CN112852921A - Nucleic acid detection method based on instant detection test strip, detection probe and kit thereof - Google Patents
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Abstract
The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof. The nucleic acid detection method is based on the combination of a CRISPR/Cas12a system and an early pregnancy test paper, the nonspecific hCG detection probe is combined with a crRNA sequence for specifically recognizing a sequence to be detected to detect nucleic acid, so that the detection cost is greatly reduced, the detection target universality is obvious, the sensitivity is high, the specificity is good, the detection sensitivity can be comparable to a fluorescent end-point characterization method, and the minimum number of copy of 2 molecules can be detected.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof.
Background
Sudden and a series of serious acute and chronic infectious diseases always have the greatest harm to human health and cause the most death. Especially, some infectious viruses with various genotypes and complex structural proteins have long development cycle of vaccines, so that Point-of-care testing (POCT) is an important prerequisite for effectively investigating and controlling various important acute and chronic infectious pathogens while searching for new drugs. At present, two methods are mainly used for detecting the paroxysmal virus infectious diseases in the market, one is to detect virus nucleic acid, and the other is to detect antigen and antibody. Because the window periods of the two methods after infection detection are different, and the window period of nucleic acid detection is obviously shorter than that of antigen-antibody detection, the method is more beneficial to early screening of infection. Therefore, the realization of sensitive, specific, portable nucleic acid detection of infectious diseases is still the goal of continuous improvement and development.
In recent years, with the vigorous development of the field of molecular diagnosis, in order to develop a portable diagnosis system, scientists have created a new sensing concept, namely, a commercial POCT product is used for detecting a non-original object to be detected; the method can not only reduce the development cost of the instrument, but also really realize the portable detection of the nucleic acid. This method is widely recognized at present and is one of the most promising portable detection methods. So far, metal ions, small molecule compounds, trans-DNA, proteins, even cancer cells can be transduced to existing POCT commercial equipment with the help of different recognition elements (aptamers, dnases, etc.). Among these innovative detection methods, the test strip-based portable detection method is rapidly developed in qualitative or quantitative analysis applications for analyzing an analyte due to a simple operation method and an intuitive detection result. At present, the existing commercial early pregnancy test strip detection technology (Angew. chem. int. Ed.,2017,56, 992-.
The CRISPR/Cas system is an immune system present in bacteria and archaea to protect against the invasion of foreign substances. In recent years, CRISPR/Cas systems have gradually become important tools in genetic engineering and are widely applied to gene editing, gene expression regulation, gene detection and the like. The CRISPR/Cas12a system is a novel CRISPR/Cas system, has RNA-guided DNase cutting activity, and can activate non-specific single-stranded DNA (ssDNA) cutting activity of Cas12a while specifically cutting double-stranded DNA (dsDNA). By utilizing the characteristic, the complementation of the CRRNA (CRISPR-derived RNA) artificially designed for the target DNA and the DNA near the PAM (protospacer-adjacent motif) region in the target DNA can be realized, and the direct detection of different target DNAs can be realized. The ssDNA with the tag is introduced by activating non-specific DNA of Cas12 a. The Doudna topic group (Science 2018,360,436-439) utilizes the CRISPR/Cas12a system to realize the detection of Human Papilloma Virus (HPV); when the DNA to be detected exists, the crRNA can specifically recognize the DNA to be detected and the Cas12a protein to form a ternary complex, so that the non-specific cleavage activity of Cas12a is activated, and the fluorescence detection is realized by cleaving single-stranded DNA with two ends respectively provided with a fluorescence group and a quenching group. Besides using fluorophores as the final output means, the method can also use electrochemical signals (Angew. chem. int. Ed.2019,58, 17399-.
The method for performing gene diagnosis by using the early pregnancy test strip is only limited to a loop-mediated isothermal amplification (LAMP) product of nucleic acid to be detected, and a three-way type nucleic acid probe (represented by P1, P2 and P3 respectively for facilitating explanation) is used, wherein the 3 ' end of P1 is marked with pre-expressed hCG protein, the 5 ' end of P2 is marked with a magnetic sphere, and the 3 ' end is a foothold sequence. The three single-stranded nucleic acids of P1, P2 and P3 form a three-way structure. When the LAMP product of the target to be detected exists, the single-stranded loop sequence LP1 can be combined with the foothold sequence in the P2 to initiate a chain substitution reaction, the single-stranded loop sequence LP3 can be combined with the P3, and the structure of the three-way probe is damaged; finally, the magnetic force of the magnet is utilized to separate the hCG which is free in the solution from the magnetic ball, and the early pregnancy test paper shows positive. On the contrary, when the LAMP product of the target to be detected does not exist, the probe keeps a three-way structure, free hCG protein does not exist in the solution theoretically, and the early pregnancy test paper shows negative. The disadvantages of this method are: 1. the probe sequence is designed in a complex way, and the combination enthalpy needs to be calculated and balanced to ensure the smooth proceeding of the chain substitution reaction; 2. three single-stranded DNAs forming the three-way probe need to optimize the system concentration, so that the low background of the detection reaction is ensured; 3. the double-labeled probe increases the complexity of experimental operation, and the magnetic balls in different batches have different stability, so that the condition needs to be optimized again when the magnetic balls are replaced each time; 4. the overall design of the experiment limits the amplification mode of the gene to be detected and can only be used for the loop-mediated isothermal amplification reaction. 5. The method can only carry out nucleic acid detection, and is not suitable for detecting small molecules, proteins and the like.
At present, a commonly adopted test strip method is used for characterizing nucleic acid detection based on a CRISPR/Cas12a system, and mainly FAM and Biotin groups are respectively marked at two ends of a detection probe; although the test strip used by the method can be commercialized and can realize final portable detection, the price of the test strip is too high, the cost of the whole detection is invisibly increased, and the large-batch sample detection is not used.
Disclosure of Invention
In view of the above, in order to overcome the defects and shortcomings of the existing nucleic acid detection technology, the invention provides a nucleic acid detection method based on a real-time detection test strip, a detection probe and a kit thereof. The detection method has obvious target universality, can realize early nucleic acid screening of various infectious diseases, has good specificity and high sensitivity, and greatly reduces the detection cost.
The invention provides a nucleic acid detection method based on a real-time detection test strip, which comprises the following steps:
dissolving a nucleic acid sample to be detected, an hCG-labeled single-stranded DNA probe, crRNA and Cas12a protein in a buffer solution, and reacting to obtain a first reaction solution;
adding a substrate substance into the first reaction solution for incubation to obtain a second reaction solution;
dripping the second reaction solution on a sample pad of the early pregnancy test strip, waiting for 5-8min, observing the color development conditions of the detection line and the quality control line, and judging whether the nucleic acid to be detected is contained;
the crRNA is a crRNA sequence which is specifically combined with the nucleic acid to be detected;
the substrate substance is a macromolecular substance capable of being combined with the hCG-labeled single-stranded DNA probe.
In the present invention, the nucleic acid sample to be detected is: the full length or the fragment of the DNA of the nucleic acid to be detected, the PCR product of the nucleic acid to be detected or the isothermal amplification product of the nucleic acid to be detected.
The detection method can be applied to early nucleic acid screening of various infectious diseases, expensive instruments and equipment and professional standard laboratories are not needed, and the detection method has important significance for early monitoring, diagnosis, prevention and control of the infectious diseases.
In some specific embodiments, the detection method provided by the invention is used for detecting HPV16 and new coronavirus samples, and has high sensitivity, and the detection limit can reach 2 molecular copy numbers.
In the present invention, the hCG-labeled single-stranded DNA probe is simply referred to as an hCG probe. The hCG probe consists of two parts, one part is hCG protein, and the other part is single-stranded DNA probe sequence. In some embodiments, the amino acid sequence of the hCG protein is modified at the 5' end of the single-stranded DNA probe by NH 2C 6. Specifically, the hCG-labeled single-stranded DNA probe comprises the following components from 5 'end to 3' end: hCG DNA fragment-NH 2C 6-Single stranded DNA Probe.
In some embodiments, the nucleotide sequence of the single-stranded DNA probe is as set forth in SEQ ID NO: 1, and the following components:
SEQ ID NO：1：5-TTATTTTATTTTATTCTCTCTGGATGATG-3′。
the 5-end NH 2C 6 of the single-stranded DNA probe is a connection end with HCG protein, the 1 st to 15nt of the 5' end is a Cas12a non-specific cleavage region, and the last 14nt (16 th to 29nt) is a LAMP substrate binding region.
In some embodiments, the base substance is a large molecular weight single-stranded DNA of any sequence bound to the single-stranded DNA probes by base complementarity, or a macromolecular substance bound to the single-stranded DNA probes by chemical bonds.
In some embodiments, the substrate material is the entire product substrate after LAMP amplification of any nucleic acid sequence.
In some embodiments, the buffer comprises 50-200mM NaCl, 10-40mM Tris-HCl, 10-40mM MgCl2And 100-.
In some embodiments, the buffer comprises 200mM NaCl,40mM Tris-HCl,40mM MgCl2And 400. mu.g/mL BSA.
In some embodiments, the reaction is specifically carried out at 25-37 ℃ for 30-60 min; the incubation is carried out at 25 ℃ for 20-40 min.
In some embodiments, the reaction is specifically a reaction at 37 ℃ for 30min or a reaction at 25 ℃ for 40-60 min; the incubation is specifically incubation at 25 ℃ for 40 min.
The invention also provides an hCG signal labeled single-stranded DNA probe for nucleic acid detection, and the nucleotide sequence of the single-stranded DNA probe is shown as SEQ ID NO: 1 is shown.
The invention also provides a nucleic acid detection kit, which comprises the hCG-labeled single-stranded DNA probe.
The nucleic acid detection kit also comprises at least one of crRNA, Cas12a protein, buffer solution, RNase inhibitor, LAMP product substrate and early pregnancy test strip.
Wherein the buffer solution comprises 50-200mM NaCl, 10-40mM Tris-HCl and 10-40mM MgCl2And 100-.
In some embodiments, the buffer comprises 200mM NaCl,40mM Tris-HCl,40mM MgCl2And 400. mu.g/mL BSA.
The nucleic acid detection method is based on the combination of the CRISPR/Cas12a system and the early pregnancy test paper, and detects the nucleic acid through the nonspecific hCG detection probe and the crRNA sequence which specifically recognizes the sequence to be detected, thereby greatly reducing the detection cost, ensuring the detection specificity and simultaneously increasing the portability of the whole detection. The detection principle is shown in figure 1: when the test nucleic acid exists, a ternary complex can be formed with the Cas12a protein and the crRNA, and the RuvC structural domain of Cas12a in the ternary complex exerts DNase activity to cut the dsDNA of the test nucleic acid. The activated Cas12a also has non-specific ssDNA cleavage activity, cleaving single-stranded DNA probes labeled with human chorionic gonadotropin (hCG) signal. The uncleaved single-stranded DNA probe is specifically combined with a single-stranded loop of a substrate (such as LAMP product) with a complex structure and a large volume, and hCG can not pass through the early pregnancy test paper by chromatography because the substrate is too large; in contrast, the cleaved single-stranded DNA probe, since hCG is completely free in solution, can pass through the early pregnancy test paper using chromatography even if the substrate is present. And judging whether the sample contains the target nucleic acid according to the color development condition of the early pregnancy test paper. The specific judging method comprises the following steps:
comparing with the color development condition of the negative sample, if the red strip of the detection area of the sample to be detected is darker than the red strip of the detection area of the negative sample, the detection result is positive, and the target nucleic acid is contained in the sample to be detected; otherwise, if the color of the detection area is consistent with or lighter than the color of the detection area of the negative sample, the detection result is negative, which indicates that the sample does not contain the nucleic acid to be detected.
Compared with the traditional test strip method for representing the CRISPR/Cas12a system nucleic acid detection system, the detection method disclosed by the invention has the advantages that the detection test strip with low price is replaced by the commercial early pregnancy test strip with low market price, so that the detection cost is greatly reduced; meanwhile, the kit has obvious detection target universality, high sensitivity and good specificity, the detection sensitivity can be compared with a fluorescence end point characterization method, and the lowest detection sensitivity can be up to 2 molecular copy numbers. In addition, the hCG probe and the LAMP substrate used in the system can be subjected to dry pulverization treatment, so that the system is convenient to store and transport.
Drawings
FIG. 1 is a schematic diagram showing the detection principle of the nucleic acid detection method of the present invention;
FIG. 2 shows the detection results of the detection method of the present invention for different concentrations of HPV-16-L1 mimicking double-stranded DNA;
FIG. 3 is a schematic diagram showing the non-specific ssDNA cleavage process triggered by the activation of CRISPR/Cas12a system by the amplification product after the isothermal amplification reaction (here, RPA reaction is taken as an example) of the gene to be detected;
FIG. 4 shows the result of HPV-16-L1 detection by the early pregnancy test paper of example 2;
FIG. 5 shows the test results of the early pregnancy test strip of example 3 for new coronavirus.
Detailed Description
The invention provides a nucleic acid detection method based on an instant detection test strip, a detection probe and a kit thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 detection of HPV Gene Using the detection method of the present invention
The test strip detection is carried out on HPV-16-L1 simulated DNA fragment sequences, and simultaneously the output method of fluorescence signals is compared, wherein the simulated DNA sequences are as follows:
ATTGGTTACAACGAGCACAGGGCCACAATAATGGCATTTGTTGGGGTAACCAACTATTTGTTACTGTTGTTGATACTACACGCAGTACAAATATGTCATTATGTGCTGCCATATCTACTTCAGAAACTACATATAAAAATACTAACTTTAAGGAGTACCTACGACATGGGGAGGAATATGATTTACAGTTTATTTTTCAACTGTGCAAAATAACCTTAACTGCAGACGTTATGACATACATACATTCTATGAATTCCAC。
the nucleotide sequence of the crRNA specifically combined with the mimic DNA sequence is as follows: 5'-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3' (SEQ ID NO: 2)
The reaction system is as follows: mu.M Cas12a, 2. mu.M crRNA,20U RNase Inhibitor (Murine),14nM hCG probe, 5nM/1nM/500pM/100pM/50pM/10pM/5pM/0pM HPV-16-L1 mock DNA double strand, for a total of 10. mu.L of mixed solution. The mixture was dissolved in a buffer system of 200mM NaCl,40mM Tris-HCl,40mM MgCl2Reacting at 37 ℃ for 30min in 400 mu g/mL BSA solution, adding 30 mu L LAMP product substrate, incubating at 25 ℃ for 40min, dropping all 40 mu L reaction solution on the bottom of the early pregnancy test strip, standing for 5min, and observing.
As can be seen from the detection results shown in fig. 2B, after the nucleic acid analytes with different concentrations are added to initiate Cas12a cleavage, the test strip detection line bands are significantly deepened; compared with the lowest detection concentration of the output mode of the fluorescent marker in the graph 2A, the early pregnancy test strip can also detect 5pM of genes to be detected, and meanwhile, as the concentration of the nucleic acid to be detected is increased, the detection line strips are increased.
Example 2 detection of Trace amount of HPV Gene Using the detection method of the present invention
First, the 16-L1 gene of artificially synthesized HPV virus is amplified by isothermal amplification reaction (for example, RPA or LAMP amplification), taking RPA amplification as an example, and the amplified product is added to the above detection system, as shown in FIG. 3.
The RPA amplification primer sequences used were as follows:
HPV-16-L1 forward primer: TTGTTGGGGTAACCAACTATTTGTTACTGTT (SEQ ID NO: 3).
HPV-16-L1 reverse primer: CCTCCCCATGTCGTAGGTACTCCTTAAAG (SEQ ID NO: 4).
RPA amplification is used in conjunction with CRISPR/Cas12a system, the reaction system is as follows: mu.M Cas12a, 2. mu.M crRNA,20U RNase Inhibitor (Murine),14nM hCG probe, 2. mu.L of RPA amplification product of different copy number templates, in total 10. mu.L of mixed solution. The mixture was dissolved in a buffer system of 200mM NaCl,40mM Tris-HCl,40mM MgCl2Reacting at 37 ℃ for 30min in 400 mu g/mL BSA solution, adding 30 mu L LAMP product substrate, incubating at 25 ℃ for 40min, dropping all 40 mu L reaction solution on the bottom of the early pregnancy test strip, standing for 5min, and observing.
FIG. 4 is a color development diagram of a test strip for pregnancy test, which is triggered by nonspecific ssDNA cleavage after the CRISPR/Cas12a system is activated by the RPA product. The result graph of the negative control is that the template concentration in the RPA reaction is 0 molecular copy number, and the positive control is that the template concentration is 2, 20, 200, 2,000 and 20,000 molecular copy numbers respectively, so that the lowest detectable molecular copy number of the invention is 2 molecular copy numbers.
Example 3 detection of Trace amount of New coronavirus Using the detection method of the present invention
In order to prove the universality of the detection method, the detection method is utilized to detect trace amount of novel coronavirus (SAR-Cov-2) reverse transcription dsDNA.
Firstly, ORF1ab gene in the novel coronavirus is subjected to isothermal amplification by using an RPA amplification method, and the amplification product directly triggers a CRISPR/Cas12a system.
The sequence of the novel coronavirus ORF1ab gene amplification probe is as follows:
ORF1ab-F:CTTGAAATTCCACGTAGGAATGTGGCAACTTTAC(SEQ ID NO：5)；
ORF1ab-R:GTATGCCAGGTATGTCAACACATAAACCTTCAG(SEQ ID NO：6)。
FIG. 5 is a color development diagram of a test strip for pregnancy test, which shows the non-specific ssDNA cleavage triggered by the activation of CRISPR/Cas12a system by the RPA product of ORF1 ab. Wherein the negative control is 0 molecular copy number of template concentration in RPA reaction, and the positive control is 2, 20, 200, 2,000, 20,000 molecular copy numbers of template concentration.
The results show that the detection method has obvious detection target universality, and the lowest detection of the test strip endpoint can be detected to 2 molecular copy numbers. Meanwhile, the RPA amplification product of the HPV-16-L1 gene is added for comparison, and the system is found to be capable of specifically detecting the ORF1ab target gene and not to generate false positive misdiagnosis even under the interference of a large amount of other gene RPA products.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Sequence listing
<110> Changchun applied chemistry research institute of Chinese academy of sciences
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Claims (12)
1. A nucleic acid detection method based on an instant detection test strip is characterized by comprising the following steps:
dissolving a nucleic acid sample to be detected, an hCG-labeled single-stranded DNA probe, crRNA and Cas12a protein in a buffer solution, and reacting to obtain a first reaction solution;
adding a substrate substance into the first reaction solution for incubation to obtain a second reaction solution;
dripping the second reaction solution on a sample pad of the early pregnancy test strip, waiting for 5-8min, observing the color development conditions of the detection line and the quality control line, and judging whether the nucleic acid to be detected is contained;
the crRNA is a crRNA sequence which is specifically combined with the nucleic acid to be detected;
the substrate substance is a macromolecular substance capable of being combined with the hCG-labeled single-stranded DNA probe.
2. The method for detecting nucleic acid according to claim 1, wherein the nucleic acid sample to be detected is: the DNA full length or the fragment of the nucleic acid to be detected, the PCR product or the isothermal amplification product of the nucleic acid to be detected.
3. The method for detecting nucleic acid according to claim 1, wherein the nucleotide sequence of the single-stranded DNA probe is as set forth in SEQ ID NO: 1 is shown.
4. The method for detecting nucleic acid according to claim 3, wherein the amino acid sequence of hCG protein in the hCG-labeled single-stranded DNA probe is modified at the 5' end of the single-stranded DNA probe by NH 2C 6.
5. The method for detecting nucleic acid according to claim 1, wherein the base substance is a high molecular weight single-stranded DNA having an arbitrary sequence that binds to the single-stranded DNA probe by base complementation,
or is
A macromolecular substance bonded to the single-stranded DNA probe via a chemical bond.
6. The method of claim 4, wherein the substrate material is the substrate of the entire product after LAMP amplification of any nucleic acid sequence.
7. The method for detecting nucleic acid according to claim 1, wherein the buffer comprises 50-200mM NaCl, 10-40mM Tris-HCl, and 10-40mM MgCl2And 100-.
8. The method for detecting nucleic acid according to claim 1, wherein the reaction is specifically a reaction at 25 to 37 ℃ for 30 to 60 min; the incubation is carried out at 25 ℃ for 20-40 min.
9. An hCG signal labeled single-stranded DNA probe for detecting nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown.
10. A nucleic acid detection kit comprising the hCG-labeled single-stranded DNA probe according to claim 9.
11. The nucleic acid detection kit of claim 10, further comprising at least one of crRNA, Cas12a protein, a buffer, an rnase inhibitor, a LAMP product substrate, and an early pregnancy test strip.
12. The nucleic acid detection kit according to claim 11, wherein the buffer comprises 200mM NaCl,40mM Tris-HCl,40mM MgCl2And 400. mu.g/mL BSA.
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| CN113699148A (en) * | 2021-07-15 | 2021-11-26 | 四川大学 | Ultrasensitive antibody detection method |
| CN115806993A (en) * | 2022-12-27 | 2023-03-17 | 中国人民解放军军事科学院军事医学研究院 | Aptamer HCG-2 for specifically recognizing human chorionic gonadotropin |
| CN115998341A (en) * | 2023-01-18 | 2023-04-25 | 珠海舒桐医疗科技有限公司 | Portable device for menstrual blood sample collection and HPV rapid detection system |
| CN116179511A (en) * | 2023-03-10 | 2023-05-30 | 之江实验室 | Application of Cpf1 protein in preparation of kit for nucleic acid detection |
| CN116656786A (en) * | 2023-03-14 | 2023-08-29 | 广州医科大学附属第一医院（广州呼吸中心） | CRISPR-lateral flow nucleic acid detection test paper based on chain hybridization and detection probe thereof |
| CN116990499A (en) * | 2023-08-03 | 2023-11-03 | 西安交通大学 | MBs-ssDNA-hCG probe, kit and application thereof in detection of IAP |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110184329A (en) * | 2019-05-31 | 2019-08-30 | 华南理工大学 | A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification |
| WO2020102709A1 (en) * | 2018-11-16 | 2020-05-22 | The Regents Of The University Of California | Compositions and methods for delivering crispr/cas effector polypeptides |
| CN111187804A (en) * | 2020-02-20 | 2020-05-22 | 国家卫生健康委科学技术研究所 | Rapid detection kit and detection method for mycoplasma pneumoniae nucleic acid based on CRISPR/Cas12a |
| CN111560482A (en) * | 2020-06-11 | 2020-08-21 | 亚能生物技术（深圳）有限公司 | Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit |
| CA3132197A1 (en) * | 2019-03-07 | 2020-09-10 | The Trustees Of Columbia University In The City Of New York | Rna-guided dna integration using tn7-like transposons |
| CN111647642A (en) * | 2020-05-19 | 2020-09-11 | 深圳市众循精准医学研究院 | Nucleic acid detection method and nucleic acid detection kit based on detection test paper display |
| 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 |
| CN112391498A (en) * | 2020-12-01 | 2021-02-23 | 中国科学院长春应用化学研究所 | Application of pregnancy test paper in on-site instant detection of hepatitis B virus drug-resistant mutant gene |
-
2021
- 2021-03-16 CN CN202110279931.XA patent/CN112852921B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020102709A1 (en) * | 2018-11-16 | 2020-05-22 | The Regents Of The University Of California | Compositions and methods for delivering crispr/cas effector polypeptides |
| CA3132197A1 (en) * | 2019-03-07 | 2020-09-10 | The Trustees Of Columbia University In The City Of New York | Rna-guided dna integration using tn7-like transposons |
| CN110184329A (en) * | 2019-05-31 | 2019-08-30 | 华南理工大学 | A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification |
| CN111187804A (en) * | 2020-02-20 | 2020-05-22 | 国家卫生健康委科学技术研究所 | Rapid detection kit and detection method for mycoplasma pneumoniae nucleic acid based on CRISPR/Cas12a |
| CN111647642A (en) * | 2020-05-19 | 2020-09-11 | 深圳市众循精准医学研究院 | Nucleic acid detection method and nucleic acid detection kit based on detection test paper display |
| CN111560482A (en) * | 2020-06-11 | 2020-08-21 | 亚能生物技术（深圳）有限公司 | Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit |
| 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 |
| CN112391498A (en) * | 2020-12-01 | 2021-02-23 | 中国科学院长春应用化学研究所 | Application of pregnancy test paper in on-site instant detection of hepatitis B virus drug-resistant mutant gene |
Non-Patent Citations (3)
| Title |
|---|
| DR. YIDAN TANG等: ""CLIPON: A CRISPR-Enabled Strategy that Turns Commercial Pregnancy Test Strips into General Point-of-Need Test Devices"", 《ANGEW CHEM INT ED ENGL》 * |
| JULIA JOUNG等: ""Point-of-care testing for COVID-19 using SHERLOCK diagnostics"", 《MEDRXIV 》 * |
| 刘园等: ""基于分子识别的免疫层析技术用于新冠肺炎感染的快速诊断"", 《高等学校化学学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113699148A (en) * | 2021-07-15 | 2021-11-26 | 四川大学 | Ultrasensitive antibody detection method |
| WO2023284514A1 (en) * | 2021-07-15 | 2023-01-19 | 四川大学 | Ultrasensitive antibody detection method |
| CN113699148B (en) * | 2021-07-15 | 2024-01-09 | 四川大学 | Ultrasensitive antibody detection method |
| CN115806993A (en) * | 2022-12-27 | 2023-03-17 | 中国人民解放军军事科学院军事医学研究院 | Aptamer HCG-2 for specifically recognizing human chorionic gonadotropin |
| CN115806993B (en) * | 2022-12-27 | 2023-11-14 | 中国人民解放军军事科学院军事医学研究院 | Aptamer HCG-2 for specifically recognizing human chorionic gonadotrophin |
| CN115998341A (en) * | 2023-01-18 | 2023-04-25 | 珠海舒桐医疗科技有限公司 | Portable device for menstrual blood sample collection and HPV rapid detection system |
| CN115998341B (en) * | 2023-01-18 | 2024-04-12 | 珠海舒桐医疗科技有限公司 | HPV rapid detection system for non-disease treatment and diagnosis |
| CN116179511A (en) * | 2023-03-10 | 2023-05-30 | 之江实验室 | Application of Cpf1 protein in preparation of kit for nucleic acid detection |
| CN116179511B (en) * | 2023-03-10 | 2023-12-22 | 之江实验室 | Application of Cpf1 protein in preparation of kit for nucleic acid detection |
| CN116656786A (en) * | 2023-03-14 | 2023-08-29 | 广州医科大学附属第一医院（广州呼吸中心） | CRISPR-lateral flow nucleic acid detection test paper based on chain hybridization and detection probe thereof |
| CN116990499A (en) * | 2023-08-03 | 2023-11-03 | 西安交通大学 | MBs-ssDNA-hCG probe, kit and application thereof in detection of IAP |
| CN116990499B (en) * | 2023-08-03 | 2024-03-01 | 西安交通大学 | MBs-ssDNA-hCG probe, kit and application thereof in detection of IAP |
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