CN107557455A - A kind of detection method of the nucleic acid specific fragment based on CRISPR Cas13a - Google Patents
A kind of detection method of the nucleic acid specific fragment based on CRISPR Cas13a Download PDFInfo
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Abstract
The present invention relates to the detection of biomaterial, specifically discloses a kind of quick determination method of nucleic acid specific fragment suitable for biological specimen based on CRISPR Cas13a.Nucleic acid fragment to be checked is expanded and obtains transcription product, for the targeted rna sequences Design guide RNA of target gene, and the signal reports RNA molecule added in detection architecture, the RNase not limited by the sequence activity activated using Cas13a albumen after targeted rna sequence is identified, the amplification and reading of signal are realized, and then realizes the detection to target gene.The detection sensitivity of the method for the invention can reach 10‑18Mol/L, i.e. A Moer ranks, specificity can reach for detecting the nucleic acid fragment with single base difference.
Description
Technical field
The present invention relates to the detection of biomaterial, specifically, it is related to and a kind of life is applied to based on CRISPR-Cas13a
The quick determination method of nucleic acid specific fragment in thing sample.
Background technology
As physianthropy develops, people gradually recognize and disease are studied and diagnosed from gene level, are real
The key of accurate this medical medical concept of existing individuation.For example, the Circulating tumor DNA in Blood of Tumor Patients is quickly examined
Survey, and mutator present in it is determined, can not only realize early diagnosis and its mutator spectrum of tumour
Draw, but also foundation can be provided to follow-up targeted drug treatment, avoid drug waste and resistance from producing；To virus or thin
After pathogen present in the biological sample of bacterium infected patient carries out separation and Extraction, its gene is entered using the method for gene diagnosis
Row Rapid identification, it can equally realize early diagnosis and guiding treatment.However, these diseases carried out on gene level are examined
It is disconnected, be required for it is a kind of can quick and precisely and high sensitivity nucleic acid detection technique, some under-developed areas or under the conditions of,
This detection technique also needs to simple and easy to operate.
At present, the nucleic acid detection technique that hospital and testing agency commonly use is PCR-based (PCR) technology hair
Exhibition, most common of which is exactly that real-time fluorescence quantitative PCR (Realtime-PCR) and amplification are hindered into mutation PCR
(ARMS-PCR) the real time fluorescent quantitative amplification that two kinds of technologies combine hinders mutation PCR (Realtime-ARMS-PCR).
Realtime-PCR technologies develop by traditional PCR technique and with reference to spectral technique, by adding in PCR reaction systems
Enter nucleic acid insertion fluorescent dye or specific quenching probes, the signal of both fluorescence probes all can be in nucleic acid amplification
Now linear enhancing, then the fluorescence detection device carried by PCR instrument send exciting light and collect detection fluorescence signal, it is possible to real
The level of amplification each circulated during Shi Fanying PCR；ARMS-PCR then make use of Taq DNA polymerase to lack 3 ' to 5 '
5 prime excision enzyme activity, the mispairing that PCR primer 3 ' is held under certain condition can cause to expand hindered, and product is drastically reduced, therefore
It is mutated for different known, designs appropriate primer, it is possible to which saltant type and wild is distinguished by this PCR method
Type gene.The Realtime-ARMS-PCR that both technologies are combined, the real-time quantitative for realizing specific gene fragment are fast
Speed detection.
Existing a variety of gene detecting kits based on Realtime-ARMS-PCR technologies are applied to clinical detection both at home and abroad
In, however, prior art still has many weak points：1.PCR technologies need accurate temperature system control, are not sent out at some
It can not be realized up to area；2. detection sensitivity and specificity still need to further improve；3.PCR technologies are only used for DNA fragmentation inspection
Survey, RNA fragments need to add the process of a step reverse transcription in advance, complex operation, and open pipe operation easily causes sample pollution.
Therefore, it is simple to need a kind of temperature control of offer badly, and sensitivity, specific high specific fragment detection method.
The content of the invention
It is simple it is an object of the invention to provide temperature control in order to solve problems of the prior art, and sensitivity,
The high specific fragment detection method of specificity, and further solve the easily contaminated problem of sample in detection process.
In order to realize the purpose of the present invention, the present invention provides a kind of nucleic acid specific fragment based on CRISPR-Cas13a
Detection method.
In a first aspect, method provided by the present invention comprises the following steps (reaction)：
(1) RPA technologies carry out nucleic acid amplification reaction to fragment to be checked.
The reagent for participating in the reaction is：RPA primers (0.48 μM), template (fragment to be checked), RPA enzyme premixed liquids (come from
Twistdx companies), magnesium acetate (14mM)；
Wherein, the length of RPA primers is usually no more than 50 nucleotides, preferably 20-35 nucleotides.RPA primers
Sense primer subsequently carries out in-vitro transcription, it is necessary in 5 ' end addition promoter sequences for amplified production.
The promoter can select T7, T3, SP6 etc., preferably T7 promoters.
G/C content is typically no less than 30% in RPA amplified production, no more than 70%, preferably 40%-60%；Length one
As be no more than 500 nucleotides, preferably 100-200 nucleotides.
It should be noted that the template (fragment to be checked) of RPA reactions of the present invention both can be DNA or RNA；When
When template is RNA, the RPA enzyme premixed liquids for RNA need to be used, to realize reversion recording function.
(2) above-mentioned amplified production is subjected to in-vitro transcription.
The reagent for participating in the reaction is：Above-mentioned RPA amplified productions (1 μ g), NTP mixed liquors (6.7mM), based on promoter
RNA polymerase premixed liquid (comes from NEB companies).
(3) the RNase activity based on the unique targeted rna activation of Cas13a albumen, carries out nucleotide sequence detection.
Participating in the reagent of the reaction includes：Above-mentioned in-vitro transcription product RNA, Cas13a albumen, guide RNA (crRNA, with
Cas13a albumen molar concentrations are identical), buffer solution (is adjusted) according to the Cas13a albumen in different bacterium source, signal reports
Reagent.
Wherein, the Cas13a albumen can come from different bacterium, including Leptotrichia shahii
(LshCas13a, dosage are 1 μM), Leptotrichia buccalis (LbuCas13a, dosage 40nM),
Leptotrichia wadei (LwCas13a, dosage 40nM) etc..Preferably LbuCas13a.
The concentration of the buffer solution needs to be adjusted according to the source of Cas13a albumen, generally there is following two selections：
Buffer A (50mM Tris-HCL pH7.5,200mM NaCl, 10mM MgCl for LshCas13a2) and be used for
LbuCas13a and LwCas13a buffer A (20mM HEPES pH7.0,50mM KCL, 5mM MgCl2)。
The structure of the guide RNA (crRNA) is 5 '-anchor series-go-ahead sequence -3 '.
Anchor series are different according to the source of Cas13a albumen, are divided into：For LshCas13a 5 '-
GGCCACCCCAAUAUCGAAGGGGACUAAAAC-3 ', for LbuCas13a 5 '-
GACCACCCCAAAAAUGAAGGGGACUAAAAC-3 ' and for LwCas13a 5 '-
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3’；
Go-ahead sequence is then 21-28 nucleotides, preferably 28 with the fragment match in targeted rna, length.
Further, the Cas13a albumen can be resistant to the mispairing of a base, therefore the present invention is used to detect single alkali
During base mutant nucleic acid fragments, it usually needs artificially add a mispairing, mismatch site one in advance in crRNA go-ahead sequence
As selection for mutating alkali yl 3 ' ends first to the 5th base, preferably second base.
The positive signal of the signal reports reagent has multiple choices, including fluorescence, light absorption value, chromogenic reaction etc., but believes
Number report condition be all based on Cas13a albumen RNase activity.Preferably, the positive signal is fluorescence signal (one
As select the RNAse Alert v2 from Thermo Scientific companies), both ends difference is added in reaction system
The RNA of fluorescent material and quencher is connected, when Cas13a albumen is with the help of crRNA, identification is with the targeting for targetting sequence
After RNA, the RNase activity that is activated can degrade RNA that this carries signal, so as to discharge fluorescence signal, realize detection.
By taking fluorescence signal as an example, it should calculate as follows：
Final fluorescence signal F=∑s【FExperimental group each time point-FExperimental group is initial-(FNegative control group each time point-FNegative control group is initial)】
Wherein, negative control group is that each experimental group is corresponding without the negative signal group for adding crRNA.
Method provided by the present invention is for qualitative, therefore, if final fluorescence signal is for more than 100 times higher than negative sample
It can determine whether that for the positive, more than 1000 times are judged as strong positive；If higher than negative sample 10 again between 100 times, it is possible to increase sample
Input amount detects again.
Further, in order to reduce the false positive in signal detection process, in the reaction system of step (3), can also draw
Enter RNase inhibitor (coming from NEB companies), and background RNA (extracting from HEK293FT cells), to exclude in reaction system
(imprudence introduces) influence of RNase to testing result beyond Cas13a albumen.
Such scheme of the present invention, the RNase not limited by sequence activated using Cas13a after targeted rna is identified are lived
Property, the signal reports RNA molecule that adds in degradation reaction system is realized final signal amplification and read.Detection sensitivity can
To reach 10-18Mol/L, i.e. A Moer ranks, specificity can reach for detecting the nucleic acid piece with single base difference
Section.
Above three step (reaction) is separately carried out, and required time is respectively 20-40 minutes, 120 minutes and 60-120 points
Clock, altogether or so about 5 hours of used time.
And preferably, when fragment to be checked is DNA, first step reaction temperature is 40 DEG C, remaining two step is 37 DEG C.
Therefore, second aspect, the present invention is three-in-one by the progress of above three step in order to simplify operating procedure, can be by instead
Significantly it is shorten between seasonable 120 minutes, records reaction signal within every 5 minutes during the course of the reaction.
When fragment to be checked is DNA, reaction temperature whole-process control is 37 DEG C；When fragment to be checked is DNA, reaction temperature
It is 37~40 DEG C to spend whole-process control.
The present invention, can be anti-at one by the way that three-step reaction is all added without complicated temperature control instrument or system
Answer in system, further streamline operation, and realize above-mentioned beneficial effect.
After " three-in-one ", the present invention further provides a kind of preferable reaction system (50 μ L)：
RPA primers (0.48 μM), the μ L of RPA enzymes premixed liquid 29.5 (coming from Twistdx companies), magnesium acetate (14mM), template
(fragment to be checked), NTP mixed liquors (2mM), the RNA polymerase premixed liquid (2 μ L, from NEB companies) based on promoter, Cas13a
Albumen (concentration is adjusted according to the Cas13a albumen in different bacterium source), guide RNA (crRNA, with Cas13a albumen mole
Concentration is identical), RNase inhibitor (3 μ L, from NEB companies), background RNA (200ng, extracts from HEK293FT cells), buffering
Liquid (is adjusted) according to the Cas13a albumen in different bacterium source, and (general select comes from Thermo to the μ L of signal reports reagent 2
RNAse the Alert v2,125nM of Scientific companies).
Final signal calculation formula and three-step reaction are identical when separately carrying out.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This area routine operation.
On the basis of common sense in the field is met, above-mentioned each optimum condition, it can be mutually combined, obtain specific embodiment party
Formula.
The beneficial effects of the present invention are：
The invention discloses a kind of nucleic acid detection technique based on CRIPSR-Cas13a, its most important mechanism is
Cas13a albumen can be identified with the help of guide RNA with targeting sequence RNA fragments, be subsequently activated not by sequence
The RNase activity of limitation, by signal reports molecule caused by adding RNA chain degradations in reaction system, final realize has
Target the signal identification of the RNA fragments of sequence.
The present invention not only expands the application of the detection technique by adding the nucleic acid amplification step based on RPA reactions
It can be detected to RNA and DNA greatly, while significantly improve the sensitivity of detection, the nucleic acid of A Moer ranks can be detected.
Because Cas13a albumen is when identification targets sequence, the mispairing of a base can be generally resistant to, the present invention passes through
Artificial base mismatch is added in guide RNA sequence in advance, reaching the specificity of this detection technique can identify with single
The nucleotide sequence of base difference.
Nucleic acid detection technique disclosed by the invention is different from the detection technique of conventional PCR-based technology, reacts whole nothing
Need complicated temperature control instrument or system；By the way that three-step reaction is all added in a reaction system, further simplify
Operating process, the nucleic acid fragment with distinguished sequence can be detected in two hours.
Inventor, as detection model, is pressed from KRAS wild type genes fragment and KRAS-G12D mutated genes fragment
Two sections of genes are have detected according to the nucleic acid detection technique of Invention Announce, about 50aM KRAS wild type gene fragments can be detected,
Simultaneously by guide RNA design, the DNA nucleotide sequences of a base difference, i.e. KRAS wild type genes piece can be distinguished
Section and KRAS-G12D mutated genes fragments.
Brief description of the drawings
Fig. 1 is the sensitivity test of the nucleic acid detection technique based on CRISPR-Cas13a.Pattern purpose nucleic acid sequence used
A fragment being classified as in KRAS genes, sequence 5 '-
ctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagct
aattcagaatcattttgtggacgaatatgatcc-3’.The crRNA sequences of synthesis be 5 '-
GACCACCCCAAAAATGAAGGGGACTAAAACccaccagctccaactaccacaagtttat-3’.Experimental result shows, with
Control group (sample is distilled water) is compared, and the nucleic acid detection technique disclosed by the invention based on CRISPR-Cas13a can detect
Go out about 50aM KRAS genetic fragments.
Fig. 2 is the specific test of the nucleic acid detection technique based on CRISPR-Cas13a.Pattern purpose nucleic acid sequence used
It is classified as KRAS wild type genes fragment and KRAS-G12D mutated genes fragments, sequence is respectively 5 '-
ctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagct
Aattcagaatcattttgtggacgaatatgatcc-3 ' and 5 '-
ctgctgaaaatgactgaatataaacttgtggtagttggagctgAtggcgtaggcaagagtgccttgacgatacagct
Aattcagaatcattttgtggacgaatatgatcc-3 ', wherein capitalization A is mutating alkali yl.Synthesis is directed to KRAS wild types
The crRNA-KRAS of genetic fragment, sequence 5 '-
GACCACCCCAAAAATGAAGGGGACTAAAACccaccTgctccaactaccacaagtttat-3’；Synthesize and be directed to KRAS
The crRNA-KRAS-G12D of mutated genes fragment, sequence 5 '-
GACCACCCCAAAAATGAAGGGGACTAAAACccatcTgctccaactaccacaagtttat-3’.Experimental result is shown, is led to
Cross the mode for artificially adding mispairing in crRNA in advance, the nucleic acid detection technique disclosed by the invention based on CRISPR-Cas13a
The DNA nucleotide sequences of a base difference, i.e. KRAS wild type genes fragment and KRAS-G12D mutated genes can be distinguished
Fragment.
Embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It is it will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this area
Art personnel can carry out various modifications and replacement in the case of without departing substantially from spirit of the invention and spirit to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1
The present embodiment is with KRAS genes, for illustrating detection of the detection method (three-in-one) to target gene, and
Test the sensitivity of this method.
First, the method that after annealing is synthesized by primer, purpose of the fragment as detection in KRAS genes is synthesized
The double-stranded DNA ,-ctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaa gagt of sequence 5 '
gccttgacgatacagctaattcagaatcattttgtggacgaatatgatcc-3’。
Secondly, RPA the primers ,-taatacgactcactataggctgctgaaaatgactgaata of sense primer 5 ' are synthesized
Taaactt-3 ' (wherein 5 ' 19 nucleotides in end are T7 promoters) ,-ggatcatattcgtccacaaaatg of anti-sense primer 5 '
attct-3’。
Again, by the method for in-vitro transcription, the crRNA for KRAS genetic fragments has been synthesized, sequence 5 '-
GACCACCCCAAAAATGAAGGGGACTAAAACccaccagctccaactaccacaagttt at-3 ', wherein capitalization are
Anchor series, lowercase are go-ahead sequence, identify 5 '-ataaacttgtggtagttggagctggt in KRAS genetic fragments
gg-3’。
Finally, 50 μ L reaction systems are built：
RPA primers (each 0.48 μM of upstream and downstream primer)；
RPA enzymes premixed liquid (29.5 μ L, from Twistdx companies)；
Magnesium acetate (14mM)；
KRAS genetic fragments (are diluted) by various concentrations；
NTP mixed liquors (2mM)；
RNA polymerase premixed liquid (2 μ L, from NEB companies) based on T7 promoters；
LbuCas13a albumen (40nM)；
For the crRNA (40nM) of KRAS genetic fragments；
RNase inhibitor (3 μ L, from NEB companies)；
Background RNA (200ng, extracts from HEK293FT cells)；
Buffer solution (20mM HEPES pH7.0,50mM KCL, 5mM MgCl2)；
The μ L of RNAse Alert v2 2 (125nM, from Thermo Scientific companies).
The KRAS genetic fragments experimental group of each concentration should set the corresponding negative control group for being not added with crRNA.
After reaction system is mixed, multi-function microplate reader is placed into, it is 37 DEG C to set reaction condition, excitation wavelength
488nm, the reaction time be 120 minutes, during the course of the reaction every 5 minutes record 520nm at fluorescence signal value, eventually through with
Lower formula calculates final signal：
Final fluorescence signal F=∑s【Each time point-F the experimental groups of F experimental groups initially-(F negative control groups each time
Point-F negative control groups are initial)】
Negative control group is that each experimental group is corresponding without the negative signal group for adding crRNA.
Experimental result is shown, disclosed by the invention to be based on CRISPR-Cas13a compared with control group (sample is distilled water)
Nucleic acid detection technique can detect about 50aM KRAS genetic fragments (Fig. 1).
Embodiment 2
The present embodiment is using KRAS genes and KRAS-G12D mutators as target gene, for illustrating detection of the present invention
Detection of the method (three-in-one) to target gene, and test the specificity of this method.
First, the method that after annealing is synthesized by primer, KRAS genetic fragments and KRAS-G12D mutation have been respectively synthesized
Purpose double-stranded DNA of the fragment as detection, sequence is respectively 5 '-
ctgctgaaaatgactgaatataaacttgtggtagttggagctggtggcgtaggcaagagtgccttgacgatacagct
Aattcagaatcattttgtggacgaatatgatcc-3 ' and 5 '-
ctgctgaaaatgactgaatataaacttgtggtagttggagctgAtggcgtaggcaagagtgccttgacgatacagct
Aattcagaatcattttgtggacgaatatgatcc-3 ', wherein capitalization A is mutating alkali yl.
Secondly, RPA primers have been synthesized, sense primer 5 '-
(wherein 5 ' 19 nucleotides in end start taatacgactcactataggctgctgaaaatgactgaatataaactt-3 ' for T7
Son) ,-ggatcatattcgtccacaaaatgattct-3 ' of anti-sense primer 5 '.
Again, because Cas13a albumen can generally be resistant to the mispairing of a base, thus we by in-vitro transcription and
The method for adding artificial mispairing in advance, has synthesized the crRNA-KRAS for KRAS wild type gene fragments, and sequence 5 '-
GACCACCCCAAAAATGAAGGGGACTAAAACccaccTgctccaactaccacaagttt at-3 ', wherein capitalization are
Anchor series and the mispairing artificially added, lowercase are go-ahead sequence, 5 ' in identification KRAS wild type gene fragments-
ataaacttgtggtagttggagctggtgg-3’；The crRNA-KRAS- for KRAS type genetic fragment is synthesized
The G12D ,-GACCACCCCAAAAATGAAGGGGACTAAAACccatcTgctccaactaccacaagttt at-3 ' of sequence 5 ',
Wherein capitalization is anchor series and the mispairing artificially added, and lowercase is go-ahead sequence, identifies KRAS-G12D saltant types
5 '-ataaacttgtggtagttggagctgatgg-3 ' in genetic fragment.
Finally, 50 μ L reaction systems are built：
RPA primers (each 0.48 μM of upstream and downstream primer)；
RPA enzymes premixed liquid (29.5 μ L, from Twistdx companies)；
Magnesium acetate (14mM)；
KRAS genetic fragments or KRAS-G12D mutant fragments (50nM)；
NTP mixed liquors (2mM)；
RNA polymerase premixed liquid (2 μ L, from NEB companies) based on T7 promoters；
LbuCas13a albumen (40nM)；
CrRNA-KRAS or crRNA-KRAS-G12D (40nM)；
RNase inhibitor (3 μ L, from NEB companies)；
Background RNA (200ng, extracts from HEK293FT cells)；
Buffer solution (20mM HEPES pH7.0,50mM KCL, 5mM MgCl2)；
The μ L of RNAse Alert v2 2 (125nM, from Thermo Scientific companies)；
The corresponding negative control group for being not added with crRNA is set.
After reaction system is mixed, multi-function microplate reader is placed into, it is 37 DEG C to set reaction condition, excitation wavelength
488nm, the reaction time be 120 minutes, during the course of the reaction every 5 minutes record 520nm at fluorescence signal value, eventually through with
Lower formula calculates final signal：
Final fluorescence signal F=∑s【Each time point-F the experimental groups of F experimental groups initially-(F negative control groups each time
Point-F negative control groups are initial)】
Wherein, negative control group is that each experimental group is corresponding without the negative signal group for adding crRNA.
Experimental result is shown, disclosed by the invention to be based on by way of artificially adding mispairing in crRNA in advance
CRISPR-Cas13a nucleic acid detection technique can distinguish the DNA nucleotide sequences of a base difference, i.e. KRAS wild types base
Because of fragment and KRAS-G12D mutated genes fragment (Fig. 2).
It should be appreciated that after equal proportion expansion is carried out to the dosage of above-described embodiment agents useful for same or raw material or is reduced
Technical scheme, it is substantially identical with above-described embodiment.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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Claims (10)
1. a kind of detection method of the nucleic acid specific fragment based on CRISPR-Cas13a, it is characterised in that by nucleic acid piece to be checked
Duan Jinhang is expanded and is obtained transcription product, for the targeted rna sequences Design guide RNA of target gene, and in detection architecture
The signal reports RNA molecule of addition, utilize not limited by sequence of being activated after targeted rna sequence is identified of Cas13a albumen
RNase activity, the amplification and reading of signal are realized, and then realize the detection to target gene.
2. according to the method for claim 1, it is characterised in that the nucleic acid fragment to be checked can be DNA or RNA：When for RNA
When, it need to first carry out reverse transcription in the amplification stage.
3. method according to claim 1 or 2, it is characterised in that the Cas13a albumen is from Leptotrichia
Shahii LshCas13a or LbuCas13a from Leptotrichia buccalis or from Leptotrichia
Wadei LwCas13a.
4. according to the method for claim 3, it is characterised in that the structure of the guide RNA is 5 '-anchor series-guide
Sequence -3 '；
Wherein, the anchor series are depending on the source of Cas13a albumen：
When Cas13a albumen is the LshCas13a ,-GGCCACCCCAAUAUCGAAGGGGACUAAAAC-3 ' of anchor series 5 '；
When Cas13a albumen is the LbuCas13a ,-GACCACCCCAAAAATGAAGGGGACTAAAAC-3 ' of anchor series 5 '；
When Cas13a albumen is the LwCas13a ,-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC- of anchor series 5 '
3’；
The go-ahead sequence and the fragment match in targeted rna, length is 21-28 nucleotides.
5. according to the method any one of claim 1,2,4, it is characterised in that the signal reports RNA molecule is tool
There is the RNA molecule of signal reports function, when RNA sequence therein is degraded, positive signal can be reported and be detected.
6. according to the method for claim 5, it is characterised in that methods described specifically comprises the following steps：
(1) RPA technologies carry out nucleic acid amplification reaction to fragment to be checked, obtain RPA amplified productions：
Reaction system is 50 μ L：RPA upstream and downstream primers each 2.4 μ L of 10 μM of concentration, final concentration of 0.48 μM；Fragment 13.2 to be checked
μL；The μ L of RPA enzymes premixed liquid 29.5；Magnesium acetate 2.5 the μ L, final concentration of 14mM of 280mM concentration；
Wherein, of length no more than 50 nucleotides of RPA primers, the end of sense primer 5 ' of RPA primers add promoter sequence, with
In-vitro transcription is subsequently carried out for amplified production；
The promoter is T7, T3 or SP6；
(2) above-mentioned amplified production is subjected to in-vitro transcription：
Reaction system is 30 μ L：The above-mentioned μ g of RPA amplified productions 1；NTP mixed liquors 10 the μ L, final concentration of 6.7mM of 20mM concentration；
The μ L of RNA polymerase premixed liquid 2 based on promoter；
(3) the RNase activity based on the unique targeted rna activation of Cas13a albumen, carries out nucleotide sequence detection：
Reaction system is 50 μ L：Above-mentioned in-vitro transcription product RNA30 μ L；The μ L of Cas13a albumen 2；The μ L of guide RNA 2；The μ of buffer solution 5
L；The μ L of signal reports reagent 2；The μ L of water 9；
Wherein, when Cas13a albumen is LshCas13a, its dosage is 1 μM, and guide RNA dosages are also 1 μM, and buffer solution is
50mM Tris-HCL pH7.5,200mM NaCl, 10mM MgCl2；
When Cas13a albumen is LbuCas13a or LwCas13a, its dosage is 40nM, and guide RNA dosages are also 40nM, buffering
Liquid is 20mM HEPES pH7.0,50mM KCL, 5mM MgCl2。
7. according to the method for claim 6, it is characterised in that when single base mutation nucleic acid fragment need to be detected, in guide
Add a mispairing in go-ahead sequence in RNA in advance, what mismatch site was typically chosen in 3 ' ends of mutating alkali yl first arrives
5th base, preferably second base.
8. the method according to claim 6 or 7, it is characterised in that in the reaction system of step (3), can also introduce RNA
Enzyme inhibitor and background RNA.
9. according to the method for claim 5, it is characterised in that methods described specifically includes：
(1) the μ L of detection architecture 50 are built：0.48 μM of RPA primers, μ L of RPA enzymes premixed liquid 29.5, magnesium acetate 14mM, fragment to be checked,
NTP mixed liquors 2mM, μ L, the Cas13a albumen of RNA polymerase premixed liquid 2 based on promoter, guide RNA, the μ of RNase inhibitor 3
L, background RNA 200ng, buffer solution, the μ L of signal reports reagent 2；
Wherein, when Cas13a albumen is LshCas13a, its dosage is 1 μM, and guide RNA dosages are also 1 μM, and buffer solution is
50mM Tris-HCL pH7.5,200mM NaCl, 10mM MgCl2；
When Cas13a albumen is LbuCas13a or LwCas13a, its dosage is 40nM, and guide RNA dosages are also 40nM, buffering
Liquid is 20mM HEPES pH7.0,50mM KCL, 5mM MgCl2；
(2) positive signal for building system is detected, judges whether measuring samples contain target gene.
10. according to the method for claim 9, it is characterised in that the molar concentration phase of the guide RNA and Cas13a albumen
Together.
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