CN115058493B - DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application - Google Patents
DNA probe for multiple nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application Download PDFInfo
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Abstract
The invention provides a DNA probe for multiplex nucleic acid detection, comprising at least four groups of probes, each group of probes respectively comprising a Bio-recognition DNA and an NH complementarily paired with the Bio-recognition DNA 2 Capture DNA. The invention also provides a CRISPR-reverse dot hybridization nucleic acid detection system for detecting multiple nucleic acids and application thereof. Meanwhile, the invention also provides a multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization technology. The invention is based on CRISPR and reverse dot hybridization technology, can greatly improve detection efficiency, reduce detection cost, save detection time, and has the advantages of high sensitivity, high specificity, universality, rapidness, no need of expensive equipment, portable detection and the like.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization technology, and specifically relates to a DNA probe for multiplex nucleic acid detection, a CRISPR-reverse dot hybridization nucleic acid detection system and application thereof.
Background
For the fields of clinical diagnosis, environmental monitoring, food safety and the like, obtaining a reliable and rapid nucleic acid diagnosis result is of great importance. In particular, multiple nucleic acid targets can be detected simultaneously in a single sample, which can significantly reduce detection costs, save detection time, and improve detection efficiency. At present, multiplex nucleic acid detection techniques rely mainly on microarray technology, next generation sequencing, and gold standard quantitative polymerase chain reaction (qPCR) technology. However, the prior art requires trained laboratory personnel, expensive equipment, and specialized laboratories.
In recent years, POCT (point of care testing) detection is a way to quickly obtain a detection result by using a portable analyzer and a matching reagent, which is performed on a sampling site. The rapid detection in time is beneficial to alleviating doctor-patient relationship, reducing unnecessary panic, being beneficial to targeted treatment of pathogens and improving accurate medical level. Therefore, POCT nucleic acid sensors have great potential in the diagnosis of a variety of diseases, and have become increasingly popular.
The CRISPR/Cas system is an immune system present in bacteria and archaea for combating the invasion of foreign substances. In recent years, CRISPR/Cas systems have become an important tool in genetic engineering, and are widely used in gene editing, gene expression control, gene detection, and the like. The CRISPR/Cas12a system is a novel CRISPR/Cas system, has RNA-guided DNase cutting activity, and can activate the non-specific single-stranded DNA (ssDNA) shearing activity of Casl2a while specifically cutting double-stranded DNA (dsDNA). By utilizing the characteristics, crRNA (CRISPR-delayed RNA) can be designed for target DNA artificially and is complementary with DNA near a PAM (protospacer-adjacent motif) region in the target DNA, so that direct detection of different target DNAs is realized. With this feature, researchers have developed a variety of powerful CRISPR/Cas biosensors, such as detect (Cas 12 a), sholock (Cas 13 a), cddetect (Cas 12 b), and Cas14 detect. Despite the advantages of rapid, good sensitivity and specificity of CRISPR diagnostic techniques, multiplex nucleic acid detection sensors based on CRISPR have been rarely reported. For example, a Recombinase Polymerase Amplification (RPA) -based dual gene amplification product simultaneously detects dual targets in a single tube by Cas12a and Cas13a accessory cleavage properties; CARMEN integrates SHERLOCK and a self-organizing micro-microfluidic technology to detect various target nucleic acids; however, the scope of its application is limited because of the need for advanced and expensive microscopes and skilled laboratory personnel. In addition, richard and colleagues designed an electrochemical microfluidic biosensor that achieved parallel detection of multiple targets by dividing the fixed area into multiple segments; but such a separation-based approach requires complex chip preparation and may introduce the risk of cross-reactions due to diffusion between the different sub-regions.
Therefore, there is an urgent need to develop a multiplex nucleic acid detection method that is convenient, rapid, efficient, and low in detection cost.
Disclosure of Invention
The invention aims to solve the technical problems of providing a DNA probe for detecting multiple nucleic acids, a CRISPR-reverse dot hybridization nucleic acid detection system and application, and the invention is based on CRISPR and reverse dot hybridization technology, can greatly improve detection efficiency, reduce detection cost and save detection time, and has the advantages of high sensitivity, high specificity, universality, rapidness, no need of expensive equipment, portability detection and the like.
The invention adopts the following technical scheme to solve the technical problems:
a DNA probe for multiplex nucleic acid detection comprising at least four sets of probes, each set comprising a Bio-recognment DNA (biotin-modified single-stranded DNA probe) and an NH complementarily paired with said Bio-recognment DNA, respectively 2 Capture DNA (amino-modified captured DNA probe); wherein, the base sequences of the Bio-recogination DNA in the four groups of probes are respectively shown as SEQ ID NO. 1-SEQ ID NO.4, and the corresponding paired NH are respectively shown 2 The nucleotide sequences of the capture DNA are shown as SEQ ID NO. 5-SEQ ID NO.8 respectively.
As one of the preferred embodiments of the present invention, in the DNA probe, the Bio-recognition DNA of each group is only combined with NH of the same group 2 Capture DNA binding in concert, not with other groups of Bio-recovery DNA or NH 2 Capture DNA pairing occurs.
As one of the preferred modes of the present invention, the Bio-recognition DNA is biotin-modified at the 5' end.
As one of the preferable modes of the invention, the NH is 2 -capture DNA, amino modified at the 5' end.
An application of the DNA probe in preparing multiple nucleic acid detection products.
A CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection comprises Cas12 protein, crRNA, bio-recogination DNA, streptavidin-horseradish peroxidase (streptavidine-HRP), and NH-binding agent 2 Nylon membrane of capture DNA, hybridization solution, substrate solution;
wherein the crRNA is a sequence that specifically binds to a target nucleic acid and is not associated with Bio-recognition DNA and NH 2 Capture DNA binding; the Bio-recognment DNA comprises at least four kinds of DNA, and the sequences of the DNA are respectively shown as SEQ ID NO. 1-SEQ ID NO. 4; the NH on nylon membrane 2 The complementary pairing of the capture DNA and the Bio-recognition DNA comprises at least four sequences shown in SEQ ID NO. 5-SEQ ID NO. 8; the streptavidin-horseradish peroxidase can be combined with Bio-recognosis DNA; the substrate solution can be catalyzed by HRP in streptavidin-horseradish peroxidase for color reaction.
As one of the preferred modes of the present invention, the Cas12 protein is one of Cas12a, cas12b, cas12c, cas12d, cas12e, and Cas12 g.
As one of the preferable modes of the invention, when the multiple nucleic acid is detected as four nucleic acids of influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus, the corresponding constructed crRNA is FA-crRNA, FB-crRNA, RSV-crRNA and Sars-2-N-crRNA, and the sequences are respectively shown in SEQ ID NO. 9-SEQ ID NO. 12.
As one of the preferable embodiments of the present invention, the nylon membrane surface is negatively charged modified.
As a preferred mode of the inventionOne of the above is combined with NH 2 The preparation method of the nylon membrane of the capture DNA is as follows:
(1) Equipped with NH 2 Capture DNA working solution: to synthesize NH 2 -capture DNA diluted to a final concentration of 10 μm for spotting;
(2) Fixing NH 2 Capture DNA: soaking nylon membrane in 3-10% EDC solution for 10-40 min, washing with pure water for 3 times and air drying at room temperature;
(3) 0.5-1. Mu.l of NH 2 -capture DNA spot on nylon membrane, left for 30min and air dried;
(4) The reaction was quenched with sodium hydroxide solution (0.1 mol/L,20 mL), washed 3 times with pure water and dried in the air.
As a preferred embodiment of the present invention, the hybridization solution includes solution A, solution B, and solution C;
the formula of the solution A is as follows:
20×SSC 100mL；
10％SDS 10mL；
890mL of purified water;
totaling 1L;
the formula of the solution B is as follows:
20×SSC 10mL；
10％SDS 4mL；
386mL of purified water;
totaling 400mL;
the formula of the liquid C is as follows:
1M sodium citrate 20mL;
180mL of purified water;
totaling to 200mL.
As one of the preferred embodiments of the present invention, a nucleic acid sample is also included; the nucleic acid sample is the DNA full length or fragment of the nucleic acid to be detected, isothermal amplification product or PCR product of the nucleic acid to be detected.
As one of the preferred modes of the present invention, the isothermal amplification includes, but is not limited to: loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase Polymerase Amplification (RPA), strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), nicking Enzyme Amplification Reaction (NEAR), rolling Circle Amplification (RCA), and the like.
Use of a CRISPR-reverse dot hybridization nucleic acid detection system as described above for the preparation of a multiplex nucleic acid detection kit for detecting multiplex target DNA and RNA in a sample.
A multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization techniques, for detecting a sample to be detected using the CRISPR-reverse dot hybridization nucleic acid detection system according to any one of claims 5 to 11.
As one of the preferable modes of the invention, the method comprises the following specific steps:
s1, dissolving a nucleic acid sample, corresponding crRNA, corresponding Bio-recogntion DNA and Cas12 protein in a buffer solution according to the detection type of target nucleic acid, and reacting at a proper temperature to obtain a plurality of first buffers;
s2, mixing all the first buffer solutions together to form a second buffer solution;
s3, combining the second buffer solution with NH 2 -hybridization reaction of nylon membrane of capture DNA with hybridization solution at a suitable temperature;
s4, incubating streptavidin-horseradish peroxidase with a nylon membrane;
s5, adding a substrate solution, and judging whether the target nucleic acid exists or not according to the color development degree of the color.
As one of the preferred embodiments of the present invention, in the step S1: the buffer solution is as follows: 10-100 mM Tris-HCl, 10-200 mM NaCl, 5-50 mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The proper temperature is 25-42 ℃; the reaction time is 10 to 60 minutes; in the step S3: the hybridization proper temperature is 25-45 ℃; the hybridization reaction time is 30-120 minutes.
The detection principle is illustrated in FIG. 1, and four nucleic acid detections are constructed as models (the invention is not limited to four in practice):
first, 4 crrnas (for different detection viruses) were designed and complementarily bound to the corresponding DNA target nucleic acids. Secondly, 4 pairs of non-interfering bio-recognition DNA and NH are designed 2 Capture DNA pairing sequence. 4 NH species 2 The capture DNA is immobilized on the same siteNylon membrane. When one or more targets are contained in the sample, i.e., crRNA/CRISPR-Cas12a complexes are formed, activated Cas12a is able to cleave one or more bio-recognition DNA out of order. The fragmented bio-recognition DNA cannot bind to capture DNA, and thus no blue spots appear or blue spots appear less strongly. Conversely, there is a strong blue spot signal. The invention designs a method for simultaneously detecting target nucleic acid, which does not need a special instrument, and the detection result shows that only one cheap nylon membrane is needed.
Compared with the prior art, the invention has the advantages that: the invention constructs a DNA probe for simultaneously detecting multiple target nucleic acid, a CRISPR-reverse dot hybridization nucleic acid detection system, a detection method and a kit based on the combination of CRISPR and reverse dot hybridization technology; compared with the conventional method, the method greatly improves the detection efficiency and greatly reduces the detection cost; meanwhile, the invention has the advantages of high sensitivity, high specificity, rapidness, universality, no need of expensive instruments, portable detection and the like, and solves the pain point of single-target nucleic acid detection of the existing CRISPR technology.
Drawings
FIG. 1 is a schematic diagram of a multiplex nucleic acid detection method of the present invention (in the figure, it means that amplified products are added to four reaction wells in equal amounts, respectively, incubated at a suitable temperature for a period of time, all of them are eluted into a reaction tank containing membrane strips using a hybridization solution, followed by hybridization reaction, color development is performed by adding a substrate, quantitative recognition is performed by APP or an interpretation result is directly observed by naked eyes);
FIG. 2 is a schematic diagram of the whole course of detecting clinical samples according to the present invention;
FIG. 3 is a graph showing the results of detecting 4 kinds of respiratory viruses in example 4.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
The invention provides a method for multiple applicationsDNA probes for heavy nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection systems and applications thereof. The flow chart of the detection clinical sample is shown in figure 2, firstly, DNA or RNA extraction is carried out by using an extraction kit; then using RPA or reverse transcription RPA kit to carry out rapid amplification; adding amplified products into a multichannel microfluidic chip, and simultaneously adding Cas12 protein, crRNA of corresponding viruses and Bio-recognition DNA into each channel, and reacting for 10-30 minutes in a reaction hole; after the reaction, the hybridization solution is used to combine all the reaction solutions of the four channels with NH 2 -hybridization of nylon membrane of capture DNA; incubating streptavidin-horseradish peroxidase with a nylon membrane; the substrate solution was added, and the result was observed and interpreted based on the signal.
It should be noted that the test materials used in the present invention are commercially available and can be directly purchased unless otherwise specified.
The invention is further illustrated by the following examples in conjunction with:
example 1
Designing a DNA probe for multiplex nucleic acid detection:
this example designed four sets of probes, each set comprising a Bio-recogination DNA (biotin-modified single-stranded DNA probe with biotin modification at the 5' end) and an NH complementary to the Bio-recogination DNA 2 Capture DNA (amino modified captured DNA probe, amino modified at the 5' end).
The sequences of the Bio-recoganism DNA-1, the Bio-recoganism DNA-2, the Bio-recoganism DNA-3 and the Bio-recoganism DNA-4 in the four groups of probes are respectively shown as SEQ ID NO. 1-SEQ ID NO. 4; NH of corresponding pairing 2 -capture DNA-1、NH 2 -capture DNA-2、NH 2 -capture DNA-3、NH 2 The sequence of the capture DNA-1 is shown as SEQ ID NO. 5-SEQ ID NO. 8.
In the present invention, the DNA probes actually include, but are not limited to, the four groups described above; when the kit is used for detecting more than four nucleic acids, several groups of matched DNA probes can be designed.
It should be noted, however, that the Bio-recovery DNA of each group is only identical to that of the same groupNH 2 Capture DNA binding in concert, not with other groups of Bio-recovery DNA or NH 2 Capture DNA pairing occurs.
Example 2
Construction of a CRISPR-reverse dot hybridization nucleic acid detection system:
CRISPR-reverse dot hybridization nucleic acid detection System comprising nucleic acid sample, cas12 protein, crRNA, bio-recovery DNA, streptavidin-horseradish peroxidase (streptavidine-HRP), and NH-conjugated 2 Nylon membrane of capture DNA, hybridization solution, substrate solution.
Wherein, the nucleic acid sample is the whole length or fragment of the DNA of the nucleic acid to be detected, the isothermal amplification product or PCR product of the nucleic acid to be detected (isothermal amplification includes, but is not limited to LAMP, HDA, RPA, SDA, NASBA, TMA, NEAR, RCA and the like).
The Cas12 protein is one of Cas12a, cas12b, cas12c, cas12d, cas12e, cas12 g.
crRNA is a sequence that specifically binds to a target nucleic acid and does not bind to Bio-recognition DNA and NH 2 Capture DNA binding.
The Bio-recognment DNA comprises at least four kinds of DNA, and the sequences of the DNA are respectively shown as SEQ ID NO.1 to SEQ ID NO. 4.
The nylon membrane surface is negatively charged. NH on Nylon Membrane 2 The complementary pairing of the capture DNA and the Bio-recognition DNA comprises at least four sequences shown in SEQ ID NO. 5-SEQ ID NO. 8.
Said binding NH 2 The preparation method of the nylon membrane of the capture DNA is as follows:
(1) Equipped with NH 2 Capture DNA working solution: to synthesize NH 2 -capture DNA diluted to a final concentration of 10 μm for spotting;
(2) Fixing NH 2 Capture DNA: the nylon membrane is soaked in 3-10% EDC solution for 10-40 min (preferably in 5% EDC solution for 25 min), washed 3 times with pure water and dried at room temperature; the method comprises the steps of carrying out a first treatment on the surface of the
(3) 0.5 to 1. Mu.l (preferably 0.8 ul) of NH 2 -capture DNA spot on nylon membrane, left for 30min and air dried;
(4) The reaction was terminated with sodium hydroxide solution (preferably 0.1mol/L,20 mL), washed 3 times with pure water and dried in the air.
The hybridization solution comprises solution A, solution B and solution C;
the formula of the solution A is as follows:
20×SSC 100mL；
10％SDS 10mL；
890mL of purified water;
totaling 1L;
the formula of the solution B is as follows:
20×SSC 10mL；
10％SDS 4mL；
386mL of purified water;
totaling 400mL;
the formula of the liquid C is as follows:
1M sodium citrate 20mL;
180mL of purified water;
totaling to 200mL.
Streptavidin-horseradish peroxidase can bind to Bio-recogination DNA.
The substrate solution can be subjected to a chromogenic reaction catalyzed by HRP in streptavidin-horseradish peroxidase.
The CRISPR-reverse dot hybridization nucleic acid detection system of the present embodiment can be used as a component of a multiplex nucleic acid detection kit for detecting multiplex target DNA and RNA in a sample.
Example 3
Multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization technique (using CRISPR-reverse dot hybridization nucleic acid detection system of example 2):
s1, according to the detection type of target nucleic acid, dissolving nucleic acid sample, corresponding crRNA, corresponding Bio-recognosis DNA and Cas12 protein in buffer solution (10-100 mM Tris-HCl, 10-200 mM NaCl and 5-50 mM MgCl) 2 ) Reacting for 10-60 minutes at the temperature of 25-42 ℃ to obtain a plurality of first buffers;
s2, mixing all the first buffer solutions together to form a second buffer solution;
s3, combining the second buffer solution withWith NH 2 The nylon membrane of the capture DNA and the hybridization solution are hybridized for 30 to 120 minutes at the temperature of 25 to 45 ℃;
s4, incubating the streptavidin-horseradish peroxidase and the nylon membrane for 5-30 minutes;
s5, adding a proper amount of substrate liquid, and judging whether the target nucleic acid exists or not according to the color development degree of the color.
In the step S1:
the buffer is preferably 50mM Tris-HCl, 100mM NaCl, 20mM MgCl 2 。
A suitable temperature is preferably 30 ℃;
the reaction time is preferably 30 minutes.
In the step S3:
the hybridization temperature is preferably 30 ℃; the hybridization reaction time is preferably 70 minutes.
In the step S4:
the incubation time is preferably 20 minutes.
Example 4
The embodiment is used for verifying the detection method of the invention:
and simultaneously detecting influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus by utilizing a recombinase isothermal amplification technology and combining CRISPR/Cas12a with a reverse dot hybridization technology.
This example describes a method for multiplex nucleic acid detection using Cas12a accessory cleavage property and reverse dot hybridization color development after amplification of a clinical sample to be detected using recombinase isothermal amplification technology.
1. Materials and methods
1. 4 pairs of RPA isothermal amplification primers (among the 4 pairs of RPA isothermal amplification primers, FA: influenza A virus, FB: influenza B virus, RSV: respiratory syncytial virus, COV: neocoronavirus) were designed:
RPA-FA-F：CAACAACCAATCCATTAATAAAACATGAGAAC；
RPA-FA-R：AAATTTTCAAGAAGATCATCCCTTAGACCAG；
RPA-FB-F：GATATACGTAATGTGTTGTCCTTGAGAGTG
RPA-FB-R:TAAGATCATCAGTAGCAACAAGTTTAGCAA；
RPA-RSV-F：CTTAGCATATGTAGTACAATTACCACTATA；
RPA-RSV-R：TACTTCACTTGGTAATGTTAAACTGTTCAT；
RPA-Sars-2-N-F：GAAATTCAACTCCAGGCAGCAGTAGGGGAAC；
RPA-Sars-2-N-R：GAAAGCTTGTGTTACATTGTATGCTTTAGTG。
2. 4 crrnas were designed:
FA-crRNA UAAUUUCUACUAAGUGUAGAUGCCUGGUGUGUGCAACAUGUGAGC (as shown in SEQ ID NO. 9);
FB-crRNA UAAUUUCUACUAAGUGUAGAUAUGAAGGACAUUCAAAGCCAAUU (shown in SEQ ID NO. 10);
RSV-crRNA UAAUUUCUACUAAGUGUAGAUUGACACAAUGAACAGUUUAACAUU (shown in SEQ ID NO. 11);
UAAUUUCUACUAAGUGUAGAUCUGCUGCUUGACAGAUUGAACCA of Sars-2-N-crRNA (shown as SEQ ID NO. 12).
3. 4 Bio-recognment DNAs (sequences shown as SEQ ID NO. 1-SEQ ID NO. 4) and 4 NH were designed 2 Capture DNA (sequence shown as SEQ ID NO. 5-SEQ ID NO. 8).
4. Preparation of Capture DNA modified Nylon Membrane:
(1) NH equipped with 4 amino modifications 2 Capture DNA working solution: will synthesize 4 kinds of NH 2 The capture DNA was diluted to a final concentration of 10. Mu.M each for spotting;
(2) Fixing NH 2 Capture DNA: the nylon membrane is soaked in 3-10% EDC solution for 10-40 min, washed 3 times with pure water and dried.
(3) 0.5 to 1 mu l of NH 2 The capture DNA is respectively spotted on a nylon membrane, and the nylon membrane is stood for 30 minutes and dried;
(4) The reaction was terminated with sodium hydroxide solution (0.1 mol/L,20 mL), and herein washed 3 times with pure water and dried in the air.
2. Detection process
1. Sample processing:
extracting RNA in a sample to be detected by using an RNA extraction kit as a detection template;
2. sample amplification:
carrying out in vitro transcription on the RPA amplification product by adopting an in vitro constant temperature RPA reverse transcription kit to obtain a transcription product;
3. preparing a constant-temperature amplification reaction system:
(1) The reaction mixture was prepared in a 200 μl tube:
gently vortex;
(2) Mu.l of 280mM magnesium acetate (MgOAc) was added and mixed well.
The amplification reaction system was amplified at 30℃for 30min.
4. Preparing a Cas12a enzyme cleavage reaction system:
a 4-tube Cas12a cleavage reaction system was prepared. Wherein each tube contains Cas12a, FA-crRNA or FB-crRNA or RSV-crRNA or Sars-2-N-crRNA (final concentration about 0.02-2. Mu.M)), and the corresponding biotin-recovery DNA (each reaction well is independent and contains a known crRNA and a known biotin-recovery DNA, and the biotin-recovery DNA can be bound with the following capture probes one by one).
The enzyme digestion buffer comprises the following components:
10～200mM NaCl；
5～100mM Tris-HCl；
2～50mM MgCl 2 ；
pH 7～9；
adding 2-10 mu l of amplification product, and placing the prepared enzyme digestion reaction system into a temperature bath at 25-42 ℃ for 5-30 min.
5. Hybridization reaction:
the 4 reaction liquids are combined with four kinds of NH 2 Nylon membrane hybridization of capture DNA, incubation at 25-45 ℃ for 30-120 min.
6. After the washing is completed, a proper amount of streptavidin-horseradish peroxidase strepavidin-HRP is added for incubation for 5 to 30 minutes.
7. The substrate was washed thoroughly, and an appropriate amount (10 to 50 mL) of the substrate solution was added thereto, followed by color development for 2 minutes and observation of the result.
The detection result of this embodiment is shown in fig. 3. In fig. 3, "full negative": blue spots appear at all sites on the membrane strip; "full positive": the sites on the membrane strip do not have blue spots; "a positive": 3 blue spots appear on the membrane strip, and no blue spots appear at the other 1 site; "two positives: 2 blue spots appear on the film strip, and no blue spots appear on the other 2 spots; 1 blue spot appears on the "three positive" membrane strip, and no blue spots appear at the other 3 sites.
The detection results were mainly specific discrimination by crRNA per well. If the amplified product only contains the influenza A nucleic acid amplified product, the biotin DNA probes in the FA channel are cleaved, while the biotin DNA probes of the other 3 channels remain intact; and then the whole reaction liquid is eluted into a reaction tank containing the membrane strips, and the biotin probes of the FA channels are not bound to the membrane strips because of enzyme digestion, and the biotin probes of other channels are bound to the corresponding membrane strips. If two, three and four of the above viruses are contained in the sample, the same will be detected.
In conclusion, the multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization technology can be used for conveniently, rapidly and efficiently detecting multiplex viral nucleic acid, and has low detection cost and strong applicability. The invention is converted into a diagnosis kit, and can be used for clinical early diagnosis, thereby improving the detection efficiency.
Meanwhile, through preliminary verification, the invention has the advantages of high sensitivity, high specificity and good detection performance.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
SEQUENCE LISTING
<110> university of Zhejiang
<120> DNA probe for multiplex nucleic acid detection, CRISPR-reverse dot hybridization nucleic acid detection system and application thereof
<130> 2022
<160> 12
<170> PatentIn version 3.3
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Claims (11)
1. A CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection is characterized in thatIncluding Cas12 protein, crRNA, bio-recognment DNA, streptavidin-horseradish peroxidase, NH-binding 2 Nylon membrane of capture DNA, hybridization solution, substrate solution;
wherein the crRNA is a sequence that specifically binds to a target nucleic acid and is not associated with Bio-recognition DNA and NH 2 Capture DNA binding; the Bio-recognment DNA comprises at least four kinds of DNA, and the sequences of the DNA are respectively shown as SEQ ID NO. 1-SEQ ID NO. 4; the NH on nylon membrane 2 The complementary pairing of the capture DNA and the Bio-recognition DNA comprises at least four sequences shown in SEQ ID NO. 5-SEQ ID NO. 8; the streptavidin-horseradish peroxidase is combined with Bio-recognosis DNA for reaction; the substrate solution is catalyzed by HRP in streptavidin-horseradish peroxidase for color reaction.
2. The CRISPR-reverse point hybrid nucleic acid detection system for multiplex nucleic acid detection according to claim 1, wherein said Cas12 protein is one of Cas12a, cas12b, cas12c, cas12d, cas12e, cas12 g.
3. The CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection according to claim 1, wherein when the multiplex nucleic acid detection is the detection of four nucleic acids of influenza a virus, influenza b virus, respiratory syncytial virus and novel coronavirus, the correspondingly constructed crrnas are FA-crRNA, FB-crRNA, RSV-crRNA, sars-2-N-crRNA, the sequences of which are shown in SEQ ID No.9 to SEQ ID No.12, respectively.
4. The CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection according to claim 1, wherein said nylon membrane surface is negatively charged modified.
5. The CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection according to claim 1, wherein said NH-binding 2 The preparation method of the nylon membrane of the capture DNA is as follows:
(1) Equipped with NH 2 Capture DNA working solution: to synthesize NH 2 -capture DNA diluted to a final concentration of 10 μm for spotting;
(2) Fixing NH 2 Capture DNA: soaking nylon membrane in 3-10% EDC solution for 10-40 min, washing with pure water for 3 times and air drying at room temperature;
(3) 0.5 to 1 mu l of NH 2 -capture DNA spot on nylon membrane, left for 30min and air dried;
(4) The reaction was terminated with sodium hydroxide solution, washed 3 times with pure water and dried in the air.
6. The CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection according to claim 1, wherein said hybridization solution comprises solution a, solution B, solution C;
the formula of the solution A is as follows:
20×SSC 100mL；
10％SDS10mL；
890mL of purified water;
totaling 1L;
the formula of the solution B is as follows:
20×SSC 10mL；
10％SDS 4mL；
386mL of purified water;
totaling 400mL;
the formula of the liquid C is as follows:
1M sodium citrate 20mL;
180mL of purified water;
totaling to 200mL.
7. The CRISPR-reverse dot hybridization nucleic acid detection system for multiplex nucleic acid detection according to claim 1, further comprising a nucleic acid sample; the nucleic acid sample is the DNA full length or fragment of the nucleic acid to be detected, isothermal amplification product or PCR product of the nucleic acid to be detected.
8. Use of a CRISPR-reverse dot hybridization nucleic acid detection system according to any one of claims 1 to 7 for the preparation of a multiplex nucleic acid detection kit for detecting multiplex target DNA and RNA in a sample.
9. A multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization techniques, characterized in that a sample to be detected is detected by using the CRISPR-reverse dot hybridization nucleic acid detection system according to any one of claims 1 to 2, or 4 to 7.
10. The multiplex nucleic acid detection method based on CRISPR and reverse dot hybridization techniques according to claim 9, comprising the specific steps of:
s1, dissolving a nucleic acid sample, corresponding crRNA, corresponding Bio-recogntion DNA and Cas12 protein in a buffer solution according to the detection type of target nucleic acid, and reacting at a proper temperature to obtain a plurality of first buffers;
s2, mixing all the first buffer solutions together to form a second buffer solution;
s3, combining the second buffer solution with NH 2 -hybridization reaction of nylon membrane of capture DNA with hybridization solution at a suitable temperature;
s4, incubating streptavidin-horseradish peroxidase with a nylon membrane;
s5, adding a substrate solution, and judging whether the target nucleic acid exists or not according to the color development degree of the color.
11. The method for multiplex nucleic acid detection based on CRISPR and reverse dot hybridization techniques according to claim 10, wherein in step S1: the buffer solution is as follows: 10-100 mM Tris-HCl, 10-200 mM NaCl, 5-50 mM MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The proper temperature is 25-42 ℃; the reaction time is 10 to 60 minutes; in the step S3: the hybridization proper temperature is 25-45 ℃; the hybridization reaction time is 30-120 minutes.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102102123A (en) * | 2009-12-22 | 2011-06-22 | 中山大学达安基因股份有限公司 | Improved reverse dot hybridization method and application thereof |
| CN110066865A (en) * | 2019-04-30 | 2019-07-30 | 常州桐树生物科技有限公司 | A kind of detection method and probe of the nucleic acid specific fragment based on CRISPR-Cas13a |
| CN111944879A (en) * | 2019-05-16 | 2020-11-17 | 华南师范大学 | Gene detection method based on CRISPR technology, kit and application |
| CN112501261A (en) * | 2021-01-29 | 2021-03-16 | 量准（上海）医疗器械有限公司 | Fluorescence detection method for nucleic acid amplified isothermally by using nano plasma resonance chip |
| CN113186257A (en) * | 2021-05-28 | 2021-07-30 | 杭州深度生物科技有限公司 | Constant-temperature hybridization method after PCR amplification based on liquid chip technology |
| CN113584134A (en) * | 2021-09-06 | 2021-11-02 | 山东启邦汇康生物技术有限公司 | Isothermal nucleic acid detection system based on CRISPR-Cas9 and method and application thereof |
| CN113755558A (en) * | 2021-08-27 | 2021-12-07 | 万子健生物技术(上海)有限公司 | Nucleic acid detection method based on liquid chip technology |
| CN114058679A (en) * | 2022-01-11 | 2022-02-18 | 深圳大学 | CRISPR cascade nucleic acid detection system and detection method and application thereof |
| CN114207145A (en) * | 2019-06-19 | 2022-03-18 | 瓦赫宁恩大学 | Type III CRISPR/Cas based diagnostics |
| WO2022060939A1 (en) * | 2020-09-16 | 2022-03-24 | President And Fellows Of Harvard College | Compositions and methods for nucleic acid preparation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8986958B2 (en) * | 2009-03-30 | 2015-03-24 | Life Technologies Corporation | Methods for generating target specific probes for solution based capture |
-
2022
- 2022-06-07 CN CN202210638261.0A patent/CN115058493B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102102123A (en) * | 2009-12-22 | 2011-06-22 | 中山大学达安基因股份有限公司 | Improved reverse dot hybridization method and application thereof |
| CN110066865A (en) * | 2019-04-30 | 2019-07-30 | 常州桐树生物科技有限公司 | A kind of detection method and probe of the nucleic acid specific fragment based on CRISPR-Cas13a |
| CN111944879A (en) * | 2019-05-16 | 2020-11-17 | 华南师范大学 | Gene detection method based on CRISPR technology, kit and application |
| CN114207145A (en) * | 2019-06-19 | 2022-03-18 | 瓦赫宁恩大学 | Type III CRISPR/Cas based diagnostics |
| WO2022060939A1 (en) * | 2020-09-16 | 2022-03-24 | President And Fellows Of Harvard College | Compositions and methods for nucleic acid preparation |
| CN112501261A (en) * | 2021-01-29 | 2021-03-16 | 量准（上海）医疗器械有限公司 | Fluorescence detection method for nucleic acid amplified isothermally by using nano plasma resonance chip |
| CN113186257A (en) * | 2021-05-28 | 2021-07-30 | 杭州深度生物科技有限公司 | Constant-temperature hybridization method after PCR amplification based on liquid chip technology |
| CN113755558A (en) * | 2021-08-27 | 2021-12-07 | 万子健生物技术(上海)有限公司 | Nucleic acid detection method based on liquid chip technology |
| CN113584134A (en) * | 2021-09-06 | 2021-11-02 | 山东启邦汇康生物技术有限公司 | Isothermal nucleic acid detection system based on CRISPR-Cas9 and method and application thereof |
| CN114058679A (en) * | 2022-01-11 | 2022-02-18 | 深圳大学 | CRISPR cascade nucleic acid detection system and detection method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout;Tao Hu等;Adv Sci.;第10卷(第2期);第1-10页 * |
| CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics;Richard Bruch等;Biosens Bioelectron;第177卷;全文 * |
| Exploiting the orthogonal CRISPR-Cas12a/Cas13a trans-cleavage for dual-gene virus detection using a handheld device;Tian Tian等;Biosens Bioelectron;第196卷;全文 * |
| Massively multiplexed nucleic acid detection with Cas13;Cheri M Ackerman等;Nature;第582卷(第7811期);第277-282页 * |
| Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6;Jonathan S Gootenberg等;Science;第360卷(第6387期);第439-444页 * |
| Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants;Nicole L Welch等;Nat Med.;第28卷(第5期);第1083-1094页 * |
| 基因魔剪CRISPR/Cas:核酸精准检测的新宠和利器;冯春凤;王云霞;蒲晓允;熊瑜;刘飞;张立群;;国际检验医学杂志(第03期);全文 * |
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