CN110387405A - A kind of (RT) RAA-CRISPR system of quick detection nucleic acid - Google Patents
A kind of (RT) RAA-CRISPR system of quick detection nucleic acid Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- 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
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
The invention discloses a kind of (RT) RAA-CRISPR protease systems of quickly detection nucleic acid, including room temperature constant-temperature amplification system and CRISPR detection system；The downstream of room temperature constant-temperature amplification system is arranged in CRISPR detection system；Wherein room temperature constant-temperature amplification system includes nucleic acid amplification system and rna transcription system；Wherein CRISPR detection system includes RNA boot sequence, detection PROTEIN C as13 and RAA probe.The present invention organically combines CRISPR-Cas system and (RT) RAA constant temperature nucleic acid amplification, in the case where guaranteeing highly sensitive trace, high specific detection, the requirement to time and environment can be greatly reduced, to establish quick, low consumption live virus nucleic acid detection technology really using gene molecule detection technique.
Description
Technical field
The present invention relates to external diagnosis reagent field, more particularly to a kind of (RT) RAA- of quickly detection nucleic acid
CRISPR protease system.
Background technique
Gene molecule detection is one of the important front edge field of contemporary medical science development, is to utilize genetic engineering and molecular biosciences
Scheduling theory and technology are learned, by directly detecting the presence or variation of nucleic acid sequence, to make diagnosis to body state and disease
Method.It is widely used in many aspects such as field diagnostic, the diagnosis of bed side, companion's diagnosis, Personalized medicine diagnosis, is disease
It predicts, diagnose, preventing, treating, prognosis and lapsing to and provide important information and decision-making foundation.
Currently, the market demand of gene molecule detection technique is continuously increased, development trend also increasingly precision, portability,
Milligram ammonia and integration.And there is drawback more in the prior art.Simply, efficiently often susceptibility is not high for diagnostic method, and Gao Min
Sensitivity detection method makes it to the high request of environment and instrument again, and there are limitations.Diagnostic environment complicated and changeable, limits base
Challenge is proposed to the development of technology because of the application of molecular detection technology, while also.
Wherein, CRISPR-Cas system is stored in about 90% archaeal and 40% bacterium, as a kind of prokaryotes
Resist the external sources such as virus invasion nucleic acid acquired immune system and exist (Van der Oost J et al., 2014；
Barrangou Ret al.,2014).CRISPR-Cas system is divided into three main Types: I type, II type and type III；It
Have a significant albumen respectively: Cas3, Cas9 and Cas10 (Makarova KS et al., 2011).Wherein CRISPR-
Cas9 system, (Deltcheva E et al., 2011；Gasiunas G et al., 2012), it is raw to be widely deployed into eukaryon
Object genome edit tool (Jinek M et al., 2012；Wang H et al.,2013).And the Cas13 egg found recently
It is white, also belong to CRISPR system II type, it have the characteristics that one it is prominent: after cutting specific RNA double-stranded sequence, remain to protect
It holds RNA enzyme and cuts activity, and do not have specificity.It can be detected for the target nucleic acids of particular sequence by this characteristic
(J.S.Gootenberg et al., 2017).Although particular sequence can be directed to however using CRISPR-Cas13 system
Target nucleic acids detected, but because whole system to target nucleic acids concentration require it is relatively high, also have led to CRISPR-
Cas13 detection limit is excessively high, and sensibility is very poor.
Recombinase-mediated amplification (Recombinase-aid Amplification, RAA) technology be also it is a kind of at a constant temperature
It can make the method for nucleic acid rapid amplifying.Unlike RPA, RAA amplification, which is used, to be obtained from bacterium or fungi
Recombinase, under 37 DEG C of constant temperature, which can combine closely with primed DNA, the condensate of enzyme and primer be formed, when primer exists
When searching the sequence of complete complementary therewith on template DNA, in single-stranded DNA binding protein (single-strandedDNA
Binding, SSB) with the help of, make template DNA unwinding, and under the action of archaeal dna polymerase, forms new DNA complementary strand, instead
Answering product is also to be increased with exponential, can obtain that the amplification piece that agarose gel electrophoresis detects can be used usually in 1h
Section.
Therefore, how CRISPR-Cas system and (RT) RAA constant temperature nucleic acid amplification technology to be organically combined, is guaranteeing trace
In the case where highly sensitive, high specific detection, can be greatly reduced the requirement to time and environment be those skilled in the art urgently
Problem to be solved.
Summary of the invention
In view of this, the present invention provides a kind of (RT) RAA-CRISPR protease system, by CRISPR-Cas system with
(RT) RAA constant temperature nucleic acid amplification technology organically combines, wherein (RT) RAA constant temperature nucleic acid amplification technology, can be avoided polymerase
The use of the large scale equipments such as thermal cycler in chain technology, while greatly improving rate of amplification；And CRISPR-Cas13
System high specific detection system solves easy, quick and accurate, sensitive incompatible situation, or even can be a large amount of
In the presence of interfering nucleic acid, the presence of microunit point mutation is detected.The present invention is guaranteeing trace high sensitivity, high specific
In the case where detection, the requirement to time and environment can be greatly reduced, so that establishing using gene molecule detection technique quick, low
The live virus nucleic acid detection technology of consumption is really possibly realized.Meanwhile in disease prevention, medical diagnosis on disease, cancer detection, Fa Yijian
Fixed, food safety, public health etc. will play an important role.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of (RT) RAA-CRISPR protease system of quick detection nucleic acid, including room temperature constant-temperature amplification system and
CRISPR detection system；The downstream of room temperature constant-temperature amplification system is arranged in CRISPR detection system；Wherein room temperature constant-temperature amplification system
System includes nucleic acid amplification system and rna transcription system；Wherein CRISPR detection system includes RNA boot sequence, detection albumen
Cas13 and RAA probe.
Preferably, RNA boot sequence can hybridize in the RNA segment containing target sequence.
Preferably, Cas13 albumen is a kind of engineered protein enzyme of dependence double-stranded RNA progress double-stranded RNA inscribe, at this
After boot sequence hybridizes with target sequence in invention, Cas13 albumen is activated；Its main feature is that: 1) it can identify double-stranded RNA point
Son, and digestion is carried out to it；2) identification of double stranded rna molecule needs to guide the guidance of RNA sequence；3) after carrying out digestion,
RNA enzyme, which cuts activity, can still retain, and can connect other RNA molecules of Non-specific cleavage；4) the optional Cas13a albumen of sequence；5)
Dosage in the reaction system is 20-50ng/ul.
Preferably, RAA probe is a two terminal modified single-stranded cRNA molecules, and length is about 25nt, in Cas13 albumen quilt
After activation, digestion can be catalyzed by it, then issue the signal that can be detected.
A kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid disclosed by the invention, can be in constant temperature room temperature item
Under part, the system and method for fast and efficiently completing detection of nucleic acids overcome the diagnosis side of current high specificity, high sensitivity
Method is big to environment, precision instrument dependence, and simply and easily diagnostic method has specific weak, poor sensitivity.
The present invention organically combines CRISPR-Cas system and (RT) RAA constant temperature nucleic acid amplification technology, not only avoids big
The use of type equipment, while greatly improving rate of amplification；And have the characteristics that it is easy, quick and accurate, sensitivity cannot
The case where coexisting, or even can still can detecte out the presence of microunit point mutation in the presence of a large amount of interfering nucleic acid.
Further, (RT) RAA-CRISPR protease system further includes providing appropriate incubation environment for reaction, and can swash
Send out and detect the instrument of signal.
Preferably, instrument be fluorogene detecting instrument, have the function of temperature control modules, the time be arranged, sample well and
Fluorescence excitation module, fluorescence detection module.
Further, nucleic acid amplification system is recombinase-mediated nucleic acid amplification system；Reverse transcriptase, recombinase, single-stranded knot
Hop protein, archaeal dna polymerase, auxilin, constant temperature room temperature amplification system chemical reagent and RAA primer sets.
Preferably, reverse transcriptase is a kind of protein carried out after genetic modification using point mutation, its main feature is that: 1) have
DNA polymerase activity can be catalyzed dNTP and be polymerized to generate complementary cDNA using RNA as template；2) make to have originally by point mutation
RNase activity inactivation, to avoid the RNA hydrolysis in DNA-RNA heterozygosis chain；3) enzyme sequence is reversed from murine leukemia
It records enzyme (M-MLV)；4) the about 8-12 of dosage in the reaction system unit/ul.
Preferably, recombinase is a kind of engineered protein enzyme, its main feature is that: 1) there is the ability in conjunction with single stranded DNA, because
This can be with the single-stranded formation initiation complex of primer tasteless nucleotide；2) have and ability that duplex DNA molecular combines, therefore could be
While forming initiation complex, also in relation with DNA double chain or DNA-RNA heterozygosis chain, to be formed in the special triple strand dna of structure
Mesosome；3) there is homologous recombination activity, when single stranded DNA invades double-stranded DNA or DNA-RNA heterozygosis chain, along chain searching homologous sequence
Qu Hou can go out homologous single-stranded exclusion, form so-called local Bubble Region, and then facilitate the intervention of subsequent archaeal dna polymerase；
4) enzyme sequence can derive from RecA the or T4 bacteriophage recombinase uvsX of Escherichia coli；5) dosage in the reaction system is about
For 100-150ng/ul.
Preferably, single strand binding protein is a kind of DNA binding protein, it is characterized in that: 1) the local Bubble Region that edge is formed,
Locally combination is replaced out homologous single-stranded, it is avoided to be degraded by nuclease；2) do not have enzymatic activity, therefore ATP knot of getting along well
It closes；3) have synergistic effect, therefore can multiple single strand binding protein adjacent bonds together in DNA it is single-stranded on, convenient for Bubble Region DNA close
At；4) this combination has short-time characteristic, therefore could constantly combine along " replication fork ", and constantly assemble again；5) the enzyme sequence
Column may be derived from the GP32 albumen of E.coli or T4；6) dosage in the reaction system is about 700-1000ng/ul.
Preferably, archaeal dna polymerase is a kind of engineered protein enzyme of dependence DNA profiling progress complementary dna sequence synthesis,
Its main feature is that: 1) with higher continuous synthesis capability；2) there is displacement chain activity, while synthesizing new chain, chain can be passed through
Diadochy cooperates single strand binding protein persistently to promote local Bubble Region along " replication fork " direction, so that homologous chain be replaced
Out；3) there is 3 ' -5 ' excision enzymes (calibration capability) activity, to guarantee the fidelity type of synthesis；4) non-refractory, thus it is very suitable
Conjunction is reacted under room temperature constant temperature, and completes the synthesis of DNA chain；5) sequence of the enzyme may originate from the DNA polymerase i of E.coli
Klenow large fragment, Bst polymerase, Phi-29 polymerase or bacillus subtilis Po1I (Bsu)；6) use in the reaction system
Amount is about 60-90ug/ul.
Preferably, auxilin be it is a kind of assistance or enhancing the active albumen of recombinase, its main feature is that: 1) promote and stablize
The combination of initiation complex and DNA double interchain；2) the uvsY albumen of the optional T4 bacteriophage of sequence；3) dosage in the reaction system
About 20-40ng/ul.
Further, rna transcription system is T7 re-recording system；T7 re-recording system includes T7 transcriptase.
Preferably, T7 transcriptase is a kind of genetic engineering that RNA synthesis is catalyzed using a DNA chain or RNA chain as template
Protease, its main feature is that: 1) promoter on RNA transcriptase energy recognition template, and continuous conjunction 2) with higher in combination
At ability, 3) non-refractory, therefore be very suitable to react under room temperature constant temperature, it completes DNA and is transcribed into RNA；4) sequence is optional；
5) dosage in the reaction system is 20-100ng/ul.
Further, constant temperature room temperature amplification system chemical reagent includes Tris, RNase inhibitor, dNTPs, rNTP, phosphoric acid
Creatine disodium salt, potassium acetate, trehalose, mannitol, polyethylene glycol, dithiothreitol (DTT), PCR primer, guidance RNA and taqman are visited
Needle.
Preferably, ATP, which is provided, continues energy；Ribonucleotide triphosphate (rNTP) and deoxyribonucleoside triphosphate (dNTP)
Synthesis unit is provided；Potassium acetate (KAc), dithiothreitol (DTT) (DTT) and polyethylene glycol (PEG) maintain reactive ion and pH value ring
Border；Trehalose (Treph lose) and mannitol (Mannitol) keep protein active in freezing dry process.
Further, T7 promoter is contained in RAA primer.
RAA primer is a pair of few chain nucleotide single-chain, respectively the upstream and downstream of specific recognition targeting regions.Primer length is about
30-35nt, and T7 promoter is separately contained at one 5 ' end in pairs of primer in the present invention.
Further, RNA boot sequence by probe region and general district's groups at；Probe region is the sequence of target sequence very high homology
Column；Contain the bond area of hair fastener shape structure, guidance detection PROTEIN C as13 identification and digestion probe region and RNA template in general area.
The single-stranded cRNA molecule that RNA boot sequence length is about 70nt, consists of two parts: 5 ' ends are general area, containing hairpin structure,
It can help Cas13 albumen identification guidance RNA；3 ' ends are probe region, energy specific recognition targeting regions, and formation in combination
Double stranded rna molecule excites the double-stranded RNA digestion activity of Cas13 albumen, its non-specific RNA enzyme is then excited to cut activity.
Further, RAA probe is rna probe, and one end of rna probe utilizes fluorescence radiation base group modification, other end benefit
It is modified with fluorescent quenching group.
Further, fluorescence radiation group be FAM, appointing in HEX, VIC, JOE, Texas Red, Cy3, Cy5 or TAMRA
It anticipates one kind.
Further, one of fluorescent quenching group BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
It is at least following the present disclosure provides a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid
Advantage:
(1) a kind of (RT) the RAA-CRISPR protease system of quickly detection nucleic acid disclosed by the invention can be in room temperature perseverance
It is detected under temperature, (RT) RAA-CRISPR protease system can utmostly simulate biological nucleic acid in vivo amplification and CRISPR
The environment of function, and all Each performs its own functions for the various toolenzymes in system, works in most suitable reaction temperature, therefore the effect that works
Rate is high.
(2) a kind of (RT) the RAA-CRISPR protease system of quickly detection nucleic acid disclosed by the invention can be in a large amount of phases
Under disturbed condition like sequence, the difference of the single base of target sequence is distinguished, therefore specificity is very strong；Meanwhile early period
(RT) amplification of the RAA to targeting regions can make the nucleic acid molecules concentration containing target sequence be promoted to higher level, therefore can be with
Detect the template of lower concentration, sensitivity is very high.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is the working principle diagram of (RT) RAA-CRISPR protease system provided by the invention.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment
A kind of (RT) RAA-CRISPR protease system of quick detection nucleic acid, including room temperature constant-temperature amplification system and
CRISPR detection system and have the function of temperature control modules, time setting, sample well and fluorescence excitation module, fluorescence detection
Module fluorogene detecting instrument；The downstream of room temperature constant-temperature amplification system is arranged in CRISPR detection system；Wherein room temperature constant temperature
Amplification system includes nucleic acid amplification system and rna transcription system；Nucleic acid amplification system is recombinase-mediated nucleic acid amplification system；It is inverse
Transcriptase, recombinase, single strand binding protein, archaeal dna polymerase, auxilin, constant temperature room temperature amplification system chemical reagent and RAA draw
Object group；Constant temperature room temperature amplification system chemical reagent include the Tris buffer of 55mM, 0.15 unit/ul RNase inhibitor,
The second of the disodium creatine phosphate of ATP, 12mM of dNTPs, rNTP, 3.5mM of 490mM, the creatine phosphokinase of 114ng/ul, 75mM
Sour potassium, 6% trehalose, 7% mannitol, 5% polyethylene glycol, 12mM dithiothreitol (DTT).Rna transcription system turns for T7
Recording system；RAA primer is a pair of few chain nucleotide single-chain, respectively the upstream and downstream of specific recognition targeting regions.Primer length is about
30-35nt, and T7 promoter is separately contained at one 5 ' end in pairs of primer in the present invention；T7 re-recording system includes T7 transcriptase.
Wherein CRISPR detection system includes RNA boot sequence, detection PROTEIN C as13 and RAA probe.RNA boot sequence
The single-stranded cRNA molecule that length is about 70nt, consists of two parts: 5 ' ends are that general area can be helped containing hairpin structure
Cas13 albumen identification guidance RNA；3 ' ends are probe region, energy specific recognition targeting regions, and formation double-stranded RNA in combination
Molecule excites the double-stranded RNA digestion activity of Cas13 albumen, its non-specific RNA enzyme is then excited to cut activity；RAA probe is
Rna probe, one end of rna probe utilize fluorescence radiation base group modification, and the other end is modified using fluorescent quenching group.Fluorescence radiation
Group is FAM, any one in HEX, VIC, JOE, Texas Red, Cy3, Cy5 or TAMRA, fluorescent quenching group BHQ1,
One of BHQ2, BHQ3, Dabcy1 or Tamra.
Working principle such as Fig. 1 of (RT) RAA-CRISPR protease system of one of quickly detection nucleic acid, can by Fig. 1
Know, (RT) RAA-CRISPR protease system will contain target sequence amplified nucleic acid molecule to corresponding DNA molecular, and start plus T7
Son；And (RT) RAA is expanded gained DNA molecular and is transcribed into corresponding RNA molecule by T7 re-recording system；It guides on rna probe area and RNA
Target coupled in series, and Cas13 proteolytic cleavage combined area is guided, then excite its non-specific nucleic acid endonuclease activity；Finally
Cas13 proteolytic cleavage rna probe, system can be inspired fluorescence signal.
In conclusion it is provided by the invention one kind (RT) RAA-CRISPR protease system, by CRISPR-Cas system with
(RT) RAA constant temperature nucleic acid amplification technology organically combines, wherein (RT) RAA constant temperature nucleic acid amplification technology, can be avoided polymerase
The use of the large scale equipments such as thermal cycler in chain technology, while greatly improving rate of amplification；And CRISPR-Cas13
System high specific detection system solves easy, quick and accurate, sensitive incompatible situation, or even can be a large amount of
In the presence of interfering nucleic acid, the presence of microunit point mutation is detected.The present invention is guaranteeing trace high sensitivity, high specific
In the case where detection, the requirement to time and environment can be greatly reduced, so that establishing using gene molecule detection technique quick, low
The live virus nucleic acid detection technology of consumption is really possibly realized.Meanwhile in disease prevention, medical diagnosis on disease, cancer detection, Fa Yijian
Fixed, food safety, public health etc. will play an important role.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid, which is characterized in that including room temperature constant-temperature amplification
System and CRISPR detection system；The downstream of the room temperature constant-temperature amplification system is arranged in the CRISPR detection system；Wherein
The room temperature constant-temperature amplification system includes nucleic acid amplification system and rna transcription system；Wherein the CRISPR detection system includes
RNA boot sequence, detection PROTEIN C as13 and RAA probe.
2. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In (RT) the RAA-CRISPR protease system further includes providing appropriate incubation environment for reaction, and can excite and detect letter
Number instrument.
3. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1 or 2, feature
It is, the nucleic acid amplification system is recombinase-mediated nucleic acid amplification system；Including reverse transcriptase, recombinase, single-stranded combination egg
It is white, archaeal dna polymerase, auxilin, constant temperature room temperature amplification system chemical reagent and RAA primer sets.
4. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In the rna transcription system is T7 re-recording system；The T7 re-recording system includes T7 transcriptase.
5. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 3, feature exist
In the constant temperature room temperature amplification system chemical reagent includes Tris, RNase inhibitor, dNTPs, rNTP, phosphocreatine disodium
Salt, potassium acetate, trehalose, mannitol, polyethylene glycol, dithiothreitol (DTT), PCR primer, guidance RNA and taqman probe.
6. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 4, feature exist
In, in the RAA primer contain T7 promoter.
7. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In, the RNA boot sequence by probe region and general district's groups at；The probe region is the sequence of target sequence very high homology；Institute
State the bond area that hair fastener shape structure, guidance detection PROTEIN C as13 identification and digestion probe region and RNA template are contained in general area.
8. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In the RAA probe is rna probe, and one end of the rna probe utilizes fluorescence radiation base group modification, and the other end utilizes fluorescence
Quenching group modification.
9. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In, the fluorescence radiation group be FAM, any one in HEX, VIC, JOE, Texas Red, Cy3, Cy5 or TAMRA.
10. a kind of (RT) RAA-CRISPR protease system of quickly detection nucleic acid according to claim 1, feature exist
In one of described fluorescent quenching group BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
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| CN111254223A (en) * | 2020-03-21 | 2020-06-09 | 上海海关动植物与食品检验检疫技术中心 | Reaction system and kit for detecting African swine fever virus nucleic acid and application of reaction system and kit |
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| WO2022246781A1 (en) * | 2021-05-28 | 2022-12-01 | 深圳华大生命科学研究院 | Nucleic acid test system and method based on electrowetting crispr |
| CN113293222A (en) * | 2021-05-31 | 2021-08-24 | 温州医科大学 | RAA primer group, crRNA sequence and RAA-CRISPR method for detecting vibrio vulnificus |
| CN113462796A (en) * | 2021-06-15 | 2021-10-01 | 浙江大学 | Method for detecting microorganisms by combining nucleic acid isothermal amplification and CRISPR/Cas13a and application |
| CN114807453A (en) * | 2022-06-27 | 2022-07-29 | 北京市疾病预防控制中心 | Specific primer group aiming at S gene of new coronavirus omitron strain and application thereof |
| CN114807453B (en) * | 2022-06-27 | 2022-09-02 | 北京市疾病预防控制中心 | Specific primer group aiming at S gene of new coronavirus omitron strain and application thereof |
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