CN110184329A - A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification - Google Patents
A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification Download PDFInfo
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Abstract
The present invention discloses a kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification.This method is for detecting nucleic acid and by fluorescence or the field fast detection method of sidestream immune chromatographic test paper presentation visual test result.The present invention is effectively ensured using the specificity of crRNA to the correctness to detection, and it whether can be determined by test paper or fluorescence in test sample within 1~2 hour containing detection nucleic acid under room temperature, compared to traditional detection method accuracy, degree easy to operate increases.The present invention can form the freeze-drying LbaCas12a equipped with purifying, crRNA and RPA system reagent box, quickly and easily the nucleic acid fragment of particular sequence can be detected at the scene, and the equipment such as complicated temperature control instrument such as PCR instrument and the centrifugation of other electrophoresis are not needed, it is only necessary to which a thermostat and fluorescence detection device even only need naked-eye observation that can detect particular sequence nucleic acid rapidly and sensitively.
Description
Technical field
The invention belongs to field of biotechnology, are related to CRISPR/cas system and RPA constant-temperature amplification system, specifically a kind of
One-step method nucleic acid detection method and kit based on CRISPR/Cas12a and constant-temperature amplification.
Background technique
For detection of nucleic acids in medical diagnosis on disease, the fields such as pathogen detection are significant.Wherein in pathogen detection field,
Because nucleic acid fragment is more stable compared with proteantigen, pathogen nucleic acid spy also is utilized vulnerable to the influence of sample background unlike RNA
Heterotactic pathogen nucleic acid detection technique is significant.
At present nucleic acid detection method detection include RT-PCR, Real-Time qPCR, genetic chip, LAMP, NASBA,
HRM, PCR, RPA and Oxford Nanopore sequencing technologies etc..When RT-PCR method carries out detection of nucleic acids, carry out first inverse
Transcription, by rna transcription at cDNA, then carries out the amplification and detection of specific DNA sequence, this is conventional RNA detection method again；It is real
When quantitative fluorescent PCR use specific marker fluorescence probe, using fluorescence signal accumulate the entire PCR process of real-time monitoring, make
Each circulation becomes " visible ", the amplification situation of real-time monitoring target；Ring mediated isothermal amplification (LAMP) technology passes through a system
Target gene can be expanded 10 in 1h by the strand displacement amplification reaction of column, this method9Times, it can efficiently, specifically, quickly
Completion under isothermal conditions；The amplification technique (NASBA) for relying on nucleic acid sequence is one using RNA as template, to transcribe principle
Based on rapid isothermal amplification technique, a kind of method of high throughput is highly suitable for the screening of extensive sample；High-resolution
Melting (HRM) analysis is an emerging technology, it can detect PCR fragment only by the melting curve analysis after PCR
Minor sequence difference, thus realize specificity nucleic acid recognizing and detection.
Although these methods can effectively and qualitative detection specific nucleic acid sequence, require special instrument greatly,
Costco Wholesale is expensive, or needs more complex operation and longer detection time.In order to solve this problem, it is developed perseverance
Warm amplification technique, and simple isothermal amplification technology often is faced with the problem that specificity is bad and testing result displaying is inconvenient.
How more quickly, simplicity, low cost, high specific, high sensitivity and specificity inspection under conditions of lacking instrument and equipment
Surveying target nucleic acid is a urgent problem to be solved.
CRISPR/Cas system is by cluster, regular interval short palindrome repetitive sequence (clustered regularly
Interspaced short palindromic repeats, CRISPR) and its auxilin (CRISPR-
Associated, Cas) it constitutes.CRISPR be life concern in history, bacterium and virus are waged a struggle the immune weapon of generation,
CRISPR/Cas system is a kind of immune defense system of prokaryotes, for resisting the invasion of foreign heredity substance, for example is bitten
Thalli virus etc..Meanwhile it provides acquired immunity (similar to the secondary immunity of mammal) for bacterium, when bacterium by
When poisoning intrusion, corresponding " memory " can be generated.When viral secondary invasion, CRISPR system can identify exogenous DNA, and
They are cut off, the expression of silencing foreign gene, resists the interference of virus.Just because of this accurate target function,
CRISPR/Cas system is developed to a kind of efficient gene editing tool, to realize the sensitive specific nucleic acid inspection of rapid and convenient
Survey provides advantageous foundation.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of based on CRISPR/
The one-step method nucleic acid detection method of Cas12a and constant-temperature amplification.This method is for detecting nucleic acid and by fluorescence or sidestream immune
The field fast detection method of chromatographic test paper presentation visual test result.Expand especially by CRISPR/cas12a and RPA constant temperature
Increasing system combines, and Visual retrieval nucleic acid is presented with sidestream immune chromatographic test paper.
Another object of the present invention is to provide a kind of detection kits of target nucleic acids molecule.
The purpose of the invention is achieved by the following technical solution:
A kind of one-step method nucleic acid detection method based on CRISPR/Cas12a and constant-temperature amplification, this method is for non-diagnostic
Or therapeutic purposes, include the following steps: that detection target nucleic acid specificity is added into the system containing target nucleic acids molecule to be measured
CrRNA sequence, LbaCas12a, report single strand dna, freeze-drying RPA react microballoon, magnesium acetate, Rehydration Buffer
(the rehydrated buffer of RPA primer free), RPA upstream and downstream primer carry out single step reaction, and reaction product is then carried out sidestream immune
Chromatographic test paper or fluorescence detection；
Specifically comprise the following steps:
(1) to target nucleic acids to be measured, the crRNA of target spot specificity is designed, according to the crRNA sequence of design, then is constructed
CrRNA is transcribed in vitro carrier and is transcribed in vitro and is purified, or directly synthesizes；
(2) for the shot design RPA upstream and downstream primer of target nucleic acids to be measured described in step (1)；
(3) above-mentioned crRNA after purification is transcribed in vitro to crRNA molecule, LbaCas12a, the report of product or synthesis
Ssdna molecule, RPA upstream and downstream primer, freeze-drying RPA react microballoon, and (RPA primer free is again by magnesium acetate, Rehydration Buffer
Hydration buffer) it is mixed in appropriate system and is reacted in appropriate proportions with target nucleic acids to be measured；
(4) reaction product obtains testing result by sidestream immune chromatographic test paper or fluorescence detection.
Preferably, target nucleic acids to be measured described in step (1) are DNA；
Preferably, by the synthetic method of the crRNA sequence of design described in step (1) are as follows: building crRNA is transcribed in vitro
Carrier is simultaneously transcribed in vitro and is purified or direct chemical synthesis, but not limited to this.
Preferably, LbaCas12a described in step (3) can be by recombinantly expressing and purifying acquisition or use NEBLbaCase12a product；
Preferably, the report single strand dna design in step (3): when for the detection of sidestream immune chromatographic test paper, both ends
It is respectively provided with 12 base random sequence ss DNA molecular, the 5 '-FAM-NNNNNNNNNNNN-Biotin- of FAM and Biotin group
3 ', but not limited to this；
When for fluorescence detection, both ends are respectively provided with 12 base random sequence ss DNA moleculars of FAM and BHQ1 group
5 '-FAM-NNNNNNNNNNNN-BHQ1-3 ', but not limited to this.
Preferably, reaction system described in step (3) be 20 μ L systems, include 50~250nM LbaCas12a, 100~
500nM crRNA, 1000nM report single strand dna, 10U RNase inhibitor (TaKaRa), 2 μ L target core to be measured
Acid, 1 × Rehydration Buffer (the rehydrated buffer of RPA primer free), freeze-drying RPA react microballoon, RPA upstream and downstream primer
Each 0.48 μM, 14mM magnesium acetate；Wherein, the molar ratio of LbaCas12a and crRNA is 1:2.
Preferably, reaction system preparation method described in step (3) is first to prepare 18 μ L to include target nucleic acids to be measured,
Rehydration Buffer (the rehydrated buffer of RPA primer free), freeze-drying RPA react microballoon, RPA upstream and downstream primer, acetic acid
The mix A of magnesium, 37 DEG C are incubated for 0.5~10 minute, and LbaCas12a, crRNA is added, and report single strand dna, RNase
Inhibitor is simultaneously settled to 20 μ L.
Preferably, the condition of reaction described in step (3) is 37 DEG C of reactions 1~1.5 hour；More preferably 1 hour.
Preferably, in step (4) sidestream immune chromatographic test paper design: flow measurement immune chromatography test paper using commercialization
MileniaHybridetect 1 (TwistDx, Cambridge, UK) test paper, successively has loading on sidestream immune chromatographic test paper
Area, Gold-NP anti-FITC antibody area, strepavidin band (i.e. control band), antiantibody band (i.e. test strip).
Preferably, the detection method of the sidestream immune chromatographic test paper in step (4) is to press step (3) detection reaction system
Volume ratio 1:5 is diluted in HybriDetect assay buffer, and test paper loading area is dipped in the sample after above-mentioned dilution,
5 minutes i.e. visible test strip band is reacted at room temperature.
Preferably, detection device used in the fluorescence detection in step (4) can for common microplate reader or it is any can be
The fluorescence detection device for carrying out fluorescence excitation on FAM fluorescence channel and detecting；
It is furthermore preferred that the testing conditions that the fluorescence detection in step (4) uses is using the excitations of wavelength 492nm
Fluorescence, the fluorescence intensity at wavelength 522nm.
A kind of detection kit of target nucleic acids molecule, including detection target nucleic acid specificity crRNA sequence,
LbaCas12a, report single strand dna, freeze-drying RPA react microballoon, magnesium acetate, Rehydration Buffer (RPA primer free
Rehydrated buffer), RPA upstream and downstream primer.
Mechanism of the invention is:
LbaCas12a albumen is one from Lachnospiraceae bacterium ND2006 bacterium CRISPR system
A DNA enzymatic.LbaCas12a enzyme with specific recognition specified sequence and can activate generation non-specific single-stranded under crRNA guidance
DNA enzymatic cuts activity.For specific nucleic acid conserved sequence, report is cut using the single stranded DNA digestion specificity of CRISPR/Cas12a
Molecule is accused, reports that the fracture of molecule separates molecule both ends fluorophor and quencher, issues fluorescence, is i.e. realization target nucleic acid
Detection.
Strand displacement enzymatic polymerization enzymatic amplification technology (RPA) is that a kind of realization condition is simple, the isothermal amplification technology of rapid sensitive.
The optimum temperature of RPA reaction, without denaturation, can carry out at normal temperature between 37 DEG C~42 DEG C.This can undoubtedly greatly speed up
The speed of PCR.Further, since not needing temperature control device, RPA can really realize portable Rapid nucleic acid detection.RPA detection
Sensitivity it is very high, can be by nucleic acid (especially DNA) template amplification of trace to the level that can be detected.And RPA is not necessarily to
Complicated sample treatment, suitable for the detection on the spot of nucleic acid can not be extracted.RPA both can be with DNA amplification or with cloning RNA.Benefit
Specific nucleic acid sequence is identified with above-mentioned Cas12a and excites the principle of non-specific single stranded DNA shear active, it can be with Cas12a
System Reports RPA constant-temperature amplification product realizes the sensitive specific nucleic acid detection of rapid and convenient.
The present invention as report molecule according to Fluorescence Quenching Principle passes through fluorescence so that suitable molecular modification DNA is single-stranded
The cutting of order report molecule is excited, and then indicates testing result.Or it is designed by decorating molecule, cooperation sidestream immune chromatography
Test paper realizes naked eyes detection without special instrument.
Reaction system is targeted by System Design combination RPA constant-temperature amplification system appropriate and Cas12a, makes constant-temperature amplification
Reaction is targeted with Cas12 while being carried out, to shorten detection time.
The present invention may be implemented lacking in fact by design specificity crRNA and RPA primer, in conjunction with LbaCas12a albumen
In the case where testing condition, quickly detection is carried out to nucleic acid and is distinguished, wherein may be implemented not needing instrument by effluent test paper method
The naked eyes result of device is read.
The present invention has the following advantages and effects with respect to the prior art:
The present invention is effectively ensured using the specificity of crRNA to the correctness to detection, and under room temperature within 1~2 hour
It can be determined by test paper or fluorescence whether containing detection nucleic acid in test sample, compared to traditional detection method accuracy, behaviour
Make simplicity degree to increase.The present invention can form freeze-drying LbaCas12a, crRNA and RPA the system reagent box equipped with purifying,
Quickly and easily the nucleic acid fragment of particular sequence can be detected at the scene, and not need complicated temperature control instrument such as PCR
The equipment such as instrument and the centrifugation of other electrophoresis, it is only necessary to which a thermostat and fluorescence detection device even only need naked-eye observation
Particular sequence nucleic acid is detected rapidly and sensitively.
Detailed description of the invention
Fig. 1 is 10-14M, 10-15Under the nucleic acid concentration of M in the present invention sidestream immune chromatographic test paper method testing result,
It is additionally provided with the blank control group BC that target nucleic acids are not added；1:BC, 2:10-14M, 3:10-15M。
Fig. 2 is 10-9M, 10-11M, 10-13M, 10-15M, 10-17The inspection of fluorescent method of the present invention under the nucleic acid concentration of M, 0M
Survey result.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system
Make experiment condition proposed by factory.
Embodiment 1
In the embodiment of the present invention, based on CRISPR/Cas12a technology scene, quickly effluent immune chromatography test paper detects nucleic acid,
This method is used for non-diagnostic or therapeutic purposes, includes the following steps:
1) it designs and transcribes crRNA: finding the crRNA targeting for meeting TTTN 18N principle in target nucleic acid sequence to be measured
Sequence (such as SEQ ID NO:1), and therefrom determine crRNA sequence.Design RPA upstream and downstream expands in targeting sequence both ends 200bp
Increase primer (such as NO:2~3 SEQ ID).
The method that crRNA is transcribed in vitro are as follows: 37 DEG C of 16 hours: Nuclease-free water of reaction in following system
Add to 20 μ L, NTP each 1.5 μ L, 10 × reaction buffer 1.5 μ L, Template DNA1 μ g, T7RNA
Polymerase Mix 1.5μL。
CrRNA purification process: product and 160 μ L nuclease-free water and 20 μ L3M acetic acid are transcribed in vitro in 20 μ L
Ammonium mixing, is extracted using 1:1 phenol chloroform mixed liquor, removes organic phase, repeats to be extracted twice with chloroform, collects water phase to new
Without enzyme 1.5mL centrifuge tube, two volumes (400 μ L) dehydrated alcohol is added and is uniformly mixed, -20 DEG C heat preservation 30 minutes to precipitate RNA,
10000g is centrifuged 10 minutes collection RNA precipitates, discards supernatant, and twice with cold 75% ethyl alcohol rinsing precipitating, precipitating is collected by centrifugation,
It discards supernatant, obtains RNA solution with 50 μ L 0.1mM EDTA solution dissolution RNA precipitate.
2) report the design of single strand dna: both ends are respectively provided with 12 base random single chain DNAs of FAM and BHQ1 group
Molecule 5 '-FAM-NNNNNNNNNNNN-Biotin-3 ' (such as SEQ ID NO:4).
3) product, target nucleic acids to be measured, NEB is transcribed in vitro in above-mentioned crRNA after purificationLbaCas12a、
Report single strand dna, which is mixed in appropriate proportions in appropriate system, to be reacted.
4) reaction system are as follows: in 20 μ L systems, 250nM LbaCas12a, 500nM crRNA, 1 μM of report single stranded DNA point
Son, 10U RNase inhibitor (TaKaRa), 2 μ L target nucleic acids to be measured, each 0.48 μM of RPA upstream and downstream primer, 1 ×
Rehydration Buffer (the rehydrated buffer of RPA primer free), freeze-drying RPA react microballoon, 14mM magnesium acetate.Reaction system
It is reacted 1 hour at 37 DEG C.
5) reaction system preparation method is first to prepare 18 μ L to include target nucleic acids to be measured, Rehydration Buffer (RPA
The rehydrated buffer of primer free), freeze-drying RPA reaction microballoon, RPA upstream and downstream primer, the mix A of magnesium acetate, 37 DEG C of incubations 0.5~
10 minutes, LbaCas12a, crRNA is added, reports that single strand dna, RNase inhibitor are simultaneously settled to 20 μ L.
6) reaction product leads to sidestream immune chromatographic test paper survey acquisition testing result: will test reaction system 20 μ L and 100 μ L
HybriDetect 1assay buffer is mixed well, and the 1 test paper loading area HybriDetect is dipped in above-mentioned mixed liquor
It is incubated at room temperature 5min, naked eyes read test strips band to obtain testing result.
As a result as shown in Figure 1, showing target nucleic acids concentration 10-15M and available above by this nucleic acid detection method
Positive findings.
Embodiment 2
In the embodiment of the present invention, nucleic acid is detected based on CRISPR/Cas12a technology scene rapid fluorescence, this method is for non-
Diagnosis or therapeutic purposes, include the following steps:
1) it designs and transcribes crRNA: finding the crRNA targeting for meeting TTTN 18N principle in target nucleic acid sequence to be measured
Sequence (such as SEQ ID NO:1), and therefrom determine crRNA sequence.Design RPA upstream and downstream expands in targeting sequence both ends 200bp
Increase primer (such as NO:2~3 SEQ ID).
The method that crRNA is transcribed in vitro are as follows: 37 DEG C of 16 hours: Nuclease-free water of reaction in following system
Add to each 1.5 μ L, Template DNA of 1.5 μ L, 10 × reaction buffer, 1 μ g, T7 RNA of 20 μ L, NTP
Polymerase Mix 1.5μL。
CrRNA purification process: product and 160 μ L nuclease-free water and 20 μ L3M acetic acid are transcribed in vitro in 20 μ L
Ammonium mixing, is extracted using 1:1 phenol chloroform mixed liquor, removes organic phase, repeats to be extracted twice with chloroform, collects water phase to new
Without enzyme 1.5mL centrifuge tube, two volumes (400 μ L) dehydrated alcohol is added and is uniformly mixed, -20 DEG C heat preservation 30 minutes to precipitate RNA,
10000g is centrifuged 10 minutes collection RNA precipitates, discards supernatant, and twice with cold 75% ethyl alcohol rinsing precipitating, precipitating is collected by centrifugation,
It discards supernatant, obtains RNA solution with 50 μ L 0.1mM EDTA solution dissolution RNA precipitate.
2) report the design of single strand dna: both ends are respectively provided with 12 base random single chain DNAs of FAM and BHQ1 group
Molecule 5 '-FAM-NNNNNNNNNNNN-BHQ1-3 ' (such as SEQ ID NO:5).
3) product, target nucleic acids to be measured, NEB is transcribed in vitro in above-mentioned crRNA after purificationLbaCas12a、
Report single strand dna, which is mixed in appropriate proportions in appropriate system, to be reacted.
4) reaction system are as follows: in 20 μ L systems, 50nM LbaCas12a, 100nM crRNA, 1 μM of report single stranded DNA point
Son, 10U RNase inhibitor (TaKaRa), 2 μ L target nucleic acids to be measured, each 0.48 μM of RPA upstream and downstream primer, 1 ×
Rehydration Buffer (the rehydrated buffer of RPA primer free), freeze-drying RPA react microballoon, 14mM magnesium acetate.Reaction system
It is reacted 1 hour at 37 DEG C.
5) reaction system preparation method is first to prepare 18 μ L to include target nucleic acids to be measured, Rehydration Buffer (RPA
The rehydrated buffer of primer free), freeze-drying RPA reaction microballoon, RPA upstream and downstream primer, the mix A of magnesium acetate, 37 DEG C of incubations 0.5~
10 minutes, LbaCas12a, crRNA is added, reports that single strand dna, RNase inhibitor are simultaneously settled to 20 μ L.
6) reaction product obtains testing result by fluorescence detection, sets a length of 492nm of excitation light wave, and detection optical wavelength is
522nm。
As a result as shown in Fig. 2, showing target nucleic acids concentration 10-17M and available above by this nucleic acid detection method
Positive findings.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>South China Science & Engineering University
<120>a kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification
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Claims (10)
1. a kind of one-step method nucleic acid detection method based on CRISPR/Cas12a and constant-temperature amplification, it is characterised in that: this method is used
In non-diagnostic or therapeutic purposes, include the following steps: that detection target core is added into the system containing target nucleic acids molecule to be measured
Acid specificity crRNA sequence, LbaCas12a, report single strand dna, freeze-drying RPA reaction microballoon, magnesium acetate,
Rehydration Buffer, RPA upstream and downstream primer carry out single step reaction, and reaction product is then carried out sidestream immune chromatography examination
Paper or fluorescence detection.
2. the one-step method nucleic acid detection method according to claim 1 based on CRISPR/Cas12a and constant-temperature amplification, special
Sign is: specifically comprising the following steps:
(1) to target nucleic acids to be measured, the crRNA of target spot specificity is designed, according to the crRNA sequence of design, then constructs crRNA body
Outer transcription vector is simultaneously transcribed in vitro and is purified, or is directly synthesized；
(2) for the shot design RPA upstream and downstream primer of target nucleic acids to be measured described in step (1)；
(3) above-mentioned crRNA after purification is transcribed in vitro to the crRNA molecule, LbaCas12a, report single stranded DNA of product or synthesis
Molecule, RPA upstream and downstream primer, freeze-drying RPA react microballoon, magnesium acetate, Rehydration Buffer and target nucleic acids to be measured with
Proper proportion, which is mixed in appropriate system, is reacted；
(4) reaction product obtains testing result by sidestream immune chromatographic test paper or fluorescence detection.
3. the one-step method nucleic acid detection method according to claim 2 based on CRISPR/Cas12a and constant-temperature amplification, special
Sign is:
Report single strand dna design in step (3): when for the detection of sidestream immune chromatographic test paper, both ends are respectively provided with FAM
With 12 base random sequence ss DNA molecular, the 5 '-FAM-NNNNNNNNNNNN-Biotin-3 ' of Biotin group.
4. the one-step method nucleic acid detection method according to claim 2 based on CRISPR/Cas12a and constant-temperature amplification, special
Sign is:
Report single strand dna design in step (3): when being used for fluorescence detection, both ends are respectively provided with FAM and BHQ1 group
12 base random sequence ss DNA molecular, 5 '-FAM-NNNNNNNNNNNN-BHQ1-3 '.
5. the one-step method detection of nucleic acids side according to claim 2,3 or 4 based on CRISPR/Cas12a and constant-temperature amplification
Method, it is characterised in that:
Target nucleic acids to be measured described in step (1) are DNA；
The condition of reaction described in step (3) is 37 DEG C and reacts 1~1.5 hour.
6. the one-step method detection of nucleic acids side according to claim 2,3 or 4 based on CRISPR/Cas12a and constant-temperature amplification
Method, it is characterised in that:
Reaction system described in step (3) be 20 μ L systems, include 50~250nM LbaCas12a, 100~500nM crRNA,
1000nM reports single strand dna, 10U RNase inhibitor, 2 μ L target nucleic acids to be measured, 1 × Rehydration
Buffer, freeze-drying RPA reaction microballoon, each 0.48 μM of RPA upstream and downstream primer, 14mM magnesium acetate；Wherein, LbaCas12a and crRNA
Molar ratio be 1:2.
7. the one-step method nucleic acid detection method according to claim 6 based on CRISPR/Cas12a and constant-temperature amplification, special
Sign is:
The reaction system preparation method is first to prepare 18 μ L to include target nucleic acids to be measured, and Rehydration Buffer freezes
Dry RPA reacts microballoon, and RPA upstream and downstream primer, the mix A of magnesium acetate, 37 DEG C are incubated for 0.5~10 minute, and LbaCas12a is added,
CrRNA reports that single strand dna, RNase inhibitor are simultaneously settled to 20 μ L.
8. the one-step method detection of nucleic acids side according to claim 2,3 or 4 based on CRISPR/Cas12a and constant-temperature amplification
Method, it is characterised in that:
The detection method of sidestream immune chromatographic test paper in step (4) is that 1:5 is dilute by volume by step (3) detection reaction system
It releases in HybriDetect assay buffer, test paper loading area is dipped in the sample after above-mentioned dilution, reacts 5 at room temperature
Minute is visible test strip band；
Detection device used in fluorescence detection in step (4) is microplate reader or any can carry out fluorescence on FAM fluorescence channel
The fluorescence detection device for exciting and detecting.
9. the one-step method nucleic acid detection method according to claim 8 based on CRISPR/Cas12a and constant-temperature amplification, special
Sign is:
The testing conditions that fluorescence detection in step (4) uses is using the excitation fluorescence of wavelength 492nm, in wavelength
Fluorescence intensity at 522nm.
10. a kind of detection kit of target nucleic acids molecule, it is characterised in that: including detecting target nucleic acid specificity crRNA sequence
Column, LbaCas12a, report single strand dna, freeze-drying RPA reaction microballoon, magnesium acetate, Rehydration Buffer, on RPA
Downstream primer.
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