CN111808931A - One-step RPA-CRISPR nucleic acid detection method, kit and application - Google Patents
One-step RPA-CRISPR nucleic acid detection method, kit and application Download PDFInfo
- Publication number
- CN111808931A CN111808931A CN202010679764.3A CN202010679764A CN111808931A CN 111808931 A CN111808931 A CN 111808931A CN 202010679764 A CN202010679764 A CN 202010679764A CN 111808931 A CN111808931 A CN 111808931A
- Authority
- CN
- China
- Prior art keywords
- rpa
- nucleic acid
- dna
- crispr
- amplification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 69
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 45
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 45
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 45
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 38
- 108020004414 DNA Proteins 0.000 claims abstract description 77
- 230000003321 amplification Effects 0.000 claims abstract description 52
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 52
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 26
- 108091033409 CRISPR Proteins 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 11
- 239000000523 sample Substances 0.000 claims description 21
- 108700004991 Cas12a Proteins 0.000 claims description 17
- 102000018120 Recombinases Human genes 0.000 claims description 8
- 108010091086 Recombinases Proteins 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 238000007689 inspection Methods 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims description 5
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 208000035473 Communicable disease Diseases 0.000 claims description 3
- 101710116602 DNA-Binding protein G5P Proteins 0.000 claims description 3
- 101710162453 Replication factor A Proteins 0.000 claims description 3
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 claims description 3
- 101710176276 SSB protein Proteins 0.000 claims description 3
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 claims description 3
- -1 dNTP Proteins 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 13
- 239000013615 primer Substances 0.000 description 23
- 238000005516 engineering process Methods 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 239000000443 aerosol Substances 0.000 description 3
- 230000004544 DNA amplification Effects 0.000 description 2
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 108700014590 single-stranded DNA binding proteins Proteins 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a one-step RPA-CRISPR nucleic acid detection method, a kit and application. The nucleic acid detection method comprises the following steps: and mixing and amplifying the solution to be detected, the RPA reagent and the CRISPR reagent, and simultaneously detecting the real-time fluorescence intensity of a reaction system in the amplification process to realize the quantitative analysis of the target DNA in the solution to be detected. According to the invention, the solution to be detected, the RPA reagent and the CRISPR reagent are mixed, so that amplification and detection are synchronously carried out, the operation steps are simple, the time consumption is short, and the detection method has high sensitivity and high specificity and can detect the target DNA with the concentration of 1 aM.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a one-step RPA-CRISPR nucleic acid detection method, a kit and application.
Background
The nucleic acid amplification technology can enable the detection sensitivity of nucleic acid to reach the angstrom molar (aM) level, and has very important value in the detection application of actual samples. PCR is the gold standard for nucleic acid detection at present, but the required denaturation-renaturation-extension three-stage program temperature control greatly increases the manufacturing cost of detection equipment, and the detection time is usually more than 1 h.
Recombinase Polymerase Amplification (RPA) is called as a nucleic acid Amplification technology which is most likely to replace PCR, and rapid Amplification of nucleic acid can be realized only by keeping the Amplification for 10-15min at constant 37-40 ℃, so that the method has wide application prospect in actual rapid field detection requirements. RPA technology relies primarily on three enzymes: recombinases that bind single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding proteins (SSBs), and strand-displacing DNA polymerases.
CRISPR technology is a rapid detection technology for nucleic acids discovered in recent two years, mainly relying on fluorescent signals to determine the concentration of target nucleic acid molecules in a detection sample, and Janice S Chen believes that CRISPR-Cas12a can be used as a powerful tool for detection of DNA from various sources (see Chen J S, Ma E, Harrington L B, et al. CRISPR-Cas12a target binding peptides single-stranded dnase activity [ J ] Science,2018,360 6387: 436 (439)).
Researchers also find that the CRISPR-Cas12a technology has very high specificity, even the single-base mismatch of crRNA and target DNA can cause huge difference of signal intensity, namely the CRISPR-Cas12a can realize the resolution of single-base difference, but the detection sensitivity of the single CRISPR-Cas12a technology is only pM-nM level.
At present, there is a method of amplifying Nucleic Acid by PCR or LAMP and then detecting CRISPR by using PCR or LAMP, i.e., firstly amplifying Nucleic Acid by PCR or LAMP, and then adding the amplification product into CRISPR system for Detection (see Li Y, Mansourh, Wang T, et al. Naked-Eye Detection of Grapevine Red-Blutch visual infection using a plasma CRISPR Cas12a Assay [ J ]. Analytical Chemistry,2019,91(18): 11510-.
The problems with the above method include: 1. the transfer process of the amplification product has the risk of causing aerosol pollution, and false positive results can easily occur in actual detection; 2. after amplification is finished, the concentrations of the nucleic acid samples are basically consistent, and quantification cannot be realized by performing CRISPR detection again.
Therefore, it is an urgent need in the art to provide a quantitative analysis method for nucleic acids with high sensitivity and accuracy and less susceptibility to contamination.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a one-step RPA-CRISPR nucleic acid detection method, a kit and application, wherein the RPA-CRISPR nucleic acid detection method can realize amplification and detection of target DNA in one step, so that the amplification and the detection are synchronously carried out, the pollution problem caused by transfer of amplification products is solved, the amplification process can be reacted in real time, and the quantitative analysis capability is realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a one-step method for detecting an RPA-CRISPR nucleic acid, which comprises the following steps: and mixing and amplifying the solution to be detected, the RPA reagent and the CRISPR reagent, and simultaneously detecting the real-time fluorescence intensity of a reaction system in the amplification process to realize the quantitative analysis of the target DNA in the solution to be detected.
In the invention, a solution to be detected, an RPA reagent and a CRISPR reagent are mixed, in the presence of the RPA reagent, the amplification of target DNA in the solution to be detected can be realized without the steps of denaturation, renaturation and extension, the amplification can be maintained for a period of time at a constant temperature, and meanwhile, in the presence of the CRISPR reagent, the fluorescence intensity in the solution to be detected can be directly and effectively detected, the content of the target DNA in the current solution can be judged in real time, so that the quantitative analysis of the target DNA is realized;
because of the uncertainty of molecular biology, amplification and other steps are easily interfered by a reaction system to cause amplification failure, in the invention, the RPA reagent and the CRISPR reagent are combined, the mutual interference is reduced as much as possible by reasonable proportioning, so that the functions of the two groups of reaction reagents can still be accurately and specifically realized, the amplification and the detection are synchronously carried out, and the high sensitivity of the RPA amplification technology and the high specificity of the CRISPR detection technology can be combined, so that the nucleic acid detection method has high sensitivity and high specificity, and has simple operation steps, short time consumption and wide application prospect.
Preferably, the amplification temperature is 26 to 42 ℃, for example, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 35 ℃,36 ℃, 38 ℃, 40 ℃ or 42 ℃, preferably 37 to 42 ℃, and more preferably 40 ℃.
Preferably, the time for amplification is 20-90 min, for example, 20min, 25min, 30min, 40min, 45min, 50min, 55min, 60min, 70min, 80min or 90min, preferably 45-90 min. The time for amplification can be adjusted depending on the concentration of the target DNA, and for low concentrations of the target DNA, a longer amplification time is required.
In a preferred embodiment of the present invention, the RPA reagent comprises DNA recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP, RPA forward primer, and RPA reverse primer.
Preferably, Cas12a, crRNA and probe DNA are included in the CRISPR agent.
The reaction system comprises reagents required by RPA amplification, including DNA recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP and primers required by target DNA amplification, and reagents required by CRISPR detection, including Cas12a, crRNA and probe DNA, wherein the crRNA and the probe DNA are sequences designed for the target DNA.
As a preferred technical scheme of the invention, the RPA amplification has strict requirements on the concentration of the primer, and if the concentration range of the primer is unreasonable, the amplification can fail; the working concentration of the RPA forward primer is 0.3-0.8. mu.M, and may be, for example, 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.5. mu.M, 0.55. mu.M, 0.6. mu.M, 0.65. mu.M, 0.7. mu.M, 0.75. mu.M, or 0.8. mu.M, and preferably 0.5. mu.M.
Preferably, the working concentration of the RPA reverse primer is 0.3-0.8. mu.M, such as 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.5. mu.M, 0.55. mu.M, 0.6. mu.M, 0.65. mu.M, 0.7. mu.M, 0.75. mu.M or 0.8. mu.M, preferably 0.5. mu.M. Preferably, the length of the RPA forward primer is 25-40 bp, for example, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 35bp, 38bp or 40bp, and preferably 30-35 bp.
Preferably, the length of the RPA reverse primer is 25-40 bp, for example, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 35bp, 38bp or 40bp, and preferably 30-35 bp.
In a preferred embodiment of the present invention, the working concentration of Cas12a is 0.01 to 0.05 μ M, and may be, for example, 0.01 μ M, 0.015 μ M, 0.02 μ M, 0.022 μ M, 0.023 μ M, 0.024 μ M, 0.025 μ M, 0.028 μ M, 0.03 μ M, 0.035 μ M, 0.04 μ M, 0.045 μ M, or 0.05 μ M, and preferably 0.025 μ M. In the invention, in order to reduce competitive interference between PRA and CRISPR, the content of Cas12a protein needs to be reduced as much as possible while ensuring the CRISPR cleavage rate, and the sensitivity is reduced sharply due to the fact that Cas12a is excessive and the RPA competes for template DNA.
Preferably, the working concentration of the crRNA is 0.1 to 0.5. mu.M, and may be, for example, 0.1. mu.M, 0.12. mu.M, 0.15. mu.M, 0.2. mu.M, 0.22. mu.M, 0.25. mu.M, 0.28. mu.M, 0.3. mu.M, 0.35. mu.M, 0.4. mu.M, 0.45. mu.M, 0.48. mu.M, or 0.5. mu.M, and preferably 0.25. mu.M.
Preferably, the working concentration of the probe DNA is 0.1 to 0.2. mu.M, and may be, for example, 0.1. mu.M, 0.12. mu.M, 0.14. mu.M, 0.15. mu.M, 0.16. mu.M, 0.18. mu.M, or 0.2. mu.M, and preferably 0.12. mu.M.
As a preferred technical scheme of the invention, both ends of the probe DNA are modified with a fluorescent group and a fluorescence quenching group.
Preferably, the fluorophore comprises any one of HEX, JOE, TET, ROX, TAMRA or FAM.
Preferably, the fluorescence quenching group comprises any one of BHQ1, BHQ2 or MGBNFQ.
As a preferred embodiment of the present invention, the nucleic acid detection method comprises the steps of:
(1) preparing a reaction system: mixing DNA recombinase, DNA polymerase, dNTP, RPA forward primer, RPA reverse primer, Cas12a, crRNA and probe DNA, and mixing with a solution to be detected to obtain the reaction system;
wherein the working concentration of the RPA forward primer is 0.2-0.8 mu M, the working concentration of the RPA reverse primer is 0.2-0.8 mu M, the working concentration of the Cas12a is 0.02-0.03 mu M, the working concentration of the crRNA is 0.1-0.5 mu M, and the working concentration of the probe DNA is 0.1-0.2 mu M;
(2) and (3) amplification and detection: and amplifying the reaction system at 37-42 ℃ for 20-40 min, and detecting the fluorescence intensity by using a fluorescence real-time quantitative detector to realize quantitative analysis of the target DNA in the solution to be detected.
Illustratively, the one-step RPA-CRISPR nucleic acid detection method disclosed by the invention adopts the following steps:
(1) designing sequences of a primer, crRNA and probe DNA according to the target DNA;
(2) preparing a reaction system: DNA recombinase, DNA polymerase, dntps, RPA forward and reverse primers, Cas12a, crRNA, probe DNA, and buffer, etc.;
wherein, the working concentration of the RPA forward primer and the reverse primer is 0.5 mu M; working concentration of Cas12a was 0.025 μ M; the working concentration of crRNA is 0.25. mu.M; the working concentration of the probe DNA is 0.12. mu.M;
(3) mixing and reacting: and mixing and reacting the RPA reagent and the CRISPR reagent at 37 ℃ to synchronously perform amplification detection, and finally detecting the fluorescence intensity by a fluorescence real-time quantitative detection device.
In a second aspect, the present invention provides a kit for nucleic acid detection using the method for nucleic acid detection according to the first aspect.
Preferably, the kit comprises an RPA agent, a CRISPR agent and a positive standard.
In a third aspect, the nucleic acid detection method according to the first aspect or the kit according to the second aspect is used for infectious disease monitoring, inspection and quarantine, food safety or drug inspection.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has at least the following beneficial effects:
according to the invention, a reaction system containing a solution to be detected, an RPA reagent and a CRISPR reagent is constructed, and is kept for a period of time at a constant temperature, so that amplification detection is synchronously carried out, and the detection method not only can remove the pollution problem caused by transfer of amplification products, but also can react the amplification process in real time, and has quantitative analysis capability; meanwhile, all components in the reaction system cannot interfere with each other, so that the detection method has high sensitivity and high specificity and can detect target DNA with the concentration of 1 aM; the one-step RPA-CRISPR nucleic acid detection method is simple in operation steps and short in time consumption, and can be widely applied to the fields of infectious disease monitoring, inspection and quarantine, food safety or drug inspection and the like.
Drawings
FIG. 1 is a schematic diagram of the principle of the one-step RPA-CRISPR nucleic acid detection method provided by the present invention;
wherein, 1-reaction system containing solution to be detected, 2-DNA amplification product, 3-crRNA, 4-CRISPR-Cas12a protein, 5-probe DNA, 6-nucleotide sequence with fluorescent group after being cut, and 7-nucleotide sequence with fluorescence quenching group after being cut.
FIG. 2 is a graph showing fluorescence kinetics at different target DNA concentrations in example 1.
FIG. 3 is a bar graph showing the signal intensity of the detection system and the amount of target DNA added in example 1.
FIG. 4 is a graph showing fluorescence kinetics at different target DNA concentrations in example 2.
FIG. 5 is a graph showing fluorescence kinetics at different target DNA concentrations in example 3.
FIG. 6 is a graph showing fluorescence kinetics at different target DNA concentrations in comparative example 1.
Detailed Description
The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.
First, the principle of the one-step RPA-CRISPR nucleic acid detection method provided by the present invention is briefly described by fig. 1:
firstly, a sample to be detected and a reagent are prepared into a reaction system 1 for nucleic acid detection, the reaction system 1 is placed in a reaction container (such as an EP tube), if the sample to be detected contains target DNA, the target DNA is amplified during an RPA amplification reaction to obtain a large amount of DNA amplification products 2, meanwhile, the amplification products are detected by a CRISPR-Cas12a system, under the action of CRISPR-Cas12a protein 4, crRNA 3 is combined with the target DNA, simultaneously, probe DNA 5 is combined with the target DNA and then cut to form a cut nucleotide sequence 6 with a fluorescent group and a cut nucleotide sequence 7 with a fluorescence quenching group, the fluorescent group emits fluorescence, a fluorescence value in the reaction system is detected while amplification is carried out through a fluorescence detection device, and further, the content of the target DNA in a solution to be detected is obtained, and quantitative detection of the target DNA is realized.
Example 1
In this example, the sequence of SEQ ID NO.1 was used as a target DNA (Template-sense), and this was quantitatively detected.
(1) Designing and synthesizing an RPA forward primer and a reverse primer aiming at target DNA;
in Table 1, the sequences of the target DNA sequence, the antisense strand of the target DNA sequence (Template-antisense), the Forward Primer (Forward Primer), the Reverse Primer (Reverse Primer), the crRNA, and the probe DNA are shown:
TABLE 1
(2) Based on a 50. mu.L reaction system, the reaction system was prepared in the amounts shown in Table 2:
TABLE 2
(3) Different DNA concentration gradients are set according to the reaction system, and the target DNA concentration is 10-1～106aM, mixing the prepared solution, reacting at 37 ℃, and detecting the fluorescence intensity of the reaction solution;
the obtained fluorescence intensity is shown in FIG. 2, and when the target DNA concentration is 1aM, the reaction proceeds to one stageAfter that, fluorescence can still be detected, which shows that the detection method provided by the invention has higher sensitivity and can detect the target DNA with the concentration of 1 aM. When the concentration of the target DNA is 106aM, the fluorescence intensity reaches the peak value when the reaction time is 30 min; the detection method provided by the invention requires a shorter time.
Meanwhile, as can be seen from FIG. 3, the signal rising rate (i.e., the slope of the rising curve) is shown in the graph, which is obtained by dividing the fluorescence intensity at a certain time point by the time, and it can be seen from the graph that the signal enhancement rate of the detection system increases as the amount of the target DNA added increases. According to this feature, the content of the target DNA in the reaction system at the initial time can be calculated using the fluorescence intensity detected at a specific time point.
Example 2
The difference from example 1 is that the contents of the components in the reaction system were adjusted, wherein the reaction system was prepared in the amounts shown in Table 3 based on 50. mu.L of the reaction system:
TABLE 3
Setting different DNA concentration gradients, the target DNA concentration is 10-1～106aM, mixing with the prepared solution, reacting at 42 ℃, and detecting the fluorescence intensity of the reaction solution; the remaining steps were as in example 1.
The obtained fluorescence intensity is shown in fig. 4, and the small amount of Cas12a causes the cutting rate of the CRISPR system to be reduced, and the signal growth rate is slower at high concentration, so that the quantitative detection of target DNA with different concentrations has lower time zone division, which is not favorable for rapid detection.
Example 3
The difference from example 1 is that the contents of the respective components in the reaction system were adjusted, wherein the reaction system was prepared in the amounts shown in Table 4 based on 50. mu.L of the reaction system:
TABLE 4
Setting different DNA concentration gradients, the target DNA concentration is 10-1～106aM, mixing the prepared solution, reacting at 30 ℃, and detecting the fluorescence intensity of the reaction solution; the remaining steps were as in example 1.
The resulting fluorescence intensity is shown in fig. 5, and due to the excess Cas12a, the competitive power with RPA is enhanced, and when the template DNA is less, the interference is greater, and finally the detection sensitivity is affected.
The experimental result shows that, in the examples 2 and 3, the amplification and detection are carried out according to the respective reaction systems, the fluorescence emitted in the solution can be detected, and the fluorescence intensity is increased along with the increase of the adding amount of the target DNA; however, the detection limit of both cannot reach 1aM, the detection sensitivity is inferior to example 1, and the detection sensitivity is significantly inferior to example 1 due to the higher Cas12a concentration in example 3.
Comparative example 1
The difference from example 1 is that in this comparative example, the amplification and detection steps were carried out in steps, specifically: mixing the solution to be detected with an RPA reagent, amplifying for 20min at 37 ℃, and adding a CRISPR reagent for fluorescence intensity detection;
however, in this comparative example, the fluorescence intensity obtained is shown in FIG. 6, and the RPA amplification kit is
The Basic kit has no obvious CT value due to the rapid amplification speed of the RPA nucleic acid, and when the concentration of the target DNA is 10-1～106and (aM), a large amount of amplification products are generated after 20min of amplification, the concentration of the amplification products is far higher than the linear range of CRISPR-Cas12a detection, and quantitative analysis cannot be performed by a CRISPR method, namely, a quantitative curve cannot be obtained because the concentrations of DNA products are close after the amplification is completed. Therefore, misjudgment may occur when non-specific amplification with small differences occurs (e.g., SNP typing detection); meanwhile, the amplification by the two-step method is also obviousThe method has the defects that if aerosol pollution exists in the laboratory environment, the nucleic acid to be detected is polluted by the aerosol due to transfer of products in the experimental step, and the accuracy of the detection result is low.
In conclusion, the nucleic acid detection method can solve the pollution problem caused by the transfer of the amplification product, can react the amplification process in real time, and has quantitative analysis capability; meanwhile, the components in the reaction system do not interfere with each other, so that the detection method has high sensitivity and high specificity, and can detect the target DNA with the concentration of 1 aM.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> southern university of science and technology
<120> one-step RPA-CRISPR nucleic acid detection method, kit and application
<130>20200706
<160>6
<170>PatentIn version 3.3
<210>1
<211>112
<212>DNA
<213> Artificial Synthesis
<400>1
aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga ttcttcttca 60
ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag ga 112
<210>2
<211>112
<212>DNA
<213> Artificial Synthesis
<400>2
tcctaggttg aagataaccc acataataag ctgcagcacc agctgtccaa cctgaagaag 60
aatcaccagg agtcaaataa cttctatgta aagcaagtaa agtttgaaac ct 112
<210>3
<211>30
<212>DNA
<213> Artificial Synthesis
<400>3
aggtttcaaa ctttacttgc tttacataga 30
<210>4
<211>30
<212>DNA
<213> Artificial Synthesis
<400>4
tcctaggttg aagataaccc acataataag 30
<210>5
<211>41
<212>RNA
<213> Artificial Synthesis
<400>5
uaauuucuac uaaguguaga uacuccuggu gauucuucuu c 41
<210>6
<211>6
<212>DNA
<213> Artificial Synthesis
<400>6
tttatt 6
Claims (10)
1. A one-step RPA-CRISPR nucleic acid detection method is characterized by comprising the following steps:
and mixing and amplifying the solution to be detected, the RPA reagent and the CRISPR reagent, and simultaneously detecting the real-time fluorescence intensity of a reaction system in the amplification process to realize the quantitative analysis of the target DNA in the solution to be detected.
2. The method for detecting a nucleic acid according to claim 1, wherein the amplification temperature is 26 to 42 ℃, preferably 37 to 42 ℃;
preferably, the time for amplification is 20-90 min, preferably 45-90 min.
3. The method for detecting nucleic acid according to claim 1 or 2, wherein the RPA reagent comprises DNA recombinase, DNA polymerase, single-stranded DNA binding protein, dNTP, RPA forward primer, and RPA reverse primer;
preferably, Cas12a, crRNA and probe DNA are included in the CRISPR agent.
4. The method for detecting nucleic acid according to claim 3, wherein the working concentration of the RPA forward primer is 0.3 to 0.8. mu.M, preferably 0.5. mu.M;
preferably, the working concentration of the RPA reverse primer is 0.3-0.8 μ M, preferably 0.5 μ M;
preferably, the length of the RPA forward primer is 25-40 bp, preferably 30-35 bp;
preferably, the length of the RPA reverse primer is 25-40 bp, preferably 30-35 bp.
5. The nucleic acid detection method of claim 3, wherein the working concentration of Cas12a is 0.01-0.05 μ M, preferably 0.025 μ M;
preferably, the working concentration of the crRNA is 0.1-0.5 μ M, preferably 0.25 μ M;
preferably, the working concentration of the probe DNA is 0.1-0.2. mu.M, and preferably 0.12. mu.M.
6. The method for detecting nucleic acid according to claim 3, wherein both ends of the probe DNA are modified with a fluorescent group and a fluorescence quenching group;
preferably, the fluorophore comprises any one of HEX, JOE, TET, ROX, TAMRA or FAM;
preferably, the fluorescence quenching group comprises any one of BHQ1, BHQ2 or MGBNFQ.
7. The method for detecting a nucleic acid according to any one of claims 1 to 6, comprising the steps of:
(1) preparing a reaction system: mixing DNA recombinase, DNA polymerase, dNTP, RPA forward primer, RPA reverse primer, Cas12a, crRNA and probe DNA, and mixing with a solution to be detected to obtain the reaction system;
wherein the working concentration of the RPA forward primer is 0.2-0.8 mu M, the working concentration of the RPA reverse primer is 0.2-0.8 mu M, the working concentration of the Cas12a is 0.02-0.03 mu M, the working concentration of the crRNA is 0.1-0.5 mu M, and the working concentration of the probe DNA is 0.1-0.2 mu M;
(2) and (3) amplification and detection: the reaction system is amplified at 37-42 ℃ for 20-90 min, and the fluorescence intensity is detected by a fluorescence real-time quantitative detector, so that the quantitative analysis of the target DNA in the solution to be detected is realized.
8. A kit for amplifying and detecting a nucleic acid by the method for detecting a nucleic acid according to any one of claims 1 to 7.
9. The kit of claim 8, wherein the kit comprises an RPA agent, a CRISPR agent, and a positive standard.
10. Use of the nucleic acid detection method according to any one of claims 1 to 7 or the kit according to claim 8 or 9 for infectious disease monitoring, inspection and quarantine, food safety or drug inspection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010679764.3A CN111808931A (en) | 2020-07-15 | 2020-07-15 | One-step RPA-CRISPR nucleic acid detection method, kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010679764.3A CN111808931A (en) | 2020-07-15 | 2020-07-15 | One-step RPA-CRISPR nucleic acid detection method, kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111808931A true CN111808931A (en) | 2020-10-23 |
Family
ID=72865490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010679764.3A Pending CN111808931A (en) | 2020-07-15 | 2020-07-15 | One-step RPA-CRISPR nucleic acid detection method, kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111808931A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553307A (en) * | 2020-12-30 | 2021-03-26 | 南方科技大学 | One-pot nucleic acid detection method based on CasRNAse and application |
| CN112980933A (en) * | 2020-12-30 | 2021-06-18 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system and application thereof |
| CN114703258A (en) * | 2022-06-06 | 2022-07-05 | 深圳大学 | One-pot type RPA-CRISPR nucleic acid detection method and system based on light activation |
| CN116083541A (en) * | 2022-12-29 | 2023-05-09 | 深圳大学 | Method for enriching low-abundance mononucleotide variant |
Citations (4)
| 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 |
| CN110453011A (en) * | 2019-07-19 | 2019-11-15 | 中山大学 | A kind of method and application based on CRISPR/Cas12a fast accurate detection African swine fever virus |
| CN110541022A (en) * | 2019-08-09 | 2019-12-06 | 福建医科大学孟超肝胆医院(福州市传染病医院) | mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system |
| CN111363842A (en) * | 2020-04-13 | 2020-07-03 | 广州医科大学附属第一医院 | Sequence, kit, method and application for rapidly detecting aspergillus fumigatus |
-
2020
- 2020-07-15 CN CN202010679764.3A patent/CN111808931A/en active Pending
Patent Citations (4)
| 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 |
| CN110453011A (en) * | 2019-07-19 | 2019-11-15 | 中山大学 | A kind of method and application based on CRISPR/Cas12a fast accurate detection African swine fever virus |
| CN110541022A (en) * | 2019-08-09 | 2019-12-06 | 福建医科大学孟超肝胆医院(福州市传染病医院) | mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system |
| CN111363842A (en) * | 2020-04-13 | 2020-07-03 | 广州医科大学附属第一医院 | Sequence, kit, method and application for rapidly detecting aspergillus fumigatus |
Non-Patent Citations (4)
| Title |
|---|
| DING X 等: "All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus", 《BIORXIV》 * |
| DING X 等: "All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus", 《BIORXIV》, 21 March 2020 (2020-03-21), pages 1 - 2 * |
| WANG Y 等: "A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level", 《ACS SENSORS》 * |
| WANG Y 等: "A One-Pot Toolbox Based on Cas12a/crRNA Enables Rapid Foodborne Pathogen Detection at Attomolar Level", 《ACS SENSORS》, vol. 5, no. 5, 27 April 2020 (2020-04-27), pages 1427 - 1434 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553307A (en) * | 2020-12-30 | 2021-03-26 | 南方科技大学 | One-pot nucleic acid detection method based on CasRNAse and application |
| CN112980933A (en) * | 2020-12-30 | 2021-06-18 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system and application thereof |
| CN112980933B (en) * | 2020-12-30 | 2023-08-29 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (CRISPR-Cas) system and application of SNP typing detection method |
| CN112553307B (en) * | 2020-12-30 | 2024-05-28 | 南方科技大学 | Cas (Cas ribonucleic acid) enzyme-based one-pot type nucleic acid detection method and application |
| CN114703258A (en) * | 2022-06-06 | 2022-07-05 | 深圳大学 | One-pot type RPA-CRISPR nucleic acid detection method and system based on light activation |
| CN114703258B (en) * | 2022-06-06 | 2022-10-11 | 深圳大学 | One-pot RPA-CRISPR nucleic acid detection method and system based on light activation |
| CN116083541A (en) * | 2022-12-29 | 2023-05-09 | 深圳大学 | Method for enriching low-abundance mononucleotide variant |
| CN116083541B (en) * | 2022-12-29 | 2024-04-02 | 深圳大学 | Method for enriching low-abundance mononucleotide variant |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111808931A (en) | One-step RPA-CRISPR nucleic acid detection method, kit and application | |
| CN107541546B (en) | Compositions, methods, systems, and kits for target nucleic acid enrichment | |
| Fraga et al. | Real‐time PCR | |
| US6238866B1 (en) | Detector for nucleic acid typing and methods of using the same | |
| US20060286558A1 (en) | Normalization of samples for amplification reactions | |
| CA2378408A1 (en) | Polynucleotide amplification method | |
| EP3568493B1 (en) | Methods and compositions for reducing redundant molecular barcodes created in primer extension reactions | |
| US9845495B2 (en) | Method and kit for detecting target nucleic acid | |
| EP1131466A1 (en) | Method for the quantitative detection of nucleic acids | |
| CN116590387B (en) | CRISPR (clustered regularly interspaced short palindromic repeats) system-based ssDNA detection method and application | |
| CN115074419A (en) | Method for isothermal amplification of nucleic acid target sequences | |
| CN111808929A (en) | Improved homogeneous phase recombinase polymerase nucleic acid amplification detection method and kit | |
| CN112553307A (en) | One-pot nucleic acid detection method based on CasRNAse and application | |
| EP1862558A1 (en) | Gene detection method | |
| US7485425B2 (en) | Methods for amplification of nucleic acids using spanning primers | |
| CN107893120B (en) | Primer group for detecting motion gene SNP, application and product thereof, and detection method and application for detecting motion gene SNP | |
| CN101443457B (en) | Method for production of pcr amplification product and use thereof | |
| CN113462821B (en) | Method and kit for monitoring amplification reaction progress based on metal organic framework material | |
| CN104087655A (en) | Single strand cleavage analysis method for restriction endonuclease based on rolling circle amplification | |
| CN114250276A (en) | MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease | |
| CN115943218A (en) | Hydrolysis-based probes and STR genotyping methods | |
| KR101628047B1 (en) | Composition for the direct nucleic acid amplification and nucleic acid amplifying method using the same | |
| CN116083655B (en) | DENV-crRNA for I-IV dengue virus detection, kit and application thereof | |
| KR102531590B1 (en) | Method for detecting target nucleic acid and composition for detecting nucleic acid by mirroring binding of nicking enzyme recognition site | |
| CN110603328A (en) | Quantitative PCR amplification primer pair and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201023 |
|
| RJ01 | Rejection of invention patent application after publication |