CN108588050A - Archaeal dna polymerase and nucleic acid detection method and kit - Google Patents
Archaeal dna polymerase and nucleic acid detection method and kit Download PDFInfo
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Abstract
The present invention provides using DNA or RNA as the archaeal dna polymerase of template, obtained to carry out following amino acid replacement by the Klenow large fragments to Escherichia coli polymerase I：G198W, V222I, E306K, Q354E, A381E and E582K.The present invention also provides the primers with loop-stem structure for constant temperature nucleic acid amplification.The present invention also provides the nucleic acid detection technique for being combined fast constant temperature nucleic acid amplification with Cas detection architectures and detection kits.They can be used for the target nucleic acid in detection sample in the case of constant temperature, has many advantages, such as that at low cost, detection time is short, easy to operate, specific high, high sensitivity, is particularly suitable for POCT and applies.
Description
Technical field
The present invention relates to nucleic acid detection techniques, and constant temperature nucleic acid amplification and Cas are especially detected the detection of nucleic acids being combined
Technology.
Background technology
Nucleic acid detection technique has great molecule diagnosis, biochemical analysis, medical diagnosis on disease application value, for example, for disease
Poison, bacterium, pathogen nucleic acid detection and the detection, etc. of nucleic acid disease markers.PCR (PCR,
Polymerase chain reaction) technology is the nucleic acid detection technique being most widely used at present.The technology by
Dr.Mullis is invented in nineteen eighty-three, is broadly divided into three basic steps, i.e.,：Denaturation, annealing and extension.Round pcr needs logical
Heating and cooling repeatedly are crossed to realize nucleic acid amplification, more demanding to instrument and operating environment, instrument precision is complicated, expensive, and
It is cumbersome, professional technician and laboratory are needed, therefore round pcr application range has larger limitation, such as limit
Its application in the fields such as detection (Point-of-care testing, POCT) immediately is made.Thus with RPA
(recombinase polymerase amplification)、RAA(recombinase-aid amplification)、
LAMP (loop-mediated isothermal amplification) etc. be representative nucleic acid constant-temperature amplification technology increasingly
The concern of people is obtained, however the specificity of these technologies is not especially high, is easy occur non-specific amplification in practice process,
Interfere the interpretation of result.
In addition, for a long time, reverse transcriptase is depended on to the detection of RNA, is needed first by reverse transcriptase by RNA reverse transcriptions
For cDNA, followed by the amplification (such as PCR etc.) of DNA sequence dna.Gulati et al. has found that DNA polymerase i can rely on widow
Nucleotide oligo-dT carries out reverse transcription to viral RNA.However, this reverse transcription system is to archaeal dna polymerase：RNA template ratios
It is required that high, reverse transcription amplification efficiency it is undesirable (Proc.Nat.Acad.Sci USA Vol.71, No.4, pp.1035-1039,
1974)。
Invention content
In order to overcome the above problem, on the one hand, the present invention provides a kind of using DNA or RNA as the DNA of template polymerizations
Enzyme obtains to carry out following amino acid replacement by the Klenow large fragments to Escherichia coli polymerase I：G198W、
V222I, E306K, Q354E, A381E and E582K.
In some embodiments, the archaeal dna polymerase has such as SEQ ID NO：Amino acid sequence shown in 18.
Correspondingly, the present invention also provides the polynucleotide sequences or its complementary series that encode the archaeal dna polymerase.
On the other hand, the present invention provides a kind of DNA primers pair for nucleic acid constant-temperature amplification, wherein the DNA primer
Any one primer or the two of centering have loop-stem structure.
In some embodiments, the loop-stem structure is by being held with the linear DNA primer 5 ' of template sequence complementation
2 to 15 bases of addition and formed, 3 ' end sequences of 2 to 15 bases and the linear DNA primer it is complementary or with
The sequence of the base of 3 ' end 1 to 10 away from the linear DNA primer is complementary.
In some embodiments, the length of any one primer of the DNA primer centering or the two is 33 to 45
Base；Or being also added with T7 promoter sequences at 5 ' ends of the linear primer, then either one or two of described DNA primer centering is drawn
The length of object or the two is 51 to 63 bases.
On the other hand, the present invention provides a kind of horizontal methods determining target nucleic acid in the sample, including：
1) target nucleic acid is detached from the sample；
2) it using the target nucleic acid of separation as template, is expanded using constant temperature nucleic acid amplification technology, obtains DNA cloning production
Object；And
3) use Cas detection combination analyte detections described in DNA cloning product amount, and by the amount of the DNA cloning product with
The level of target nucleic acid described in the sample is associated.
In some embodiments, the target nucleic acid is DNA or RNA.
In some embodiments, the constant temperature nucleic acid amplification technology includes using the DNA primer pair with loop-stem structure.
In some embodiments, the constant temperature nucleic acid amplification technology includes using poly- containing unwindase, recombinase and DNA
The amplification reaction system of synthase.
In some embodiments, the unwindase is selected from RecQ unwindases, UvrD unwindases, DnaB unwindases and CMC
Unwindase；The recombinase is selected from UvsX systems, eucaryote Rad systems, yeast or the Escherichia coli recA systems of bacteriophage；
The archaeal dna polymerase is selected from Deep VentRTM archaeal dna polymerases, Deep VentRTM (exo-) archaeal dna polymerase, Klenow pieces
Section (3 ' -5 ' exo-), DNA polymerase i, Klenow large fragments, phi29DNA polymerases,Archaeal dna polymerase, VentR
(exo-) archaeal dna polymerase.
In preferred embodiments, the archaeal dna polymerase is to be carried out to the Klenow large fragments of Escherichia coli polymerase I
Following amino acid replacement and obtain：G198W, V222I, E306K, Q354E, A381E and E582K.
In preferred embodiments, the archaeal dna polymerase has such as SEQ ID NO：Amino acid sequence shown in 18.
In some embodiments, the Cas detection combinations object includes Cas12a or Cas13a albumen.
In other embodiments, the Cas detection combinations object includes Cas13a albumen and Csm6 albumen.
In some embodiments, the target nucleic acid is originated from virus or bacterium.
In some embodiments, it is described virus or bacterium be selected from Inf.A, Inf.B, Inf.C, HPV, Strep.A, RSV,
PTB, MP, CP, AdV, EV, BoV and HRV.
On the other hand, the present invention provides a kind of kits for detecting target nucleic acid in the sample, including are used to carry out
Unwindase, recombinase and the archaeal dna polymerase of constant temperature nucleic acid amplification and for being detected to DNA cloning product Cas detection
Composition.
In some embodiments, the unwindase is selected from RecQ unwindases, UvrD unwindases, DnaB unwindases and CMC
Unwindase；The recombinase is selected from bacteriophage UvsX systems, eucaryote Rad systems and yeast or Escherichia coli recA systems；
The archaeal dna polymerase is selected from Deep VentRTM archaeal dna polymerases, Deep VentRTM (exo-) archaeal dna polymerase, Klenow pieces
Section (3 ' -5 ' exo-), DNA polymerase i, Klenow large fragments, phi29DNA polymerases,Archaeal dna polymerase and
VentR (exo-) archaeal dna polymerase.
In preferred embodiments, the archaeal dna polymerase is to be carried out to the Klenow large fragments of Escherichia coli polymerase I
Following amino acid replacement and obtain：G198W, V222I, E306K, Q354E, A381E and E582K.
In preferred embodiments, the archaeal dna polymerase has such as SEQ ID NO：Amino acid sequence shown in 18.
In some embodiments, the Cas detection combinations object includes Cas12a or Cas13a albumen.
In some embodiments, the Cas detection combinations object includes Cas13a albumen and Csm6 albumen.
In some embodiments, the kit further includes the DNA primer pair for constant temperature nucleic acid amplification, the DNA
Any one primer or the two in primer pair have loop-stem structure.
In some embodiments, the loop-stem structure is by the linear DNA primer 5 ' with the complementary target
End 2 to 15 bases of addition and formed, 3 ' end sequences of 2 to 15 bases and the linear DNA primer it is complementary or
It is complementary with the sequence of the base of 3 ' end 1 to 10 away from the linear DNA primer.
In some embodiments, the length of any one primer of the DNA primer centering or the two is 33 to 45
Base；Or being also added with T7 promoter sequences at 5 ' ends of the linear primer, then either one or two of described DNA primer centering is drawn
The length of object or the two is 51 to 63 bases.
In some embodiments, the kit is used to detect the detection of influenza B virus, and the DNA primer is to dividing
It Ju You not SEQ ID NO：Nucleotide sequence shown in 6 and 7 or SEQ ID NO：Nucleotide sequence shown in 9 and 10；Or
The kit is used for the detection of HPV viruse, and the DNA primer is to being respectively provided with SEQ ID NO：Nucleotide shown in 14 and 15
Sequence.
In some embodiments, the kit is used to detect the detection of influenza B virus, further includes with SEQ
ID NO：The crRNA of nucleotide sequence shown in 8 or 11；Or the kit is used for the detection of HPV viruse, further includes having
SEQ ID NO：The crRNA of nucleotide sequence shown in 16.
Archaeal dna polymerase provided by the invention, loop-stem structure primer and detect the method for nucleic acid and kit can be used for
The target nucleic acid in sample is detected in the case of constant temperature, at low cost, detection time is short, easy to operate, specific high, high sensitivity
The advantages that, it is particularly suitable for POCT applications.
Description of the drawings
Fig. 1 is RINA-CAS technologies schematic diagram of the present invention
Fig. 2 is the Klenow large fragments (MT-Klenow) and wild type Klenow large fragments (WT- that the present invention is transformed
Klenow the comparing result figure of constant temperature nucleic acid amplification) is carried out.Wherein,Indicate bent using the fluorescence of MT-Klenow amplifications
Line,Indicate the fluorescence curve using WT-Klenow amplifications.
Fig. 3 is the comparing result figure that normal linear primer is used for constant temperature nucleic acid amplification with loop-stem structure primer of the present invention.Its
InIndicate three groups of parallel fluorescent curves of loop-stem structure primer, It corresponds to respectivelyNegative control,Indicate common
Three groups of parallel fluorescent curves of linear primer,It corresponds to respectively Negative control.
It is testing result figure of the detection combination object to Influenza B samples for detecting albumen that Fig. 4, which is with LwCas13a,.Its
InIndicate three groups of parallel fluorescent curves to Influenza B sample detections, It is respectively correspondingNegative control.
It is testing result figure of the detection combination object to Influenza B samples for detecting albumen that Fig. 5, which is with LbCas12a,.Its
InIndicate three groups of parallel fluorescent curves to Influenza B sample detections, It is respectively correspondingNegative control.
Fig. 6 is using quantitative fluorescent PCR to the testing result figure of Influenza B samples.Wherein Indicate three groups of parallel fluorescent curves to Influenza B sample detections,Respectively
It is correspondingNegative control.
It is testing result figure of the detection combination object to HPV samples for detecting albumen that Fig. 7, which is with LwCas13a,.WhereinIndicate three groups of parallel fluorescent curves to HPV sample detections,
It is respectively correspondingNegative control.
Specific implementation mode
Unless otherwise indicated, technical and scientific terms used herein has those skilled in the art institute is normally understood to contain
Justice.Unless otherwise indicated, the basic operations such as the molecular biology, the genetic engineering that use herein are known to those skilled in the art
Conventional biology technology.Unless otherwise indicated, test material used in the present invention is commercially available from common biochemical Reagent Company
.
When used in this paper, term " Klenow large fragments " refers to the DNA polymerase i of Escherichia coli through trypsase or withered grass bar
The segment for 605 amino acid residues of C-terminal that mycoproteinase partial hydrolysis generates.The segment remains the 5 '-of DNA polymerase i
3 ' polymerase activities and 3 ' -5 ' 5 prime excision enzyme activities, but lack 5 ' -3 ' 5 prime excision enzyme activities of complete enzyme.In addition, as described above,
The Klenow large fragments also have the DNA polymerase activity that weaker RNA is relied on.
When used in this paper, term " constant temperature nucleic acid amplification " or " constant temperature nucleic acid amplification technology " are used interchangeably, and are referred in isothermal
In the case of the amplification process that carries out.That is, without thermal denaturation repeatedly in amplification procedure, this is different from common round pcr.
Over nearly more than 20 years, constant temperature nucleic acid amplification technology is in during continuous development, emerges the subdivision skills using different amplification principles
Art expands for example, loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP), chain substitute
Increase (strand displacement amplification, SDA), amplification (rolling circle are changed in rolling
Amplification, RCA), rely on unwindase isothermal DNA amplification (helicase-dependent isothermal DNA
Amplification, HDA), recombinase polymeric enzymatic amplification (recombinase polymerase amplification,
RPA), recombinase-mediated amplification (recombinase-aid amplification, RAA), etc..Due to these constant-temperature amplifications
Method need not thermal denaturation repeatedly, amplification rate is much faster than pcr amplification reaction, usually in 30 minutes, even at 15 points
It is completed in clock, is also referred to as fast constant temperature nucleic acid amplification (rapid isothermal nucleic-acid herein
Amplification, RINA).
When used in this paper, refers to that the term " loop-stem structure " used when DNA primer or " hairpin structure " are used interchangeably, refer to
The DNA primer holds base pairings with 3 ' self by 5 ' end bases and forms secondary facility.The double-strand formed by base pairing
Part is " stem ", and the sequence matched between base is then formed " ring portion ".In some cases, stem can not be flat end,
Such as with 3 ' protruding terminus or 5 ' protruding terminus, it is properly termed as the loop-stem structure with foothold.It is matched in loop-stem structure
Base quantity is usually 2 to 15 bases, for example, 5,6,7,8,10,12 bases etc.." ring portion " then usually by ten a few to tens of
A base is formed.
When used in this paper, term " Cas detection architectures " or " Cas detection combinations object " refer to the CRISPR (rules using bacterium
The short palindrome repetitive sequence of intermittent cluster, clustered regularly interspaced short palindromic
Repeats) GAP-associated protein GAP (CRISPR associated proteins, Cas) is come the system that carries out detection of nucleic acids.CRISPR systems
System is to have now been found that a kind of bacterial immune system being present in most of bacteriums, is used to identify and destroys bacteriophage and its
Its pathogen is invaded.CRISPR is the unique dna region in bacterial genomes, viral dna fragment is stored, to allow bacterium
Cell, which can identify, attempts again subinfection its virus.The short rna sequence that CRISPR regional sequences generate after transcription (is referred to as
CrRNA) after identifying and combining viral nucleic acid, cause the Cas albumen (or being Cas enzymes) combined with crRNA to viral nucleic acid
It is sheared.Hitherto it is found that a variety of CRISPR/Cas systems, such as CRISPR/Cas9, CRISPR/Cas13a, CRISPR/
Cas12a etc..There is corresponding nuclease upon activation different from Cas9, Cas13a and Cas12a, in addition to can be to target nucleus
Except acid cutting, also there is subsidiary cutting (collateral cleavage) activity, can continue to cut other non-target nearby
Single stranded DNA (Cas12a) or RNA (Cas13a).It can be by these features for the detection to target nucleic acid in sample.For example, in order to
It detects in sample that target nucleic acid is horizontal, the crRNA of specific bond target nucleic acid sequence can be designed and with fluorophor and is quenched
The short rna or DNA reporter molecule of group are realized using this subsidiary cleavage activity of Cas13a or Cas12a to reporting molecule
The cutting of nucleic acid sequence generates fluorescence signal.Finally, pass through the power for detecting this fluorescence signal or the situation that changes with time
To reflect the level of target nucleic acid in sample.This system is referred to herein as " Cas detection architectures ".Certainly, Cas detection architectures are not
It is only limitted to Cas13a or Cas12a, can also be realized using with other albumen similar to Cas13a or Cas12a enzymatic activitys
This detection.
When used in this paper, term " report molecule (reporter) " refers to short single stranded DNA or single strand RNA molecule, for example, 6 to
20 bases longs, 5 ' ends are connected with fluorophor (for example, FAM, HEX, cy3, JOE or ROX), and 3 ' ends are connected as that base is quenched
Group's (such as BHQ2, BHQ3 etc.).In completely report molecule, fluorophor is spatially approached with quenching group, and base is quenched
Fluorescence signal generates caused by group inhibits exciting light irradiation；And it is single-stranded in such as Cas13a or Cas12a cutting report molecules
In the case of DNA or single stranded RNA, fluorophor is detached with quenching group, then can detect fluorescence signal under exciting light irradiation
It generates.
Transformation to Klenow large fragments
Klenow large fragments lack the remainder of 5 ' -3 ' exonuclease domains, including 605 for DNA polymerase i
Amino acid residue, amino acid sequence are：VISYDNYVTILDEETLKAWIAKLEKAPVFAFDTETDSLDNISANLVGLSFAIE
PGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGQNLKYDRGILANYGIELRGIAFDTMLESYILNSVA
GRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIE
MPLVPVLSRIERNGVKIDPKVLHNHSEELTLRLAELEKKAHEIAGEEFNLSSTKQLQTILFEKQGIKPLKKTPGGAP
STSEEVLEELALDYPLPKVILEYRGLAKLKSTYTDKLPLMINPKTGRVHTSYHQAVTATGRLSSTDPNLQNIPVRNE
EGRRIRQAFIAPEDYVIVSADYSQIELRIMAHLSRDKGLLTAFAEGKDIHRATAAEVFGLPLETVTSEQRRSAKAIN
FGLIYGMSAFGLARQLNIPRKEAQKYMDLYFERYPGVLEYMERTRAQAKEQGYVETLDGRRLYLPDIKSSNGARRAA
AERAAINAPMQGTAADIIKRAMIAVDAWLQAEQPRVRMIMQVHDELVFEVHKDDVDAVAKQIHQLMENCTRLDVPLL
VEVGSGENWDQAH(SEQ ID NO：17).Structurally, the P61-Q194 structural domains of Klenow large fragments have 3 ' -5 '
Exonuclease check and correction activity, L422-D570 structural domains are polymerase domain.It has the DNA polymerase activity for relying on DNA
With the DNA polymerase activity (i.e. reverse transcriptase activity) of weak dependenc RNA.
In order to improve its activity, especially reverse transcriptase activity, we are transformed the Klenow large fragments, utilize fixed point
Which part amino acid is replaced in mutation (site-directed mutagenesis).Based on structure biology data
(PDB:1KFD_A), and pass through test of many times, we design and introduce the displacement of amino acid residue following six：G198W,
V222I, E306K, Q354E, A381E and E582K.The sequence of modified Klenow large fragments is：
Use IUPAC-IUB biochemical here
The one-letter symbol that naming committee is recommended indicates each amino acid, for example, wherein G represents glycine, W represents color ammonia
Acid, V represents valine, I represents isoleucine, E represents glutamic acid, K represents lysine, Q represents glutamine, A represents the third ammonia
Acid.198th glycine in the Klenow large fragments is replaced with tryptophan by G198W expressions, and so on.In the above sequence
Amino acid for replacing is shown with the runic added with underscore.The variation of this 6 amino acid residues improves Klenow sheets
Activity of the section in reverse transcription and follow-up isothermal amplification reactions (see below embodiment 1).It is contemplated that those skilled in the art
It can also be on the basis of this modified Klenow large fragments (being known as archaeal dna polymerase of the present invention) provided by the invention to other
Amino acid does not cause the forfeiture of its DNA polymer active (including reverse transcriptase activity), such as silence to be set into line replacement, this displacement
It changes, then this displacement is also included in the range of polymerase of the present invention.
Correspondingly, the present invention also provides the polynucleotide sequences for encoding archaeal dna polymerase of the present invention.The polynucleotide sequence
Such as can by being detached from genome of E.coli, the nucleotide sequence of amplification coding Klenow large fragments, and expanding
When specific site introduce nucleotide subsitution mutation and obtain.Alternatively, can be according to the amino of present invention provided herein polymerase
Acid sequence (SEQ ID NO：18) coding nucleotide sequence, is designed based on the correspondence of amino acid and trinucleotide codons
Row are then obtained by the methods of chemical synthesis.These operations are the conventional methods of this field, are known to technical staff.It needs
It is noted that being based on Codon degeneracy, there can be multiple polynucleotide sequences all to encode the archaeal dna polymerase of the present invention, it
Be intended to be included within the scope of this invention.
Primer for nucleic acid constant-temperature amplification
In order to improve the specificity of nucleic acid constant-temperature amplification technology, the present invention provides with hairpin structure (or stem ring knot
Structure) amplimer.These hairpin structures are only when authentic and valid combination occurs for amplimer and target target sequence, just meeting
It opens；At the same time, the hairpin structure of amplimer intramolecular also effectively prevents the appearance of mispairing between amplimer, prevents
The generation of false positive results, to solving the problems, such as poor specificity in isothermal amplification technology.This draws with common PCR
Object is on the contrary, the complementary series for generally requiring primer itself that cannot have continuous 4 bases or more in PCR primer designs.
It is identical or complete complementary that 3 ' ends of the loop-stem structure primer of the present invention, which are with nucleic acid target sequence,；In primer
The base of the additional addition 2-15nt in 5 ' ends is complementarily shaped to loop-stem structure with the end of primer 3 '；Or the special outer addition 2-15nt's in 5 ' ends
Base forms the loop-stem structure with foothold with the base complementrity away from 3 ' end 1-10nt.
(it can connect with the part of target nucleic acid for example, a pair of common linear primer can be designed according to target nucleic acid sequence
Continuous sequence complete complementary or identical), primer length is preferably 30-35nt, and G/C content is in 40%-60%, and in primer or primer
Between without or there is only simple secondary structure, avoid the end of primer 3 ' and itself or another Primers complementary.Then, it is designing
Linear primer 5 ' end addition 2-15nt base, preferably 6-8nt, the base complementrity at the base of addition and the primer 3 ' end, or
With several base complementrities after 1-10nt bases are held away from the primer 3 ', and pass through its structure of software simulated determination and stem ring knot
The Tm values of structure primer.NUPACK (http can be used://www.nupack.org/partition/new) carry out secondary structure
Simulated determination, and use Quikfold (http://unafold.rna.albany.edu/Q=DINAMelt/
Quickfold loop-stem structure primer Tm measurement) is carried out.Loop-stem structure primer optimum state is single stable state and stem ring knot
Structure primer Tm optimum values are higher than reaction temperature (reaction temperature≤65 DEG C, such as 25 DEG C).The best Tm values of loop-stem structure primer with
Optimum state should consider when can not be compatible with, e.g., when it is best to design loop-stem structure primer Tm, loop-stem structure primer list
Once ring portion has, can to choose Tm values in the presence of simple secondary structure be best；When it is best to design loop-stem structure primer Tm,
In the presence of loop-stem structure primer is not single or/and ring portion has complicated secondary structure, loop-stem structure primer state should be mainly considered,
Ensure loop-stem structure primer unicity and stability.
Fast constant temperature nucleic acid amplification is combined with Cas detection architectures is used for detection of nucleic acids
As described above, Cas detection architectures can be used for detecting target nucleic acid sequence in sample.However, its sensitivity is frequent
It is difficult to meet testing requirements.For example, in the case of target nucleic acid sequence horizontal relatively low for example several to hundreds of copies, cannot produce
Raw effective fluorescence signal.In addition, although the DNA binding dyes such as EvaGreen, SybrGreen or PNA can also be used
Opener, DNA Beacon, PNA Beacon reflect the case where amplified production in sample during monitoring constant-temperature amplification in real time
The level of target nucleic acid, but specificity aspect cannot usually be met the requirements.It is provided by the invention to use fast constant temperature nucleic acid amplification
With the nucleic acid detection technique (hereinafter also referred to RINA-CAS technologies) that Cas detection architectures are combined, sensitivity on the one hand can be improved,
Such as can detect down to several copies or even 1 target nucleic acid molecule copied, on the other hand pass through drawing during constant-temperature amplification
Object matching is matched in conjunction with the crRNA in detection process to improve the specificity of detection.
Fig. 1 shows the schematic diagram of RINA-CAS technologies of the present invention.In the presence of forward and reverse primer of loop-stem structure, pass through
Constant temperature nucleic acid amplification obtains amplified production DNA in general 15 minutes.For the detection architecture using Cas13a, first by the amplified production
DNA is transcribed into single stranded RNA (ssRNA).The crRNA combined with Cas13a by identifying and in conjunction with the complementary series on single stranded RNA,
The subsidiary cleavage activity (single stranded RNA nucleic acid enzymatic activity) for activating Cas13a, leads to the cutting to RNA fluorescent reporter molecules, discharges
Fluorescence signal.For the detection architecture using Cas12a, is identified by the crRNA combined with Cas12a and combine amplified production
Complementary series on DNA activates the subsidiary cleavage activity (single stranded DNA nucleic acid enzymatic activity) of Cas12a, causes to the single-stranded fluorescence of DNA
It reports the cutting of molecule, discharges fluorescence signal.The fluorescence signal can be detected by fluorescence detector.
The present invention is improved existing fast constant temperature nucleic acid amplification technologies, uses and untwists simultaneously in constant-temperature amplification
Enzyme, recombinase and archaeal dna polymerase can improve amplification efficiency and specificity, usually at 25-45 DEG C (such as 37 DEG C) in 3 to 20
Amplification procedure is completed in minute.Unwindase used for example can be selected from RecQ unwindases, UvrD unwindases, DnaB unwindases,
CMC unwindases or other similar unwindases；Recombinase can be for example selected from bacteriophage UvsX systems, Eukaryotic Rad
Recombinase in system, yeast or the recA of Escherichia coli systems or other prokaryotic systems；Archaeal dna polymerase can for example selected from
Deep VentRTM archaeal dna polymerases, Deep VentRTM (exo-) archaeal dna polymerase, Klenow segments (3 ' -5 ' exo-), DNA
Polymerase I, Klenow large fragment, phi29DNA polymerases,Archaeal dna polymerase, VentR (exo-) archaeal dna polymerases or
Other similar polymerases.
In the presence of amplimer, recombinase is combined the Protein-DNA mixtures to be formed can be in target nucleic acid with amplimer
Upper searching homologous sequence；DNA double chain is unlocked in unwindase assistance, forms stable D ring structures；DNA single strand binding proteins simultaneously
(SBB) it is single-stranded to stablize the DNA unlocked.Pass through the synergy of recombinase and unwindase so that amplimer and complementary target
Sequence combine, archaeal dna polymerase effect under polymerize extend, under 25-45 DEG C of isothermy, these reaction can constantly repeat into
Row, such as in 3 to 20 minutes, complete the exponential type amplification of nucleic acid.
In some embodiments, amplification system component used by fast constant temperature nucleic acid amplification of the present invention can be found in as follows
Table 1.
The component of 1 fast constant temperature nucleic acid amplification system of table
The pH value of Tris-HCl buffer solutions be 7.0-8.5, preferably 7.4.The molecular weight ranges of polyethylene glycol are 1000-
50000, preferably 20000.Dithiothreitol (DTT) is for keeping each protein component reproducibility.
In some embodiments, the combination of UvsX enzymes and auxilin UvsY may be used in recombinase, they be from
The albumen of T4 or T6 bacteriophages, strand displacement when can mediate template strand unwinding and starting reaction between template strand and primer strand.
This process needs ATP to provide energy.After unwinding and strand displacement, single strand binding protein (for example, Gp32) can with it is therein
Single-stranded combination, prevents double-strand from being formed.
ATP can be regenerated by phosphocreatine, and creatine kinase can realize the regeneration of phosphocreatine, be recycled.
In preferred embodiments, archaeal dna polymerase used is that Klenow large fragments can with strand-displacement activity
Specific extension is carried out after amplimer and template strand carry out strand displacement, to realize that target fragments expand.Preferred
In embodiment, archaeal dna polymerase used is the archaeal dna polymerase of the present invention, i.e., modified Klenow large fragments described above.
Archaeal dna polymerase using the present invention can not only be expanded using single-stranded or double-stranded DNA molecular as template, additionally it is possible to RNA points
Son is that template is expanded.When being expanded as template using RNA molecule, closed first with the reverse transcriptase activity of DNA of the present invention
At the cDNA molecules that can be used as template, subsequent amplified reaction is then carried out as template using the cDNA molecules.It is using the present invention
When archaeal dna polymerase expands target RNA, reverse transcription operation is carried out without additional reverse transcriptase, which greatly simplifies expansions
Increasing process.
In preferred embodiments, used upstream and downstream primer is drawing with loop-stem structure provided by the invention
Object.Primer length is generally 33nt-45nt, to ensure that recombinase carries out the recognition sequence specificity in chain replacement process, is adopted
Primer number can increase with the number of target nucleic acid sequence to be detected and be increased.In addition, when using Cas13a detection architectures
When, T7 promoter sequences TAATACGACTCACTATAG (SEQ ID NO can be introduced at 5 ' ends of positive amplimer：22),
For the transcription of subsequent DNA cloning product and the detection of Cas13a proteolytic cleavages.In this way, including successively from 5 ' ends to 3 ' ends in primer
With sequence, T7 promoter sequences and the 5 ' terminal sequences for being used to be formed loop-stem structure with 3 ' ends of template pairing, total length is about
51-63nt。
DNTPs used includes：DATP, dTTP, dCTP, dGTP, dUTP, wherein dTTP may exist or be not present, and
The use of dUTP is conducive to eliminate by UNG enzymes or high degree eliminates pollution problem.
In addition, also using additives such as DMSO, PEG, DTT, Betaine, Proline, Formamide, BSA to this expansion
Increasing system advanced optimizes.
Fast constant temperature nucleic acid amplification step generate DNA cloning product can by the detection architecture comprising Cas12a or comprising
The detection architecture of Cas13a detects.
The crRNA used in these detection architectures includes two-part structure sequence：With CRISPR-Cas12a/Cas13a
Protein bound sequence and the sequence matched with complementary target.It is wherein 24nt- with the sequence length of complementary target pairing
30nt, this can further increase the specificity and sensitivity of detection.
In Cas12a detection architectures, the Cas12a-RNP compounds specific recognition that crRNA is formed with Cas12a albumen is simultaneously
Target dna fragment (for example, DNA cloning product) is cut, while activating the ssDNase activity of Cas12a albumen so that DNA reports point
Sub (reporter) is degraded.DNA reporter molecule is that 5 ' ends carry fluorophor, 3 ' oligonucleotides of the end with quenching group
SsDNA segments.Finally, fluorophor is detached with quenching group, under exciting light irradiation, is generated fluorescence signal and is read by instrument
It takes.
In some embodiments, the Cas12a detection architectures that the present invention uses include component as shown in Table 2.
2 Cas12a detection architecture components of table
In Cas13a detection architectures, first by T7 RNA polymerases by the amplified production DNA containing T7 promoter sequences
It is transcribed into RNA, the Cas13a-RNP compounds then formed by crRNA and Cas13a albumen carry out the RNA of transcription special
Property identification cutting, and then activate Cas13a albumen RNA nucleases so that RNA report molecule be degraded, send out fluorescence letter
Number.RNA reports that molecule is that 5 ' ends carry fluorophor, 3 ' oligonucleotides RNA chains of the end with quenching group.
Specifically, which for example may include following process：(1) T7 promoter sequences will be carried by T7 RNA polymerases
The DNA cloning product transcription of row becomes target RNA chain；(2) it allows Cas13a albumen to be combined with crRNA, it is multiple to form Cas13a-RNP
Object is closed, the target RNA under crRNA guiding after selectively targeted transcription；(3) it is compound to form target RNA-Cas13a-crRNA
Object activates its non-specificity RNA nuclease to make Cas13a protein conformations change；(4) Cas13a albumen is non-
Specific RNA nuclease can report fluorogenic substrate RNA to molecule digestion, and fluorophor is made to be detached with group is quenched,
Exciting light irradiation is lower to generate fluorescence signal, is tested and analyzed by instrument.Although being to describe to detect in a step-by-step manner here
Journey, but they can be carried out in same reaction system when practical operation.
In some embodiments, the Cas13a detection architectures that the present invention uses include component as shown in table 3 below.
3 Cas13a detection architecture components of table
The pH value of Tris buffer solutions is preferentially selected as 7.4 in the detection architecture.
In some embodiments, another CRISPR-Cas PROTEIN Cs sm6 and its activation can be added in the system
Factor precursor (rUrUrUrUrUrArArArArArA- (2,3- cycli phosphate)), after Cas13a is activated, can cut the precursor
It cuts, to generate the activity factor of Csm6, by the RNase activity activators of Csm6, RNA report molecules can be hydrolyzed, so as to
Effectively improve the sensitivity of CRISPR-Cas13a detection architectures.It therefore, in some embodiments, can be to CRISPR-
Cas13a detection architectures are improved, before Csm6 albumen and the 500nM Csm6 activity factors of final concentration of 10nM is added in supplement
Body.
The RINA-CAS technologies of the present invention can make target nucleic acid complete amplification and detection in a reaction system, and right
Amplicon (i.e. DNA cloning product) carries out qualitative and quantitative analysis.
Detection kit
Detection kit provided by the invention can be used in invention described above RINA-CAS technologies.The kit
May include the main agents for the technology, for example, enzyme for carrying out constant temperature nucleic acid amplification reaction and for amplified production into
The enzyme of row detection.Enzyme for carrying out constant temperature nucleic acid amplification reaction includes mainly unwindase, recombinase and archaeal dna polymerase.Excellent
In the embodiment of choosing, which is archaeal dna polymerase provided by the invention.The enzyme that amplified production is detected for example including
Cas12a and/or Cas13a.For specific bacterium or viral diagnosis, kit of the invention may also include for carrying out constant temperature
The primer of nucleic acid amplification reaction and the crRNA being used cooperatively with Cas12a and/or Cas13a.In addition, in many places this paper with table
Case form lists the component for carrying out constant temperature nucleic acid amplification reaction and the component for being detected to amplified production, these components
All or part may include the present invention detection kit in.
RINA-CAS technologies provided by the invention even can just complete reaction under room temperature at 37 DEG C, it is only necessary to a kind of
Miniature constant temperature and fluorescence signal detection device, the PCR thermal cyclers without using accurate costliness, are very suitable for examining immediately
Test (POCT) application.
Explanation is explained in more detail to the present invention below in conjunction with specific embodiments, makes the purpose of the present invention, technology
Scheme and effect are apparent clear.Following embodiment only as illustrating, does not constitute the scope of the present invention any limit
It is fixed.
The activity of 1 archaeal dna polymerase of the present invention of embodiment and original Klenow large fragments
The present embodiment is to the Klenow large fragments (MT-Klenow) that (introduce amino acid replacement described herein) after transformation
It is compared with the activity of the Klenow large fragments (WT-Klenow) of wild type.Using identical RNA templates and primer, profit
Constant temperature nucleic acid amplification is carried out under the same conditions with MT-Klenow and WT-Klenow.Used RNA template sequences are：
CAGGGAGGUGCCUUGAUGACAUAGAAGAAGAACCAGAUGAUGUUGAUGGCCCAACUGAAAUAGUAUUAAGGGACAUG
AACAACAAAGAUGCAAGGCAAAAGAUAAAGGAGGAAGUAAACACUCAGAAAGAAGGGAAGUUCCGUUUGACAAUAAA
AAGGGAUAUGCGUAAUGUAUUGUCCCUGAGAGUGUUAGUAAACGGAACAUUCCUCAAACACCCCAAUGGAUACAAGU
CCUUAUCAACUCUGCAUAGAUUGAAUGCAUAUGACCAGAGUGGAAGGCUUGUUGCUAAACUUGUUGCUACUGAUGAG
CUUACAGUGGAGGAUGAAGAAGAUGGCCAUCGGAUCCUCAAUUCACUCUUCGAGCGUCUUA(SEQ ID NO：19)；
Forward primer sequence is CAGGGAGGTGCCTTGATGACATAGAAGAAGAACCA (SEQ ID NO：20)；
Reverse primer sequences are TAAGACGCTCGAAGAGTGAATTGAGGATCCGATG (SEQ ID NO：21).
Used reaction system volume is 50 μ L, and component therein is as shown in table 4 below.
Table 4 is used for the constant temperature nucleic acid amplification reaction system component of WT-Klenow and MT-Klenow activity contrast tests
Amplification procedure is monitored by luminescent dye molecule Eva Green, as a result sees Fig. 2, in figureIt indicates to use
The fluorescence curve of MT-Klenow amplifications,Indicate the fluorescence curve using WT-Klenow amplifications.As can be seen from Figure 2, through this
Inventing improved MT-Klenow ratios WT-Klenow has better reverse transcription and amplification efficiency.
Embodiment 2：Loop-stem structure primer and linear primer contrast test
It is identical or complete complementary that 3 ' ends of loop-stem structure primer of the present invention, which are with nucleic acid target sequence,；Primer 5 '
The base of the additional addition 2-15nt in end is complementarily shaped to loop-stem structure with the end of primer 3 '；Or 5 ' the special outer addition 2-15nt in end base,
The loop-stem structure with foothold is formed with the base complementrity away from 3 ' end 1-10nt.
According to the above loop-stem structure design of primers principle, the present invention is to a pair of of normal linear primer and its loop-stem structure primer
Isothermal amplification reactions carried out Experimental comparison.Wherein DNA template sequence is：5’-
GACAGACTGCACAGGGCATGGATTACTTACACGCCAAGTCAATCATCCACAGAGACCTCAAGAGTAATAATATATTT
CTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGAGTGGGTCCCATCA
GTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGCACCAGAAGTCATCAGAATGCAAGATAAAAATCCATACAGCT
TTCAGTCAGATGTATATGCATTTGGAATTGTTCTGTATGAATTGATGACTGGACAGTTACCTTATTCAAACATCAGA
CGGGA-3’(SEQ ID NO：1), normal linear primer is：(sequence is BRAF-F：5’-
CGCCAAGTCAATCATCCACAGAGACCTCAAGAG-3’(SEQ ID NO：2)), (sequence is BRAF-R：5’-
CCAAATGCATATACATCTGACTGAAAGCTGTATGG-3’(SEQ ID NO：3))；Loop-stem structure primer is：JH-BRAF-F
(sequence is：5’-CTCTTGAGCGCCAAGTCAATCATCCACAGAGACCTCAAGAG-3’(SEQ ID NO：4)), JH-
(sequence is BRAF-R：5’-CCATACACCAAATGCATATACATCTGACTGAAAGCTGTATGG-3’(SEQ ID NO：5)).
Here the base for pairing that 5 ' ends are additionally added in loop-stem structure primer and hereafter is added with solid underline.The amplification is anti-
It is 50 μ L to answer system volume, and the component of use is as shown in table 5 below.
Table 5 is used for the constant temperature nucleic acid amplification reaction system component of primer contrast test
Whole experiment process is repeated three times, and amplification procedure is monitored by luminescent dye molecule Eva Green, experiment
As a result it shows in figure 3.In figureIndicate that three groups of parallel fluorescents of loop-stem structure primer are bent
Line,It is respectively correspondingNegative control,Indicate three groups of parallel fluorescent curves of normal linear primer, It corresponds to respectivelyNegative control.It can be seen from the results that the stem ring knot of the present invention
Structure primer can significantly reduce non-specific amplification.
Embodiment 3 detects (Influenza B) containing influenza B virus using RINA-CAS (using LwCas13a) technology
Sample
First, nucleic acid extraction, nucleic acid extraction are carried out to Influenza B (influenza B virus yamagata hypotypes) sample
Kit is the viral RNA extracts kit (QIAamp Viral RNA Mini Kit) of Qiagen.Using without
The sample of Influenza B is as negative control.
It takes the 1 μ L of RNA of extraction to be added in nucleic acid amplification reaction system, and is added a concentration of 10-5Two amplifications of M are drawn
(sequence is object Influenza Primer F：5’-ACCAACTTAATACGACTCACTATAGGTGAAACTGCGGTGGGAGTCT
TATCCCAAGTTGGT-3’(SEQ ID NO：6)), (sequence is Influenza Primer R：5’-TGGTTGTCACAAGCACTGCCTGCTGTACACTTCAACCA-3’(SEQ ID NO：7)) each 2.4 μ L, are incubated at 37 DEG C
It educates, time 15min.Designed primer is the conservative gene Influenza for influenza B virus yamagata hypotypes
B NS1.The amplification reaction system volume is 50 μ L, and each component is as shown in table 6 below.
Constant-temperature amplification system of the table 6 for Influenza B sample amplifications forms
Then, 4 μ L amplified productions are pipetted, with the 20 μ L (LwCas13a- wherein included of detection liquid containing LwCas13a
CrRNA sequences are： (SEQ ID NO：8) it, is used to be combined with LwCas13a added with the sequence of wave underline, added with void
The sequence of line underscore with amplified production for being combined) and 1 μ L mixing of RNA report molecules (FAM-5 '-UUUUU-3 '-TAMAR),
It is incubated at 37 DEG C, time 30min.The detection architecture total volume is 25 μ L, and each component is as shown in table 7 below.
Table 7 forms the detection architecture that amplified production is detected using LwCas13a
Whole experiment process is repeated three times, is as a result shown in Fig. 4.In figureTable
Show the three groups of parallel fluorescent curves detected to Influenza B actual samples,It is respectively correspondingNegative control.As can be seen from Figure 4, it by first carrying out constant-temperature amplification to sample, then uses
Detection architecture containing LwCas13a is detected, and RINA-CAS technologies of the invention can be successfully to Influenza B samples
It is detected.
Embodiment 4：(Influenza B) containing influenza B virus is detected using RINA-CAS (using LbCas12a) technology
Sample
First, nucleic acid extraction, nucleic acid extraction are carried out to Influenza B (influenza B virus yamagata hypotypes) sample
Kit is the viral RNA extracts kit (QIAamp Viral RNA Mini Kit) of Qiagen.It uses and is free of simultaneously
The sample of Influenza B is as negative control.
It takes the 1 μ L of RNA of the Influenza B of extraction to be added in isothermal amplification reactions system, and is added a concentration of 10-5M
Two amplimer Influenza Primer F (sequence is：5’-ACCAACTGAAACTGCGGTGGGAGTCTTATCCCAAGTTGGT-3’(SEQ ID NO：9))、Influenza Primer R
(sequence is：5’-TGGTTGTCACAAGCACTGCCTGCTGTACACTTCAACCA-3’(SEQ ID NO：10)) each 2.4 μ L,
37 DEG C are incubated, time 15min.The amplification reaction system volume is 50 μ L, and each reactive component is as shown in table 8 below.
Constant-temperature amplification system of the table 8 for Influenza B sample amplifications forms
Then, 4 μ L amplified productions are pipetted, it is (wherein included with the 20 μ L of detection liquid containing LbCas12a
LbCas12acrRNA sequences are： (SEQ ID NO：11) it, is used for added with the sequence of wave underline
Combined with LbCas12a, be used to be combined with amplified production added with the sequence of dotted line underscore) and DNA reporter molecule (FAM-5 '-
TTTTT-3 '-TAMAR) 1 μ L, it is incubated at 37 DEG C, time 15min.The detection architecture total volume is 25 μ L, and each component is such as
Shown in the following table 9.
Table 9 forms the detection architecture that amplified production is detected using LbCas12a
Whole experiment process is repeated three times, and the results are shown in Figure 5.In figureIt indicates
To three groups of parallel fluorescent curves of Influenza B sample detections,It is respectively correspondingNegative control.
As can be seen from Figure 5, it by first carrying out constant-temperature amplification to sample, is then examined using the detection architecture containing LwCas13a
It surveys, RINA-CAS technologies of the invention can successfully be detected Influenza B samples.
Embodiment 5：Identical Influenza B samples are detected using quantitative fluorescent PCR
The RNA extracted by RNA extracts kits (QIAamp Viral RNA Mini Kit) in embodiment 3 is used as
Template carries out PCR amplification, wherein there is influenza B virus to verify.Two PCR amplification primers are respectively：PCR-F (sequences
For：5’-GGGAGTCTTATCCCAAGTTGGT-3’(SEQ ID NO：12)), (sequence is PCR-R：5’-
TGCCTGCTGTACACTTCAACCA-3’(SEQ ID NO：13)).PCR reaction systems and program are as follows：
Reverse transcription reaction system
PCR reaction systems
PCR response procedures
Fluorescence signal is acquired at the end of each cycle.It tests in triplicate, using the sample conduct without Influenza B
Negative control.Reaction result is shown in figure 6.WhereinIt indicates to Influenza B samples
Three groups of parallel fluorescent curves of detection,It is respectively corresponding
Negative control.It can be seen that there are influenza B virus in sample used, the RINA-CAS skills used with embodiment 3
The result that art obtains is consistent.
Embodiment 6：HPV samples are detected using RINA-CAS (using LwCas13a) technology
First, nucleic acid extraction is carried out to HPV samples, nucleic acid extraction kit is Tiangeng Fast DNA extraction detection kit
(being purchased from TIANGEN Biotech (Beijing) Co., Ltd.).Simultaneously using the sample without HPV as negative control.
It takes the 1 μ L of DNA of the HPV of extraction to be added in reaction system, and is added a concentration of 10-5Two amplimers of M
(sequence is HPV Primer F：5’-ACAGTAATACGACTCACTATAGGTTTGTTGGGGTAACCAACTATTTGTTACTGT
-3’(SEQ ID NO：14)), (sequence is HPV Primer R：5’-ACTGTGACGTCTGCAGTTAAGGTTATTTTGCACAG
T-3’(SEQ ID NO：15)) each 2.4 μ L, are incubated at 37 DEG C, time 15min.The constant-temperature amplification system volume is 50 μ
L, each component are as shown in the following table 10.
The constant-temperature amplification system of the HPV sample amplifications of table 10 forms
Then, 4 μ L amplified productions are pipetted, with the 20 μ L detection liquid (LwCas13a- wherein included containing LwCas13a
CrRNA sequences are： (SEQ ID NO：16) it, is used to be combined with LwCas13a added with the sequence of wave underline, added with dotted line
The sequence of underscore with amplified production for being combined) and 1 μ L mixing of RNA report molecules (FAM-5 '-UUUUU-3 '-TAMAR),
37 DEG C are incubated, time 30min.The detection architecture total volume is 25 μ L, and each component is as shown in table 11 below.
Table 11 forms the detection architecture that amplified production is detected using LbCas13a
Whole experiment process is repeated three times, and the results are shown in Figure 7.In figureIt indicates
To three groups of parallel fluorescent curves of HPV sample detections,It is respectively correspondingNegative control.
As can be seen from Figure 7, it by first carrying out constant-temperature amplification to sample, is then examined using the detection architecture containing LwCas13a
It surveys, RINA-CAS technologies of the invention can successfully be detected HPV samples.
Embodiment 7：Sensitivity of the RINA-CAS technologies of the present invention to a variety of virus/bacteria samples
By the plasmid of a variety of viruses/Bacteria Detection target/genome standard items gradient dilution at about 105Copy/μ L, 104It copies
Shellfish/μ L, 103Copy/μ L, 102Copy/μ L, 101Copy/μ L, 100Copy/μ L.
Constant temperature nucleic acid amplification is carried out respectively to the template after above-mentioned dilution, amplification temperature is 37 DEG C, time 15min, with
Result is compared with negative control afterwards, is judged as criterion, detection sensitivity result is as shown in table 11.
11 RINA-CAS technologies of the present invention of table are to virus not of the same race/bacterium sensitivity technique result
"+" indicates that positive findings, "-" indicate negative findings in table.
Correlation abbreviation：Inf.A, Flu-A；Inf.B, influenza B；Inf.C, influenza C；HPV, human papilloma virus
Poison；Strep.A, streptococcus；RSV, Respiratory Syncytial Virus(RSV)；PTB, pulmonary tuberculosis, pulmonary tuberculosis；MP, lung
Scorching mycoplasma；CP, chlamydia pneumoniae；AdV, adenovirus；EV, Epstein-barr virus epstein-Barr virus；BoV, bocavirus；
HRV, ERC group virus.
The RINA-CAS technologies of the present invention have high detection sensitivity, such as are limited to 5 to the detection of Inf.A etc. and copy
Shellfish, detection limit even as low as 1 copy of HPV etc..
Claims (28)
1. it is a kind of using DNA or RNA as the archaeal dna polymerase of template, to pass through the Klenow large fragments to Escherichia coli polymerase I
It carries out following amino acid replacement and obtains：G198W, V222I, E306K, Q354E, A381E and E582K.
2. archaeal dna polymerase as described in claim 1 has such as SEQ ID NO：Amino acid sequence shown in 18.
3. the polynucleotide sequence or its complementary series of coding archaeal dna polymerase as claimed in claim 1 or 2.
4. a kind of DNA primer pair for nucleic acid constant-temperature amplification, wherein any one primer or the two of the DNA primer centering
With loop-stem structure.
5. DNA primer pair as claimed in claim 4, wherein the loop-stem structure is by linear with template sequence complementation
It adds 2 to 15 bases and is formed, 3 ' end sequences of 2 to 15 bases and the linear DNA primer in the end of DNA primer 5 '
Complementary or sequence with the base of 3 ' end 1 to 10 away from the linear DNA primer is complementary.
6. DNA primer pair as claimed in claim 5, wherein the length of any one primer of the DNA primer centering or the two
Degree is 33 to 45 bases；Or also it is added with T7 promoter sequences, the then DNA primer pair at 5 ' ends of the linear primer
In any one primer or the two length be 51 to 63 bases.
7. a kind of horizontal method determining target nucleic acid in the sample, including：
1) target nucleic acid is detached from the sample；
2) it using the target nucleic acid of separation as template, is expanded using constant temperature nucleic acid amplification technology, obtains DNA cloning product；
And
3) use Cas detection combination analyte detections described in DNA cloning product amount, and by the amount of the DNA cloning product with it is described
The level of target nucleic acid described in sample is associated.
8. the method for claim 7, wherein the target nucleic acid is DNA or RNA.
9. the method for claim 7, wherein the constant temperature nucleic acid amplification technology includes using the DNA with loop-stem structure
Primer pair.
10. the method for claim 7, wherein the constant temperature nucleic acid amplification technology includes using containing unwindase, recombination
The amplification reaction system of enzyme and archaeal dna polymerase.
11. method as claimed in claim 10, wherein the unwindase is selected from RecQ unwindases, UvrD unwindases, DnaB solutions
Revolve enzyme and CMC unwindases；The recombinase is selected from UvsX systems, eucaryote Rad systems, yeast or the large intestine bar of bacteriophage
Bacterium recA systems；The archaeal dna polymerase is selected from Deep VentRTM archaeal dna polymerases, Deep VentRTM (exo-) DNA polymerize
Enzyme, Klenow segments (3 ' -5 ' exo-), DNA polymerase i, Klenow large fragments, phi29DNA polymerases,DNA is poly-
Synthase, VentR (exo-) archaeal dna polymerase.
12. method as claimed in claim 10, wherein the archaeal dna polymerase is big to the Klenow of Escherichia coli polymerase I
Segment carries out following amino acid replacement and obtains：G198W, V222I, E306K, Q354E, A381E and E582K.
13. method as claimed in claim 10, wherein the archaeal dna polymerase has such as SEQ ID NO：Amino acid shown in 18
Sequence.
14. the method for claim 7, wherein the Cas detection combinations object includes Cas12a or Cas13a albumen.
15. the method for claim 7, wherein the Cas detection combinations object includes Cas13a albumen and Csm6 albumen.
16. the method for claim 7, wherein the target nucleic acid is originated from virus or bacterium.
17. the method described in claim 16, wherein the virus or bacterium be selected from Inf.A, Inf.B, Inf.C, HPV,
Strep.A, RSV, PTB, MP, CP, AdV, EV, BoV and HRV.
18. a kind of kit for detecting target nucleic acid in the sample includes for carrying out the unwindase of constant temperature nucleic acid amplification, again
Group enzyme and archaeal dna polymerase and the Cas detection combination objects for being detected to DNA cloning product.
19. kit as claimed in claim 18, wherein the unwindase is selected from RecQ unwindases, UvrD unwindases, DnaB
Unwindase and CMC unwindases；The recombinase is selected from bacteriophage UvsX systems, eucaryote Rad systems and yeast or large intestine
Bacillus recA systems；It is poly- that the archaeal dna polymerase is selected from Deep VentRTM archaeal dna polymerases, Deep VentRTM (exo-) DNA
Synthase, Klenow segments (3 ' -5 ' exo-), DNA polymerase i, Klenow large fragments, phi29DNA polymerases, DNA
Polymerase and VentR (exo-) archaeal dna polymerase.
20. kit as claimed in claim 18, wherein the archaeal dna polymerase is the Klenow to Escherichia coli polymerase I
Large fragment carries out following amino acid replacement and obtains：G198W, V222I, E306K, Q354E, A381E and E582K.
21. kit as claimed in claim 18, wherein the archaeal dna polymerase has such as SEQ ID NO：Amino shown in 18
Acid sequence.
22. kit as claimed in claim 18, wherein the Cas detection combinations object includes Cas12a or Cas13a albumen.
23. kit as claimed in claim 18, wherein the Cas detection combinations object includes Cas13a albumen and Csm6 eggs
In vain.
24. kit as claimed in claim 18, wherein further including the DNA primer pair for constant temperature nucleic acid amplification, the DNA
Any one primer or the two in primer pair have loop-stem structure.
25. kit as claimed in claim 24, wherein the loop-stem structure is by the line with the complementary target
Property DNA primer 5 ' end addition 2 to 15 bases and formed, 3 ' end sequences of 2 to 15 bases and the linear DNA primer
The complementary or sequence with the base of 3 ' end 1 to 10 away from the linear DNA primer of row is complementary.
26. DNA primer pair as claimed in claim 25, wherein any one primer of the DNA primer centering or the two
Length is 33 to 45 bases；Or 5 ' ends of the linear primer are also added with T7 promoter sequences, the then DNA primer pair
In any one primer or the two length be 51 to 63 bases.
27. kit as claimed in claim 24, wherein the kit is used for the detection of influenza B virus, the DNA
Primer pair is respectively provided with SEQ ID NO：Nucleotide sequence shown in 6 and 7 or SEQ ID NO：Nucleotides sequence shown in 9 and 10
Row；Or the kit is used for the detection of HPV viruse, the DNA primer is to being respectively provided with SEQ ID NO：Shown in 14 and 15
Nucleotide sequence.
28. kit as claimed in claim 24, wherein the kit is used for the detection of influenza B virus, further include tool
There are SEQ ID NO：The crRNA of nucleotide sequence shown in 8 or 11；Or the kit is used for the detection of HPV viruse, also wraps
It includes with SEQ ID NO：The crRNA of nucleotide sequence shown in 16.
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| CN112941155A (en) | 2021-06-11 |
| CN112941155B (en) | 2023-07-14 |
| CN108588050B (en) | 2021-06-25 |
| CN112899350B (en) | 2023-01-24 |
| CN112899350A (en) | 2021-06-04 |
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