CRISPR-Cas protein trans-cleavage activity is silenced, an initiator is reserved, the initiator is combined with a magnetic bead, and an HCR reaction is triggered through the initiator, so that hairpin DNA modified with a label is combined to the surface of the magnetic bead, and the fluorescence of the magnetic bead is enhanced;
under the condition of a nucleic acid target, a Cas-crRNA binary complex formed by the CRISPR-Cas protein and the crRNA is specifically combined with the nucleic acid target to activate the trans-cleavage activity of the CRISPR-Cas protein, so that an initiator is cracked and cannot trigger HCR reaction, hairpin DNA modified with a marker cannot be combined to the surface of a magnetic bead, and the fluorescence of the magnetic bead cannot be enhanced; therefore, the concentration of the nucleic acid target can be calculated by establishing the relation between the concentration of the nucleic acid target to be detected and the fluorescence intensity of the magnetic beads after reaction and detecting the fluorescence intensity result of the magnetic beads after reaction by using flow cytometry, and the detection of the nucleic acid target is realized.
The method for rapidly detecting the DNA is constructed by combining CRISPR-Cas, HCR (hybrid chain reaction) and flow cytometry, can efficiently identify the target in a short time, and has the advantages of high specificity, high sensitivity and the like.
In a preferred embodiment, the crRNA consists of a direct repeat of a stem-loop structure and a spacer sequence complementary to a portion of the nucleic acid target, and the T in the crRNA is replaced by U.
In a preferred embodiment, the hairpin DNA comprises hairpin DNA H1 and hairpin DNA H2, and the label is modified on the hairpin DNA H1 and/or the hairpin DNA H2. In further preferred embodiments, the label is a quantum dot or a fluorophore, including but not limited to: FAM, CY3, TAMRA, CY 5.
In preferred embodiments, the hairpin DNA is bound to the magnetic bead in a manner including, but not limited to, the following: covalent binding, electrostatic adsorption, antigen-antibody binding, aptamer-ligand binding, enzyme-substrate binding; among them, biotin and streptavidin, or antigen and antibody, or thiol and gold, or amino and carboxyl groups are preferably condensed. . In a further preferred embodiment, the hairpin DNA and the magnetic bead are bound to streptavidin through biotin, the streptavidin is modified on the magnetic bead, and the biotin is labeled at the 3' end of the initiator.
The following examples are provided to further illustrate the present invention.
Example 1
In this example, all nucleic acids were synthesized and purified by Shanghai. Reaction buffer (PBS buffer) was also purchased from shanghai workers. Cas protease was purchased from Nelumben Biotechnology NEB, USA, RNase inhibitor was purchased from Takara Biotechnology, Inc., Dalian, China, 1 XNEB buffer 2.1 (containing 10mM magnesium ion) was purchased from Samorfeishel technology, Inc., MgSO4And streptavidin-magnetic beads were obtained from Shanghai Aladdin Biotechnology GmbH. The other chemicals were of analytical grade and used without further purification. All reagents were analytical grade and no further purification was required. All nucleic acid sequence syntheses are provided by the Producer (Biotechnology engineering (Shanghai) Co., Ltd.) and the purification method is HPLC. Ultrapure water (resistivity > 18.2 M.OMEGA.. multidot.cm) was used for the preparation of all solutions. All nucleic acid sequences are shown in table 1.
TABLE 1
Figure BDA0003326755520000061
Figure BDA0003326755520000071
Firstly, nucleic acid and protein pretreatment:
preparing a nucleic acid solution mother solution: firstly, Initiator DNA powder (called Initiator for short) and nucleic acid Target DNA (called Target for short) powder are centrifuged at 4000rpm for 30-60 s, and 1 XNEB buffer solution 2.1 (containing 10mM magnesium ions) is added to dissolve the powder to 10 MuM for standby. ② the hairpin DNA H1 (abbreviated as H1) and DNA H2 (abbreviated as H2) powder is centrifuged at 4000rpm for 30-60 s, and 10mM PBS buffer solution is added to dissolve the powder to 10 μ M for standby.
Preparing a protein solution mother liquor: CRISPR-Cas12a protein (Cas 12a for short) solution (100. mu.M) was diluted to 1. mu.M by adding 1 XNEB buffer solution 2.1 (containing 10mM magnesium ions).
Hair clip loop formation: the solutions containing the sequences of hairpin DNA H1 (abbreviated as H1) and hairpin DNA H2 (abbreviated as H2) were incubated at 95 ℃ for 5 minutes, respectively, and then slowly cooled to room temperature to anneal to form the hairpin loop structure.
Secondly, a nucleic acid detection method based on gene editing and flow analysis technology, which comprises the following steps:
1) target, 50nM Cas12a protein, 60nM crRNA, 200nM initiator and 20U RNase inhibitor were mixed, made up to 40. mu.L using 1 XNEB buffer 2.1 (containing 10mM magnesium ions), incubated for 0.5h at 37 ℃;
2) the reaction system was expanded to 100. mu.L with 10mM PBS, and 1. mu.L of magnetic beads (magnetic beads or MB for short) was added for incubation (three washes before addition) for magnetic bead modification;
3) after reaction, separating and taking out the magnetic beads, washing the magnetic beads for 3 times by using 10mM PBS, mixing the magnetic beads with hairpin DNA H1 and hairpin DNA H2, and incubating for 2 hours at 37 ℃; wherein the concentrations of the hairpin DNA H1 and the hairpin DNA H2 are the same, and the volumes are both 50 mu L;
4) the magnetic beads after the reaction were separated and removed, washed 3 times with 10mM PBS, and diluted to 300. mu.L with 10mM PBS (the final concentration of the magnetic beads was constant at 0.1mg/mL), and the fluorescence intensity of the magnetic beads was measured by flow cytometry; under the excitation of 488nm laser, 10000 nanoparticles are counted through a FL1(FAM/FITC) channel to calculate the average fluorescence intensity (MFI) of the tested magnetic beads, and the data analysis is carried out by using FlowJo software V10; and finally, calculating the concentration of the nucleic acid target to be detected in the sample to be detected according to the fluorescence detection result.
Referring to FIG. 1, a schematic diagram of the method of the present invention is shown, in which a Biotin-Initiator represents an Initiator, Strep MNP represents magnetic beads, and Hairpin DNA H1 and Hairpin DNA H2 represent Hairpin DNA 1 and Hairpin DNA 2, respectively.
Example 2 verification of CRISPR-Cas Trans-cleavage Activity
crRNA consists of a direct repeat and a spacer. In the CRISPR/Cas12a system, the direct repeat sequence forming the stem-loop structure is "UAAUUUCUACUAAGUGUAGAU" and the spacer sequence is complementary to the detection sequence. The spacer sequence of crRNA is complementary to the Target sequence, and T is replaced by U. The crRNA sequence is shown in Table 1. In the CRIPSR-Cas12 cleavage system, 1 XNEB buffer 2.1 (containing 10mM magnesium ions) was used as reaction buffer. For the trans-cleavage activity validation, 50nM cas12 a; 60nM crRNA; different concentrations of Target (0,20,40,80nM) were mixed with 60nM FQ-labeled probe and fluorescence signals were detected with a BMG-CLARIOstar microplate reader (BMG-Labtech, UK) at 480nM excitation wavelength and 520nM emission wavelength.
To verify CRISPR-Cas trans-cleavage activity, this example designed FQ-labeled ssDNA fluorescent probes (probes in table 1), Cas protein recognized target, Cas protein as DNA helicase, and target DNA was cleaved in cis. After the target strand is cleaved, the CRISPR-Cas trans-cleavage activity is further activated by the Cas12 a/CrRNA/target ternary complex, the probe is cleaved, so that the marker is released, the quenched fluorescence is recovered, and the solution fluorescence signal is enhanced; fig. 2A is a schematic diagram thereof. Referring to FIG. 2B, the results of the experiment are shown, wherein the target concentrations are 0nM, 20nM, 40nM, and 80nM, respectively. The results show that the rate of change of the fluorescence signal values increases significantly with time and increasing target concentration, demonstrating the successful design of crRNA and the trans-cleavage activity of CRISPR-Cas12 a.
Example 3 feasibility verification of HCR (hybrid chain reaction) System
In this example, the feasibility of HCR was verified by FCBA and 20% SDS-PAGE gel electrophoresis (FIG. 3C) using high concentrations (200nM) of FAM-modified H1 and H2. In addition, the respective functions of the initiator and hairpin loop were also confirmed. Solutions containing either the H1 or H2 sequences were incubated at 95 ℃ for 5 minutes, then slowly cooled to room temperature to anneal and form hairpin loops. Different concentrations of initiator were mixed with the same concentrations of H1 and H2, the control group was added with the same volume of PBS, reacted at 37 ℃ for 2 hours, and the magnetic beads were separated after the reaction. Measurements were performed by flow cytometry.
The results show that the HCR reaction only occurs in the presence of an Initiator (denoted Initiator in fig. 3), and when H1 and H2 coexist, the fluorescence intensity on the surface of the magnetic beads is significantly increased (fig. 3A, 3B), which means that long DNA hybrid strands are formed, indicating that the fluorescence signal is efficiently amplified. FIG. 3C demonstrates the feasibility of this strategy from a molecular point of view, and HCR occurs only when the initiator, H1, H2 are present together, forming a specific HCR band. The feasibility of HCR was thus demonstrated at the molecular level.
FIG. 3A shows fluorescence intensity of the surface of a magnetic bead, wherein the reference numerals 1, 2, 3, 4, 5, and 6 respectively represent:
1:0nMInitiator+500nM H1,2:0nMInitiator+500nM H1+500nM H2,3:5nMInitiator+500nM H1,4:5nMInitiator+500nM H1+500nM H2,5:10nMInitiator+500nM H1,6:10nMInitiator+500nM H1+500nM H2。
FIG. 3B shows the flow results of different concentrations of the initiator and hairpin loop incubated with magnetic beads;
FIG. 3C shows the result of SDS-PAGE electrophoresis of reacted DNA, wherein each reference numeral is 1: Initiator; h1; 3: H2; 4: H1+ H2; initiator + H1+ H2. The Initiator, H1 and H2 concentrations were all 2. mu.M.
Example 4 validation of the feasibility of the reaction System
After the CRISPR-Cas trans-cleavage activity verification succeeds and the HCR reaction feasibility is verified, the strategy verification of the whole system is performed in this example, the specific steps refer to example 1, and the result is shown in fig. 4. The Mean Fluorescence Intensity (MFI) of the tested magnetic beads was calculated by counting 10000 nanoparticles through FL1(FAM/FITC) channel under 488nm laser excitation. In the presence of the target sequence, the fluorescent signal on the surface of the magnetic beads was significantly different from that in the absence of the target sequence (Δ F in fig. 4B represents the difference in fluorescence), and thus the feasibility of the entire reaction system was confirmed.
Example 5 evaluation of detection sensitivity of reaction System
In this example, 9 different target concentrations of 10pM, 15pM, 25pM, 40pM, 60pM, 80pM, 100pM, 120pM, 150pM were selected for the experiment (each concentration was repeated three times), and the relative fluorescence intensities (IF (520)/IF (580)) at 520nm for FAM and 580nm for TAMRA were recorded by a fluorescence spectrophotometer, respectively. The results are shown in FIG. 5, where the relative fluorescence intensity increases with increasing concentration of target DNA. The result shows that the absorbance of the magnetic beads and the target concentration have good linear relation in the range of 25pM to 120pM, and the detection limit is 2 pM; linear equation of y 0.58692x-3.81533 where y represents percent fluorescence difference, x represents target concentration, linear correlation coefficient R2Is 0.99.
FIG. 5A shows the mean fluorescence intensity of the magnetic bead surface, wherein the curves show the results of target concentrations of 0pM, 10pM, 15pM, 25pM, 40pM, 60pM, 80pM, 100pM, 120pM, and 150pM, respectively.
Fig. 5B is a target detection linear calibration curve where the percent fluorescence difference, Δ F% — background fluorescence-target fluorescence)/background fluorescence.
Example 6 evaluation of the selectivity and specificity of detection of the reaction System
In this embodiment, targets with concentrations of 25pM, 50pM, and 100pM are selected respectively in the presence/absence of serum to perform an experiment (each concentration is repeated three times) to evaluate the interference ability of the detection method of the present invention on serum, as shown in fig. 6A, the average fluorescence intensity of the surfaces of the magnetic beads of the experimental group added with serum is similar to that of the control group without serum, which indicates that the present solution has a certain resistance to serum and can resist the interference of serum in clinical samples. Furthermore, we spiked different target standard solutions into 10% serum to determine sample recovery, and the results are shown in table 2: the recovery of the target ranged from 95.6-105.3%.
TABLE 2
Figure BDA0003326755520000101
Further, in this example, the average fluorescence intensities of the magnetic bead surfaces of the single-base mismatch target sequence (MT1), the double-base mismatch target sequence (MTT2), the triple-base mismatch target sequence (MTT3), the non-complementary target sequence (NCT), and the target sequence were measured and compared, respectively. As shown in fig. 6B, the same concentrations of MT1, MT2, MT3, and NCT produced significantly lower fluorescence responses compared to the target dna (t). These results indicate that the method of the present invention has an ability to recognize a target in which a base mutation has occurred, and that this ability gradually increases as the number of mutated bases increases.
The invention provides a novel fluorescent sensor development strategy, namely, on the basis of flow cytometry measurement, CRISPR-Cas12a and HCR are combined to detect DNA, the CRISPR-typev system Cas12a (cpf1) is used as a high-efficiency biosensing system, HCR reaction is reduced through a non-specific cutting initiator, a fluorescent signal is reduced, and finally a nucleic acid detection method with high sensitivity, specificity and selectivity is constructed, and the detection limit is as low as 2 pM.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Suzhou Institute of Biomedical Engineering Technology, Chinese Academy of Sciences
The invention name is as follows: nucleic acid detection method based on gene editing and flow analysis technology
Name Sequence (5’→3’)
Target: CTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCG
crRNA: UAAUUUCUACUAAGUGUAGAUGCAUGAGCUGCAUGAUGAGCUG
Biotin-Initiator: AGTCTAGGATTCGGCGTGGGTTAATTTTTT(Biotin)
IF : (Fam)AGTCTAGGATTCGGCGTGGGTTAATTTTTT(Biotin)
Hairpin DNA H1: (Fam) TTAACCCACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG
Hairpin DNA H2: AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACTACTTTG
Probe : 5’6-FAM-TTA TT-3’BHQ1
MT1: CTCCACCGTGCAGCACATCATGCAGCTCATGCCCTTCG
MT2: CTCCACCGTGCAGCAGATCATGCAGCTCATGCCCTTCG
MT3: CTCCACCGTGCAGCAGTTCATGCAGCTCATGCCCTTCG
NCT: GCAGCTCATGCCCATATGGCAAGAGCTGCAGTTAGCTC

Claims (10)

1. A nucleic acid detection method based on gene editing and flow analysis technology is characterized by comprising a CRISPR-Cas protein enzyme digestion system and a hybridization chain reaction system; the method comprises the following steps: mixing a sample to be detected, CRISPR-Cas protein, crRNA and an initiator, and incubating to obtain an enzyme digestion solution A, wherein the CRISPR-Cas is Cas12, Cas13, Cas9 or Cas14 protein; then adopting the processing mode of (1) or (2):
(1) adding magnetic beads into the enzyme digestion solution A for reaction, separating and taking out the magnetic beads after reaction, mixing the magnetic beads with probe hairpin nucleic acid after washing, and incubating; finally, separating and taking out the magnetic beads after reaction;
(2) adding probe hairpin nucleic acid into the enzyme digestion solution A, mixing and incubating; then adding magnetic beads, and finally separating and taking out the magnetic beads after reaction;
and (3) detecting fluorescence by using the washed and separated magnetic bead solution through flow cytometry, and analyzing the type and the concentration of the nucleic acid target to be detected in the sample to be detected.
2. The method for detecting nucleic acid based on gene editing and flow analysis technology as claimed in claim 1, wherein the initiator is a nucleic acid chain, namely: a DNA strand or an RNA strand; the combination mode of the magnetic beads and the magnetic beads comprises: covalent binding, electrostatic adsorption, antigen-antibody binding, aptamer-ligand binding, enzyme-substrate binding.
3. The method for detecting nucleic acid based on gene editing and flow analysis technology of claim 1 or 2, wherein the combination of the initiator and the magnetic beads is: biotin is bound to streptavidin, or antigen is bound to antibody, or sulfhydryl is bound to gold, or amino is condensed with carboxyl.
4. The method for detecting nucleic acid based on gene editing and flow analysis technology of claim 1, wherein the hairpin nucleic acid comprises hairpin H1 and hairpin H2, and the hairpin H1 and/or the hairpin H2 are modified with a label.
5. The method of claim 4, wherein the label is a quantum dot or a fluorophore.
6. The nucleic acid detection method based on gene editing and flow analysis technology as claimed in any one of claims 1-5, wherein an RNase inhibitor is added to the reaction system when the sample to be detected, CRISPR-Cas protein, crRNA and initiator are mixed.
7. The method for detecting nucleic acid based on gene editing and flow analysis technology as claimed in claim 1, wherein the incubation temperature is-10 ℃ to 100 ℃.
8. The method for detecting nucleic acid based on gene editing and flow analysis technique according to claim 1, wherein the incubation time is 0 to 24 hours.
9. The method for detecting nucleic acid based on gene editing and flow analysis technique according to claim 1, 7 or 8, wherein the incubation temperature is 32 to 40 ℃ and the incubation time is 0.5 to 2.5 hours.
10. The method for nucleic acid detection based on gene editing and flow analysis technique of claim 1, wherein the sequence of the crRNA can be complementarily paired with the nucleic acid target or partial target sequence or target related sequence.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116024313A (en) * 2022-09-20 2023-04-28 华南农业大学 Programmable nucleic acid molecule detection method and platform

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116024313A (en) * 2022-09-20 2023-04-28 华南农业大学 Programmable nucleic acid molecule detection method and platform

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