CN113324956A - CRISPR technology and framework nucleic acid coupling-based modular detection platform, construction method and application thereof - Google Patents
CRISPR technology and framework nucleic acid coupling-based modular detection platform, construction method and application thereof Download PDFInfo
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention provides a CRISPR technology and framework nucleic acid coupled modular detection platform, a construction method and application thereof. The detection platform comprises the following 2 modules: (1) constructing an identification module by using a CRISPR technology, wherein the identification module is used for identifying nucleic acid (new coronavirus, human papilloma virus, hepatitis B virus nucleic acid and the like) and non-nucleic acid (protein, antibiotics, metal ions and the like) targets; (2) and constructing signal modules with different valence states by adopting frame nucleic acid, and outputting the detection signals in a controllable amplification manner. And finally, integrating the identification module and the signal module on an electrochemical chip or a paper chip simultaneously. The invention realizes the sensitive, rapid, simple, convenient and timely detection of nucleic acid and non-nucleic acid targets in a complex matrix by utilizing the high-sensitivity and high-specificity recognition of the CRISPR/Cas on the targets and combining the signal amplification of a frame nucleic acid signal group, and has good medical diagnosis and food safety monitoring application prospects.
Description
Technical Field
The invention belongs to the field of biotechnology and detection technology, and particularly relates to construction of a modular detection platform based on coupling of CRISPR technology and frame nucleic acid and detection application of the modular detection platform to nucleic acid and non-nucleic acid targets.
Background
CRISPR (clustered regulated short palindromic repeats) -Cas (CRISPR-associated) systems were originally derived from prokaryotic adaptive immune systems to recognize and degrade invading nucleic acids, an important invention in the field of gene editing in the 21 st century. Due to the excellent specificity and sensitivity of the CRISPR/Cas system, the application of the CRISPR/Cas system in the field of biosensing besides the gene editing field has attracted great interest. Compared with CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14 have both targeted specific DNA/RNA cutting activity and collateral non-specific DNA/RNA cutting activity under the mediation of crRNA, and when the binding of a Cas-crRNA complex and target DNA/RNA is activated, the target DNA/RNA is specifically cut and any adjacent DNA/RNA is non-specifically cut. Due to the existence of the attribute, the proteins such as CRISPR/Cas12a, CRISPR/Cas13 and CRISPR/Cas14 can be applied to construct a detection platform by marking a signal molecule on a single chain as a report chain, so that the detection and capture of nucleic acid, metal ions, proteins and even cells are realized.
For CRISPR technology, achieving amplification of a signal in a complex real sample is a breakthrough in the technology. The number of single-stranded DNA/RNA modified signal molecules is limited, and the complex matrix does not have the capacity of resisting nonspecific adsorption. Therefore, the use of a single-stranded DNA/RNA-modified signal molecule as a reporter strand limits the sensitivity and specificity of target detection in complex matrices, and is difficult to put into practical use. The characteristics of editable, addressable and rigid DNA nano material and the like enable the DNA nano material to be applied to solving the technical problem. To achieve amplification and transmission of signals in practical complex samples, we will use frame nucleic acids (e.g. DNA tetrahedron, TDF) as signal clusters to extend different numbers of arm chains at their vertex positions and modify the signal molecules, thereby achieving controllable amplification output of complex matrix detection signals. By coupling the CRISPR technique with the framework nucleic acids, highly sensitive and highly specific detection of nucleic and non-nucleic acid targets is ultimately achieved.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to solve the problem of signal output in actual complex sample detection by the CRISPR technology, and the controllable amplification output of the signal can be realized by using the rigid framework nucleic acid as the signal group. Multiple signal outputs are achieved by modifying redox indicators or fluorophore-quenching groups on the arm strands of the DNA tetrahedron, and multiplexing can be achieved. The modular detection platform has good medical diagnosis and food safety monitoring application prospects.
In order to solve the technical problems, one of the technical schemes adopted by the invention is as follows:
providing a modular detection platform coupled to a framework nucleic acid based on CRISPR technology, the platform comprising:
(1) for the nucleic acid target, mediated crRNA aiming at the nucleic acid target is contained, a Cas-crRNA complex is obtained by pre-incubation with a Cas protein and serves as a recognition module, and a framework nucleic acid signal module with different valence states is additionally contained; the different valence frame nucleic acid signal module is a DNA Tetrahedral (TDF) signal group which extends 1, 2, 3 or 4 arm chains from the top and modifies an electric signal or fluorescent signal molecule at the tail end of the arm chain; or
(2) For the non-nucleic acid target, an aptamer containing the non-nucleic acid target specifically combined is paired with a complementary strand aDNA in advance to form a double-stranded aptamer-aDNA compound; the aDNA and the crRNA are complementary strand aDNA and mediated crRNA designed according to a non-nucleic acid target and a specific aptamer. Mediating the incubation of crRNA with Cas protein in advance to obtain a Cas-crRNA complex which is used as a recognition module and comprises a framework nucleic acid signal module with different valence states; the different valence frame nucleic acid signal module is a DNA Tetrahedral (TDF) signal group which extends 1, 2, 3 or 4 arm chains from the top and modifies an electric signal or fluorescent signal molecule at the tail end of the arm chains.
The platform is used for capturing a nucleic acid target by a recognition module Cas-crRNA complex to form a Cas-crRNA-target complex, activating the side-cutting activity of a Cas protein, cutting a TDF signal group and generating a changed current signal or a fluorescence signal; or
For non-nucleic acid targets, the target competes with the aptamer-aDNA duplex, forming an aptamer-target complex, releasing aDNA. The released aDNA is captured by a Cas-crRNA complex of a recognition module to form a Cas-crRNA-aDNA complex, and meanwhile, the side cleavage activity of the Cas protein is activated to cleave a TDF signal group to generate a changed current signal or a fluorescence signal.
Wherein, the nucleic acid target can be neocoronavirus, human papilloma virus or hepatitis B virus nucleic acid;
the non-nucleic acid target can be protein, antibiotic or metal ion, etc.
The Cas-crRNA complex is usually present in a buffer solution, 1 XNEBuffer 2.1 (containing 10U of ribonuclease inhibitor) is commonly used.
The concentration of the Cas-crRNA compound is 50 nM-1 μ M.
The framework nucleic acid in the present invention is DNA Tetrahedron (TDF).
The DNA tetrahedron buffer is a TM buffer (50mM MgCl2, 20mM Tris, pH 8.0).
The side length of the DNA tetrahedron is 7bp, 13bp, 17bp, 26bp or 37 bp.
The DNA tetrahedron arm chain is an oligonucleotide sequence extending from the vertex of the DNA tetrahedron and has the length of 10 nt-50 nt.
The redox indicator is methylene blue MB, ferrocene Fc, biotin or a fluorescent group FAM-quenching group DABCYL.
The invention also provides a construction method of the modular detection platform based on coupling of CRISPR technology and frame nucleic acid, which comprises the following steps:
(1) constructing a recognition module Cas-crRNA compound by using a CRISPR/Cas technology to realize high-sensitivity and high-specificity capture on nucleic acid and non-nucleic acid targets;
(2) constructing different valence frame nucleic acid signal modules by using a DNA nanotechnology to realize controllable amplification output of detection signals;
(3) and (3) integrating the identification module obtained in the step (1) and the signal module obtained in the step (2) on an electrochemical chip or a paper chip to realize high-sensitivity and high-specificity detection on the target object.
Wherein the step (1) is as follows: a recognition module Cas-crRNA compound is constructed by using a CRISPR/Cas technology, so that high sensitivity and high specificity capture of nucleic acid and non-nucleic acid targets are realized.
The method for constructing the recognition module Cas-crRNA complex comprises the following steps: designing a crRNA hybridized with the gene sequence aiming at the nucleic acid target; for non-nucleic acid targets, specific aptamers (aptamers) are selected first, and then aDNA and crRNA which are hybridized with the aptamers are designed according to the aptamers. And then, mixing the Cas protein and the crRNA in a concentration ratio of 1:1 in a 1 XNEBbuffer 2.1, 10U RNase inhibitor solution, and incubating for 10 minutes at room temperature to obtain the recombinant protein.
Wherein the Cas proteins are Cas12a, Cas13, Cas 14.
Wherein the nucleic acid target is neocoronavirus, human papilloma virus or hepatitis B virus nucleic acid.
Wherein the non-nucleic acid target is protein, antibiotic or metal ion, etc.
Wherein the concentration ratio of the Cas protein to the crRNA is 1: 1. The concentration of the Cas-crRNA complex is 50nM to 1 μ M. The Cas-crRNA captures a target, and the reaction time is 5-200 minutes. And heating and annealing the aptamer and the aDNA at the concentration ratio of 1:1 to form a double chain.
Wherein the step (2) is as follows: and (3) constructing different valence frame nucleic acid signal modules by using a DNA nanotechnology to realize controllable amplification output of detection signals.
The method for constructing the different valence state frame nucleic acid (TDF signal group) signal module comprises the following steps: TDF semaphore in different valence states As described in example 3, TDF-1 (an arm chain) consists of TDF ID NO 1 TDF ID NO 8; TDF-2 (two arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 6-TDF ID NO 9; TDF-3 (three arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 7-TDF ID NO 10; TDF-4 (four arm chains) consists of TDF ID NO:1 TDF ID NO:4 and TDF ID NO:8 TDF ID NO: 11. Mixing 8 DNA strands constituting each TDF at an equal ratio in TM buffer solution (pH 8.0), heating to 95 deg.C for 10 min, rapidly cooling to 4 deg.C, and maintaining at 4 deg.C for 5 min.
The TDF blob sides may be 7bp, 13bp, 17bp, 26bp, and 37bp in length.
The DNA tetrahedron arm chain is an oligonucleotide sequence extending from the vertex of the DNA tetrahedron and has the length of 10 nt-50 nt.
The TDF signaling group may be modified with a redox indicator Methylene Blue (MB), ferrocene (Fc) or biotin (biotin), a fluorophore FAM-quenching group DABCYL, or the like.
The incubation concentration of the TDF signal group at the interface is 25nM to 500 nM. Of these, the rate of change of signal was maximal at 50 nM.
Wherein the step (3) is as follows: the recognition module and the signal module are integrated on an electrochemical chip or a paper chip, and after the recognition module is specifically combined with a target, the activated Cas protein cuts the signal module, so that a changed current signal or a fluorescence signal is generated, and high-sensitivity and high-specificity detection on a target is realized.
Wherein the nucleic acid target is recognized and captured by a recognition module Cas-crRNA complex to form a Cas-crRNA-target complex, and simultaneously activates the side cutting activity of the Cas protein. The non-nucleic acid target, competes with aptamer-aDNA for binding, releasing aDNA. The released aDNA is recognized and captured by the recognition module Cas-crRNA complex to form a Cas-crRNA-aDNA complex, and the Cas protein side cleavage activity is activated. The activated Cas protein cleaves the signaling module TDF signal bolus, generating an altered current signal or a fluorescent signal. The cutting time is 5-200 minutes. The signal may be a current signal or a fluorescent signal.
According to the modular detection platform based on coupling of the CRISPR technology and the frame nucleic acid, the identification module is constructed based on the CRISPR technology, amplification of a target is not required in advance, and controllable signal amplification output is realized by utilizing the signal module constructed by the frame nucleic acid, so that high-sensitivity and high-specificity detection is realized, and the problem of false positive/false negative caused by target amplification is effectively avoided; based on the frame nucleic acid as a signal module, the sequence design and the change are not required to be carried out according to the target, the universality is realized, the effect of nonspecific adsorption resistance is achieved, the signal can be controllably amplified, and the accurate and rapid detection of a complex actual sample is realized. The advantages of the two modules are combined, and the detection platform has good medical diagnosis and food safety monitoring application prospects.
Drawings
FIG. 1 is a representation of polyacrylamide gel electrophoresis of TDF signal groups in different valencies after purification;
FIG. 2 is an AFM topographic representation of the TDF-4 signal bolus (containing 4 arm chains) after purification;
FIG. 3 is a representation of agarose gel electrophoresis of cleavage of TDF signalophores in different valencies between a target captured by the recognition module and a target not captured;
FIG. 4 is a graph of AC voltammetric current before addition of target HPV viral nucleic acid, and after addition of 1nM and 100nM HPV viral nucleic acid, wherein HPV is human papilloma virus;
FIG. 5 is a graph of the rate of change of signal for different valency signallings incubated by the recognition module.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of Cas12a-crRNA Complex
crRNA was designed against human papillomavirus HPV16 nucleic acid sequence. The crRNA can be incubated with Cas12a at room temperature to form a Cas12a-crRNA complex. The HPV16 nucleic acid sequence and the crRNA sequence are respectively shown in HPV ID NO 1-HPV ID NO 3.
DNA and RNA sequences referred to in the examples of Table 1
Example 2 preparation of the recognition Module Cas12a-crRNA Complex
Cas12a protein was purchased from New England Biolabs (NEB) technologies, Inc., USA. crRNA was mixed with Cas12a protein at a concentration ratio of 1:1 in 1 × NEBbuffer 2.1, 10U rnase inhibitor solution and incubated at room temperature for 10 minutes.
Example 3 Synthesis and purification of TDF signalophores
The oligonucleotide sequences required for TDF signal group synthesis and the extended arm chain were synthesized by Shanghai Producens, Inc. The sequences of the oligonucleotides are listed as TDF ID NO 1 to TDF ID NO 11, respectively. TDF-1 (an arm chain) is composed of TDF ID NO 1-TDF ID NO 8; TDF-2 (two arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 6-TDF ID NO 9; TDF-3 (three arm chains) consists of TDF ID NO 1-TDF ID NO 4 and TDF ID NO 7-TDF ID NO 10; TDF-4 (four arm chains) consists of TDF ID NO:1 TDF ID NO:4 and TDF ID NO:8 TDF ID NO: 11. Mixing 8 DNA strands constituting each TDF at an equal concentration in TM buffer (pH 8.0), heating to 95 deg.C for 10 min, rapidly cooling to 4 deg.C, and maintaining at 4 deg.C for 5 min.
The TDF signal mass was purified using an Agilent 1260HPLC system, configured with a size exclusion chromatography column (Phenomenex BioSec-SEC-4000,300mm-7.8mm) and chromatograms recorded at 260 nm. The buffer was replaced with a TM buffer suitable for stable structure by centrifugation at 3000g for 10 min using an Amicon Ultra-0.5mL ultrafiltration tube (MWCO 100kDa), followed by replenishment of the TM buffer in the ultrafiltration tube to a total volume of 500uL and further centrifugation at 3000g for 10 min. The concentration of the TDF bolus was then determined using an ultraviolet-visible absorption spectrophotometer. The results of TDF synthesis were verified by Agarose Gel Electrophoresis (AGE) and modified methylene blue TDF synthesis by polyacrylamide gel electrophoresis (PAGE).
For comparison with the experimental TDF signal bolus, TDF-0 without arm chain was synthesized as a control group.
The experimental result is shown in fig. 1, and the mobility of the band becomes slow with the increase of the valence state, which proves that the TDF signal groups with different valence states are successfully synthesized and can be used for further experimental study.
The morphology of TDF-4 (valence 4 TDF signal cluster, containing 4 arm chains) was imaged using an atomic force microscope, as shown in FIG. 2, demonstrating that the TDF-4 signal cluster morphology is tetrahedral.
Table 2 DNA sequence referred to TDF in example
DNA sequences related to TDF in different valences in the example of Table 3
Example 4 preparation of Cas12a-crRNA-target Complex
The experimental result is shown in fig. 3, after the Cas12a-crRNA complex captures the target and forms the Cas12a-crRNA-target complex, the activity of the Cas12a protein is successfully activated, the TDF signalase with different valence states is cleaved, and the band mobility of the TDF signalase band becomes fast after the cleavage.
Example 5 integration of an identification Module and a Signal Module on an electrochemical chip
To achieve low cost detection, the identification module and the signal module are integrated on an electrochemical chip. The electrode chip was cleaned and purged with nitrogen (N)2) Blow-dry, incubation of the TDF bolus on the working electrode surface followed by placing the chip in a wet box overnight. Wash the electrode chip with 1 XPBS and use N2And (5) drying. The experimental group incubate the recognition module (containing target DNA), set the control group (containing no target DNA), rinse the electrode chip with 1 × PBS 30 minutes later, and finally drop 7 μ L PBS buffer for electrochemical measurements. Measuring an electric signal by adopting square wave voltammetry and alternating current voltammetry: the frequency of the square wave voltammetry is 10 Hz; the AC voltammetry frequency was 50 Hz.
The experimental result is shown in fig. 4, after the control group (containing no target DNA), the recognition module (containing 10nM target DNA), and the recognition module (containing 100nM target DNA) are respectively incubated on the signal module, the current signal gradually decreases, and the signal change rate increases, indicating that the recognition module successfully cuts the signal group.
Example 6 examination of the Change Rate of TDF semaphore Signal in different valence states
TDF signaling (modified MB) was incubated overnight on gold electrodes (2 mm diameter) at concentrations of 50nM each with different valencies, the electrodes were washed with 1 XPBS, and N was used2And (5) drying. The recognition modules (containing 100nM target DNA) were incubated on different valency signal groups, and after 30 minutes the electrode chip was washed with 1 XPBS and finally electrochemically measured in PBS buffer. The electrical signal was measured using square wave voltammetry at a frequency of 50 Hz. And comparing the signal change rates of the signal groups with different valence states.
The experimental results show in fig. 5 that the TDF-4 signal change rate is maximized as the signal change rate becomes larger as the valence state increases.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and various modifications may be made to the above-described embodiment of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present invention. The invention has not been described in detail in order to avoid obscuring the invention.
Claims (10)
1. A modular detection platform based on CRISPR technology and frame nucleic acid coupling is characterized in that the platform contains a recognition module constructed by the CRISPR technology and a signal module with different valence states constructed by the frame nucleic acid:
(1) for the nucleic acid target, mediated crRNA aiming at the nucleic acid target is contained, a Cas-crRNA complex is obtained by pre-incubation with a Cas protein and serves as a recognition module, and a framework nucleic acid signal module with different valence states is additionally contained; the different valence frame nucleic acid signal module is a DNA Tetrahedral (TDF) signal group which extends 1, 2, 3 or 4 arm chains at the top and modifies an electric signal or fluorescent signal molecule at the tail end of the arm chains; or
(2) For the non-nucleic acid target, an aptamer containing the non-nucleic acid target specifically combined is paired with a complementary strand aDNA in advance to form a double-stranded aptamer-aDNA compound; the aDNA and the crRNA are complementary strand aDNA and mediated crRNA designed according to a non-nucleic acid target and a specific aptamer. Mediating the incubation of crRNA with Cas protein in advance to obtain a Cas-crRNA complex which is used as a recognition module and comprises a framework nucleic acid signal module with different valence states; the different valence frame nucleic acid signal module is a DNA Tetrahedral (TDF) signal group which extends 1, 2, 3 or 4 arm chains from the top and modifies an electric signal or fluorescent signal molecule at the tail end of the arm chains.
2. The modular detection platform coupled to framework nucleic acids based on CRISPR technology of claim 1, wherein the platform:
(1) for a nucleic acid target, the target reacts with a Cas-crRNA complex to form a Cas-crRNA-target complex, the side-cut activity of a Cas protein is activated, a TDF signal group is cut, and a changed current signal or a fluorescence signal is generated; or
(2) For non-nucleic acid targets, the target competes with the aptamer-aDNA duplex, forming an aptamer-target complex, releasing aDNA. The released aDNA reacts with the Cas-crRNA complex to form a Cas-crRNA-aDNA complex, and meanwhile, the Cas protein collateral cleavage activity is activated to cleave the TDF signal group to generate a changed current signal or fluorescence signal.
3. The platform of claim 1, wherein said nucleic acid is a neocoronavirus, human papilloma virus or hepatitis b virus nucleic acid; the non-nucleic acids are proteins, antibiotics or metal ions.
4. The platform of claim 1, wherein the Cas-crRNA complex is at a concentration of 50nM to 1 μ Μ.
5. The platform of claim 1, wherein the TDF signaling moiety-modified signaling molecule is a redox indicator methylene blue MB, ferrocene Fc, biotin, or a fluorophore FAM-quenching group DABCYL.
6. The platform of claim 1, wherein the TDF bolus is a DNA tetrahedron of 7bp, 13bp, 17bp, 26bp, or 37bp in side length.
7. The platform of claim 1, wherein the DNA tetrahedron arms are 10nt to 50nt in length.
8. A method for constructing a modular detection platform based on CRISPR technology and frame nucleic acid coupling according to claims 1-7, which comprises the following steps:
(1) constructing a recognition module Cas-crRNA compound by using a CRISPR/Cas technology to realize high-sensitivity and high-specificity capture on nucleic acid and non-nucleic acid targets;
(2) constructing different valence frame nucleic acid signal modules by using a DNA nanotechnology to realize controllable amplification output of detection signals;
(3) and (3) integrating the identification module obtained in the step (1) and the signal module obtained in the step (2) on an electrochemical chip or a paper chip to realize high-sensitivity and high-specificity detection on the target object.
9. Use of the modular detection platform based on CRISPR technology coupled with framework nucleic acids of claims 1-7 in target detection.
10. The use of the modular assay platform of claim 9, wherein the use comprises the assay of a pathogenic nucleic acid, protein, small molecule or antibiotic.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113774114A (en) * | 2021-09-13 | 2021-12-10 | 上海交通大学 | Nucleic acid analysis method and application |
| CN114621998A (en) * | 2022-03-09 | 2022-06-14 | 深圳大学 | CRISPR-SPR biosensor chip and preparation method and application thereof |
| CN115873991A (en) * | 2022-11-02 | 2023-03-31 | 中创科瑞(北京)生物科技有限公司 | Block crRNA, function of enabling crRNA to switch and control binding with Cas12a and function verification method thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017189870A1 (en) * | 2016-04-27 | 2017-11-02 | Massachusetts Institute Of Technology | Stable nanoscale nucleic acid assemblies and methods thereof |
| CN107488710A (en) * | 2017-07-14 | 2017-12-19 | 上海吐露港生物科技有限公司 | A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule |
| CN111378722A (en) * | 2019-11-04 | 2020-07-07 | 江苏大学 | Specific nucleic acid fragment nano-fluorescence trace rapid detection method based on CRISPR-Cas12g |
| CN111549177A (en) * | 2020-04-27 | 2020-08-18 | 广州再生医学与健康广东省实验室 | gRNA and kit for detecting SARS-CoV-2 |
| CN112301016A (en) * | 2020-07-23 | 2021-02-02 | 广州美格生物科技有限公司 | Application of novel mlCas12a protein in nucleic acid detection |
| CN112342273A (en) * | 2020-11-11 | 2021-02-09 | 军事科学院军事医学研究院环境医学与作业医学研究所 | MOF-DNA hydrogel colorimetric detection kit and method based on CRISPR-Cas12a |
| CN112378971A (en) * | 2020-09-22 | 2021-02-19 | 华南师范大学 | CRISPR/Cas13 a-driven catalytic renewable electrochemical biosensor and application thereof |
| CN112595766A (en) * | 2020-10-16 | 2021-04-02 | 南京邮电大学 | Electrochemical sensor based on CRISPR/Cas13a and application thereof |
| AU2021100190A4 (en) * | 2021-01-13 | 2021-04-08 | Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine | CRISPR-Cas12a-based MOF-DNA Hydrogel Colorimetric Assay Kit and Method |
-
2021
- 2021-04-22 CN CN202110435381.6A patent/CN113324956B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017189870A1 (en) * | 2016-04-27 | 2017-11-02 | Massachusetts Institute Of Technology | Stable nanoscale nucleic acid assemblies and methods thereof |
| CN107488710A (en) * | 2017-07-14 | 2017-12-19 | 上海吐露港生物科技有限公司 | A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule |
| CN111378722A (en) * | 2019-11-04 | 2020-07-07 | 江苏大学 | Specific nucleic acid fragment nano-fluorescence trace rapid detection method based on CRISPR-Cas12g |
| CN111549177A (en) * | 2020-04-27 | 2020-08-18 | 广州再生医学与健康广东省实验室 | gRNA and kit for detecting SARS-CoV-2 |
| CN112301016A (en) * | 2020-07-23 | 2021-02-02 | 广州美格生物科技有限公司 | Application of novel mlCas12a protein in nucleic acid detection |
| CN112378971A (en) * | 2020-09-22 | 2021-02-19 | 华南师范大学 | CRISPR/Cas13 a-driven catalytic renewable electrochemical biosensor and application thereof |
| CN112595766A (en) * | 2020-10-16 | 2021-04-02 | 南京邮电大学 | Electrochemical sensor based on CRISPR/Cas13a and application thereof |
| CN112342273A (en) * | 2020-11-11 | 2021-02-09 | 军事科学院军事医学研究院环境医学与作业医学研究所 | MOF-DNA hydrogel colorimetric detection kit and method based on CRISPR-Cas12a |
| AU2021100190A4 (en) * | 2021-01-13 | 2021-04-08 | Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine | CRISPR-Cas12a-based MOF-DNA Hydrogel Colorimetric Assay Kit and Method |
Non-Patent Citations (7)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113774114A (en) * | 2021-09-13 | 2021-12-10 | 上海交通大学 | Nucleic acid analysis method and application |
| CN114621998A (en) * | 2022-03-09 | 2022-06-14 | 深圳大学 | CRISPR-SPR biosensor chip and preparation method and application thereof |
| CN115873991A (en) * | 2022-11-02 | 2023-03-31 | 中创科瑞(北京)生物科技有限公司 | Block crRNA, function of enabling crRNA to switch and control binding with Cas12a and function verification method thereof |
| CN115873991B (en) * | 2022-11-02 | 2024-03-01 | 中创科瑞(北京)生物科技有限公司 | Block crRNA, function enabling crRNA to switch and control binding with Cas12a and function verification method thereof |
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