CN109097448A - A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme - Google Patents
A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme Download PDFInfo
- Publication number
- CN109097448A CN109097448A CN201810922821.9A CN201810922821A CN109097448A CN 109097448 A CN109097448 A CN 109097448A CN 201810922821 A CN201810922821 A CN 201810922821A CN 109097448 A CN109097448 A CN 109097448A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- enzyme
- isothermal duplication
- nicking enzyme
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of isothermal duplication nucleic acid detection method based on unwindase and nicking enzyme includes the following steps: that S1. according to target nucleic acid sequence designs specificity amplification primer and with the Taqman probe of nicking enzyme recognition site；S2. by sample nucleic acid, enzyme treated to obtain single-stranded target nucleic acid-templated；S3. by the nucleic acid-templated specific binding of single-stranded target obtained by Taqman probe obtained by S1 and S2, specific binding product is obtained, specifically binds product, and real-time detection fluorescence signal using nicking enzyme digestion.Unwindase and nicking enzyme are used in combination the present invention, design has the Taqman probe of nicking cleavage sites, without amplifying a large amount of nucleic acid products, it only needs to generate appropriate single-stranded template, then the probe in conjunction with target site is constantly cut with nicking enzyme to be detected, it is the shortcomings that laboratory pollution being be easy to cause to avoid isothermal duplication, easy to operate, quick, it is suitable for field quick detection.
Description
Technical field
The present invention relates to molecular biology nucleic acid detection technique fields, and in particular, to one kind is based on unwindase and nicking
The isothermal duplication nucleic acid detection method and kit of enzyme.
Background technique
Nucleic acid detection technique is widely used to prevention and control of food inspection, environment measuring and disease etc..Its
It is most wide that the high sensitivity of middle polymerase chain reaction (Polymerase Chain Reaction, PCR) becomes current use
General DNA cloning method, however existing Standard PCR technology needs thermal denaturation repeatedly to unlock DNA double chain, in application according to
Rely the thermal cycle in high quality, and expanding effect is vulnerable to multifactor impact, reaction time length etc..Isothermal amplification technique is nucleic acid body
Outer amplification technique, reaction process maintains at a constant temperature always, by the enzyme and respectively specificity that add different activities
Primer come achieve the purpose that Rapid nucleic acid expand.Isothermal amplification technique greatly simplifies instrument requirements, and the reaction time compares PCR skill
Art greatly shortens, and specificity is high, low in cost, is more able to satisfy the demand quickly detected.Common nucleic acid isothermal amplification technology
There are loop-mediated isothermal amplification technique (LAMP), strand displacement amplification (SDA), the isothermal amplification technique (HAD) for relying on unwindase, rolling ring
Amplification technique (RCA) etc..
The constant-temperature amplification (LAMP) that ring mediates is although method amplification efficiency is high, and high specificity, this method is to design of primers
Requirement it is especially high, design of primers is complicated, four primers that can identify six specific regions of target sequence is needed, in same reaction
The difficulty that Multiple detection is carried out in pipe is bigger；The target position sequence length that LAMP is identified is not too much, can not to long segment into
Row amplification；Because having higher sensitivity, strict partition need to be required operation, pollution is otherwise highly prone to and generates false positive results
Deng.
Rolling circle amplification (Rolling Circle Amplification, RCA) is under steady temperature with single stranded circle DNA
Pass through the rolling of primer and the annealing progress of template ring in the case where the Phi29DNA for having strong strand-displacement activity polymerize enzyme effect for template
Ring type DNA synthesis, RCA high amplification efficiency make its can become signal amplification means, but in RCA reaction process not at
There may be some background signals for the template DNA or RNA of the padlock probe of ring and unbonded probe.
Strand displacement isothermal duplication (SDA) method is based on having in the restriction nuclease that is modified by sulphation at target DNA both ends
Enzyme recognition sequence is cut, endonuclease makes a breach chain DNA in its recognition site, and archaeal dna polymerase extends notch 3 ' and holds and replace
A DNA chain is changed, is set to that the DNA changed is single-stranded to extend into double-strand in conjunction with primer and by archaeal dna polymerase, to realize
Amplification.But the technology, which combines primer and target sequence to form 5 ' and hold, particular/special requirement.SDA needs one before isothermal duplication
Heat denatured opens the step of double-strand, since Klenow Fragment exo- is without thermal stability, it is necessary to after target DNA denaturation
It can be added in system, easily cause pollution.In order to introduce the distinguished sequence of nicking restriction endonuclease identification, it is usually also required to 3 or 3
The above primer of item.
In addition, also there is the method for relying on unwindase or recombinase to carry out isothermal duplication, using unwindase or recombinase by DNA
Double-strand is opened, and into amplification cycles, these methods require to carry out exponential amplification to target sequence, to detect, this just and
Other PCR amplifications are identical, can all generate a large amount of pcr amplification products, in negligence of operation or without the experiment of advanced ventilation equipment
Room or the diffusion without easily causing nucleic acid aerosol in the small-size laboratory of functional areas segmentation, so as to cause serious dirt
Dye.This point also limit isothermal duplication at the scene quickly detect (POCT) in application.
Therefore, it needs to develop a kind of new method suitable for field quick detection nucleic acid at present, to reduce operating process
The pollution of middle generation influences detection of nucleic acids accuracy and result in turn.
Summary of the invention
The purpose of the invention is to overcome the above-mentioned deficiency of the prior art, provide a kind of based on unwindase and nicking enzyme
Isothermal duplication nucleic acid detection method, this method can effectively solve laboratory pollution caused by a large amount of nucleic acid products of conventional amplification and ask
Topic, principle is simple, easy to operate, high specificity, high sensitivity.
Another object of the present invention is to provide a kind of isothermal duplication nucleic acid detection reagent based on unwindase and nicking enzyme
Box.
To achieve the goals above, the present invention is achieved by following scheme:
A kind of isothermal duplication nucleic acid detection method based on unwindase and nicking enzyme, includes the following steps:
S1. it is visited according to target nucleic acid sequence design specificity amplification primer and the Taqman with nicking enzyme recognition site
Needle；
S2. by sample nucleic acid, enzyme treated to obtain single-stranded target nucleic acid-templated；
S3. by the nucleic acid-templated specific binding of single-stranded target obtained by Taqman probe obtained by S1 and S2, specific knot is obtained
Product is closed, specifically binds product, and real-time detection fluorescence signal using nicking enzyme digestion.
The present invention has the specificity T aqman probe of nicking enzyme recognition site, probe and target single-stranded template by design
It can be cut by nicking digestion in conjunction with after, because Tm value reduces after probe is cut off, will fall off, fluorophor and quench from target site
The group that goes out separation, generates fluorescence signal.And new probe will continue to be specifically bound to target single-stranded template, again by nicking enzyme
Cutting generates fluorescence, and signal persistently carries out, and generates a large amount of fluorescence signals.
Unwindase and nicking enzyme are used in combination the present invention, and specificity T aqman of the design with nicking cleavage sites is visited
Needle does not need to amplify a large amount of nucleic acid products, it is only necessary to generate appropriate single-stranded template, then constantly be cut and target position with nicking enzyme
The shortcomings that probe that point combines can be detected, can easily cause laboratory pollution to avoid isothermal duplication, easy to operate,
Quickly.
When sample is double-strandednucleic acid, step S2 double center chain sample of nucleic acid is enzyme treated to obtain the list for expanding and detecting
Chain target nucleic acid template.Preferably, enzyme described in step S2 is one of helicase, strand displacement enzyme, recombinase or a variety of.More
Preferably, enzyme described in step S2 is UvrD unwindase and strand displacement archaeal dna polymerase.
When sample is single stranded RNA, it is preferable that RNA sample forms RNA- under the action of reverse transcriptase in step S2
DNA hybridization double-strand, then the RNA chain in RNA-DNA hybridization system is decomposed using RNaseH, form the single-stranded target of amplification and detection
It is nucleic acid-templated.I.e. enzyme described in step S2 is reverse transcriptase and RNaseH at this time.
Preferably, when single-stranded target is nucleic acid-templated very few, by into reaction system be added specificity amplification primer and
Polymerase, under the enzyme alternating action described in specificity amplification primer, polymerase and step S2 that keeps single-stranded target nucleic acid-templated constantly
Amplification.Generate target single-chain fragment by enzymatic treatment double-strandednucleic acid described in step S2, target single-chain fragment in specific primer and
Further synthesize double-strandednucleic acid under the action of polymerase, the double-strandednucleic acid of synthesis again constantly the enzyme described in step S2 under the action of produce
Raw target single-chain fragment；It moves in circles, constantly generates single-stranded target segment.The generation of target single-stranded template need to only lead at a constant temperature
It crosses a pair of of specific primer and enzyme carries out effect and can be obtained, technical principle is simple, easy to operate, high specificity.
Preferably, the detailed process of the step S3 are as follows: Taqman probe and the nucleic acid-templated specific binding of single-stranded target,
In the nicking enzyme recognition site of probe, probe falls off from single stranded nucleic acid template nicking enzyme effect after being cut off, and generates fluorescence
Signal；New probe in conjunction with single stranded nucleic acid template, is broken by nicking digestion again, falls off from single-stranded template again, generates fluorescence
Signal；The combination-of probe cuts off-and the process of falling off repeats, continuous generation fluorescence signal.It is fixed by ABI7500 real-time fluorescence
It measures PCR instrument and real-time monitoring is carried out to the fluorescence signal of generation, detect first order fluorescence signal per minute.
Preferably, the condition of the detection is 50~60 DEG C of reaction temperature, detection time 90min.
The present invention is also claimed a kind of isothermal duplication kit for detecting nucleic acid based on unwindase and nicking enzyme, including with
The each component of lower final concentration: 8~12 μM of specificity amplification primers, 8~12 μM of Taqman probes, 180~220ng/ μ L enzyme, 4~
6 μ g/ μ L T4 gene, 32 albumen, 5~10U/ μ L polymerase, 8~12U/ μ L nicking enzyme, 8~12mM dNTP.
Preferably, the isothermal duplication kit for detecting nucleic acid further includes 5 × buffer；5 × the buffer include with
The each component of lower final concentration: 25mM Tris-HCl, pH7.8,125mM NaCl, 50mM (NH4)2SO4, 250mM MgCl2,
2.5mg/mL BSA, 1M glycine betaine, 0.5%Tween20.
Preferably, the polymerase is the Klenow segment of e. coli dna polymerase I, T7DNA polymerase, BST polymerize
Any one of enzyme.
It is highly preferred that the polymerase is BST polymerase.
Preferably, the nicking enzyme be Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI,
Any one of Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII.
It is highly preferred that the nicking enzyme is Nt.BstNBI.
Compared with prior art, the invention has the following advantages:
(1) detection method of the present invention does not need to carry out massive amplification to target sequence, as long as obtaining appropriate single-stranded template,
It can produce a large amount of fluorescence signal by continuous cutting by Taqman probe to be detected, avoid conventional fluorescent detection method
In background segment interference problem, greatly reduce a possibility that laboratory pollution generates, laboratory that can be common in condition
Carry out experimental implementation.
(2) detection kit principle of the present invention is easily understood, and it is convenient to operate, and by the sample nucleic acid of extraction and need to only be somebody's turn to do
Kit reaction system mixes, and can be reacted automatically at 55 DEG C, real-time detection fluorescence signal, rapid sensitive, can be used for existing
Field quickly detection.
Detailed description of the invention
Fig. 1 is the basic schematic diagram that detection method of the present invention detects DNA.
Fig. 2 is the basic schematic diagram that detection method of the present invention detects RNA.
Fig. 3 is the testing result of human genome GAPDH gene in the embodiment of the present invention 1.
Fig. 4 is the testing result that clostridium difficile TcdB produces virus gene in the embodiment of the present invention 2.
Fig. 5 is that detection of pathogens specificity analyzes result in the embodiment of the present invention 3.
Fig. 6 is the mRNA testing result of source of people GAPDH gene in the embodiment of the present invention 4.
Specific embodiment
With reference to the accompanying drawings of the specification and specific embodiment is made the present invention and is further elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
1 human genome GAPDH genetic test of embodiment
1, prepared by detection kit
(1) specific primer design
For human genome GAPDH gene, comprehensively considers melting temperature, G/C content, primer length, primer construction, draws
The multiple parameters such as object dimer design specific primer pair, specific as follows shown.
Amplified fragments (SEQ ID NO:1):
CGGGTGATGCTTTTCCTAGATTATTCTCTGGTAAATCAAAGAAGTGGGTTTATGGAGGTCCTCTTGTGT
CCCCTCCCCGCAGAGGTGTGGTGGCTGTGGCATGGTGCCAAGCCGGGAGAAGCTGAGTCATGGGTAGTTGGAAAAGG
ACATTTCCACCGCAAAATGGCCCCTCTG
Upstream primer Primer F (SEQ ID NO:2): 5 '-CGGGTGATGCTTTTCCTAGATT-3 '
Downstream primer Primer R (SEQ ID NO:3): 5 '-CAGAGGGGCCATTTTGCGG-3 '
(2) specificity T aqman probe designs
Position is identified containing nicking enzyme according to the nucleic acid-templated design specificity T aqman probe of single-stranded target, and in probe sequence
Point.5 ' ends of probe sequence are connected with fluorophor, and 3 ' ends are connected with quenching group.When probe is in normal length, excitation
The transmitting light of fluorophor can be quenched group absorptions when light irradiates, and can't detect fluorescence signal at this time；When probe is cut off
Afterwards, it because Tm value reduces, will fall off from target site, fluorophor and quenching group separation generate fluorescence signal.Nicking enzyme
The characteristics of be to be cut in recognition site, but a chain in double-strand can only be cut off.I.e. when probe specificity is integrated to list
After in chain target nucleic acid template, double-strand is formed, nicking enzyme recognition site cuts off probe, and probe is nucleic acid-templated de- from single-stranded target
From generation fluorescence signal.
Probe sequence probe (SEQ ID NO:4): 5 '-FAM-CTGAGTCATGGGTAGTTGGAAA-BHQ-3 '
(3) reaction system is prepared
It is poly- containing 45 × buffers of μ L, 2 μ L 10mM dNTP, 1 μ L 8U/ μ L BST in the amplification reaction system of every 20 μ L
Synthase, 0.5 μ L 200ng/ μ L UvrD unwindase, 0.5 μ L, 5 μ g/ μ L T4 gene, 32 albumen (gp32), 0.6 μ L 10U/ μ L
Nt.BstNBI, 1.5 μ L, 10 μM of Primer F, 1.5 μ L, 10 μM of Primer R, 1 μ L, 10 μM of probe, 2 μ L sample DNAs,
5.4μL ddH2O。
Wherein 5 × buffer is by 25mM Tris-HCl (pH7.8), 125mM NaCl, 50mM (NH4)2SO4, 250mM
MgCl2, 2.5mg/mL BSA, 1M glycine betaine, 0.5%Tween20 composition.
2, human genome GAPDH genetic test, specific steps are as follows:
(1) the human archeocyte HEK293 for collecting culture, conventionally extracts genomic DNA.
(2) by extracting genome DNA liquid be diluted to 1 μ g/ μ L of various concentration, 100ng/ μ L, 10ng/ μ L, 1ng/ μ L,
0.1ng/ μ L takes 2 μ L DNA dilutions to be added in the amplification system of 20 μ L respectively, and the DNA concentration in each amplified reaction is
2 μ L ddH are added in 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, blank control group2O。
(3) amplification reaction system for preparing 20 μ L, mixes on the oscillator, is put into ABI7500 real-time fluorescence quantitative PCR instrument
In, setting reaction temperature is 55 DEG C, and the reaction time is 90 minutes, detects first order fluorescence signal per minute.
Testing result is as shown in figure 3, detection method of the present invention can be detected from human genome as the result is shown
GAPDH gene, Monitoring lower-cut are 0.1ng/ μ L DNA.
2 clostridium difficile of embodiment produces virus gene TcdB detection
1, prepared by detection kit
(1) specific primer and probe design
Virus gene TcdB design specific primer and probe, institute specific as follows are produced for detection target sequence clostridium difficile
Show.
Amplified fragments (SEQ ID NO:5):
CACAAGTGGTAGAAGAAAGGATTGAAGAAGCTAAAAGCTTAACTTCTGACTCTATTAATTATATAAAGA
ATGAATTTAAACTAATAGAATCTATTTCTGATGCACTATACGATTTAAAACAACAGAATGAATTAGAAGAGTCTCAT
TTTATATCTTTTGAGGATATATCGGAGACTGATGAAGGCT
Upstream primer Primer F (SEQ ID NO:6): 5 '-CACAAGTGGTAGAAGAAAGGATTG-3 '
Downstream primer Primer R (SEQ ID NO:7): 5 '-AGCCTTCATCAGTCTCCGATA-3 '
Probe sequence probe (SEQ ID NO:8): 5 '-FAM-AAGAGTCTCATTTTATATCTTTT-BHQ-3 '
(2) reaction system is prepared
It is poly- containing 45 × buffers of μ L, 2 μ L 10mM dNTP, 1 μ L 8U/ μ L BST in the amplification reaction system of every 20 μ L
Synthase, 0.5 μ L 200ng/ μ L UvrD unwindase, 0.5 μ L, 5 μ g/ μ L T4 gene, 32 albumen (gp32), 0.6 μ L 10U/ μ L
Nt.BstNBI, 1.5 μ L, 10 μM of Primer F, 1.5 μ L, 10 μM of Primer R, 1 μ L, 10 μM of probe, 2 μ L sample DNAs,
5.4μL ddH2O。
Wherein 5 × buffer is by 25mM Tris-HCl (pH7.8), 125mM NaCl, 50mM (NH4)2SO4, 250mM
MgCl2, 2.5mg/mL BSA, 1M glycine betaine, 0.5%Tween20 composition.
2, clostridium difficile produces virus gene TcdB detection, specific steps are as follows:
(1) clostridium difficile culture is carried out in anaerobic box, then conventionally extracts the genome of clostridium difficile
DNA, be detected clostridium difficile bacterium solution concentration be respectively 150CFU/mL, 300CFU/mL, 500CFU/mL, 1500CFU/mL,
5000CFU/mL。
(2) 2 μ L DNA are taken to be added to the amplification body of 20 μ L respectively the clostridium difficile extracting genome DNA liquid of various concentration
In system, 2 μ L ddH are added in blank control group2O。
(3) amplification reaction system for preparing 20 μ L, mixes on the oscillator, is put into ABI7500 real-time fluorescence quantitative PCR instrument
In, setting reaction temperature is 55 DEG C, and the reaction time is 90 minutes, detects first order fluorescence signal per minute.
Testing result as shown in figure 4, Monitoring lower-cut be 300CFU/mL, illustrate that the method for the invention can meet pathogen
The primary demand of detection.
The analysis of 3 detection of pathogens specificity of embodiment
Using kit described in embodiment 2 and detection method, select Escherichia coli, salmonella, staphylococcus aureus,
Shigella bogdii, shigella dysenteriae, clostridium bifermentans, C.perfringens and atoxigenic clostridium difficile are inspection
Test sample sheet, detection clostridium difficile produce virus gene TcdB, analyze kit described in embodiment 2 and detection method in detection of pathogens side
The specificity in face, specific experiment setting are as shown in table 1.
1 detection of pathogens specificity of table analyzes experimental setup
| Experimental group | Pathogen | Concentration |
| 1 | Produce malicious clostridium difficile | 5000CFU/mL |
| 2 | Produce malicious clostridium difficile | 500CFU/mL |
| 3 | Escherichia coli, salmonella | 106CFU/mL |
| 4 | Staphylococcus aureus, Shigella bogdii | 106CFU/mL |
| 5 | Shigella dysenteriae, clostridium bifermentans | 106CFU/mL |
| 6 | C.perfringens, atoxigenic clostridium difficile | 106CFU/mL |
It after amplification reaction system prepares, mixes, is put into ABI7500 real-time fluorescence quantitative PCR instrument on the oscillator, is arranged
Reaction temperature is 55 DEG C, and the reaction time is 90 minutes, detects first order fluorescence signal per minute.
As a result as shown in figure 5, being added in the detection pipe for producing malicious clostridium difficile, there is hyperfluorescence signal, and other bacteriums are added
Basic unstressed configuration signal in the pipe of DNA, wherein also include atoxigenic difficile dna because atoxigenic clostridium difficile without
TcdB gene, therefore be not in fluorescence signal.The above result shows that detection method of the present invention has good specificity,
Special target gene can be identified.
Embodiment 4RNA detection
1, prepared by detection kit
(1) specific primer design
For the mRNA of human source gene GAPDH, design specific primer to and probe, it is specific as follows shown in.
Amplified fragments (SEQ ID NO:9):
ACCCATTCATTGACCTTGATTACATGGTTTACATGTTCCAGTACGACTCCACCCATGGAAAGTACAAGG
GTGAGGTTAAGGCAGAAGGCGGCAAACTGGTCATTGATGGTCATGCAATCACAGTCTATAGCGAGAGGGACCCAGCC
AACATTAAGTGGGGTGATGCAGGTGCTACTTATGTTGTGGAGTCTAC
Upstream primer Primer F (SEQ ID NO:10): 5 '-ACCCATTCATTGACCTTGAT-3 '
Downstream primer Primer R (SEQ ID NO:11): 5 '-GTAGACTCCACAACATAAGT-3 '
Probe sequence probe (SEQ ID NO:12): 5 '-FAM-TGGAGTCGTACTGGAACATGTA-BHQ-3 '
(2) amplification reaction system is prepared
It is poly- containing 45 × buffers of μ L, 2 μ L 10mM dNTP, 1 μ L 8U/ μ L BST in the amplification reaction system of every 20 μ L
Synthase, 0.5 μ L 200ng/ μ L UvrD unwindase, 0.5 μ L, 5 μ g/ μ L T4 gene, 32 albumen (gp32), 0.6 μ L 10U/ μ L
Nt.BstNBI, 1.5 μ L 10U/ μ L AMV reverse transcriptases, 1 μ L 5U/ μ L RNaseH, 1.5 μ L, 10 μM of Primer F, 1.5 μ
L10 μM of Primer R, 1 μ L, 10 μM of probe, 2 μ L sample DNAs, 2.9 μ L ddH2O。
Wherein 5 × buffer is by 25mM Tris-HCl (pH7.8), 125mM NaCl, 50mM (NH4)2SO4, 250mM
MgCl2, 2.5mg/mL BSA, 1M glycine betaine, 0.5%Tween20 composition.
2, the mRNA detection of human source gene GAPDH, specific steps are as follows:
(1) the human archeocyte HEK293 for collecting culture, conventionally extracts the total serum IgE of HEK293 cell.
(2) RNA extracting solution is diluted to various concentration 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, total serum IgE is heated in 70 DEG C
5min is placed on 3min on ice, and 2 μ L is taken to be added in the amplification system of 20 μ L respectively, and the rna content in each amplified reaction is
2 μ L ddH are added in 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, blank control group2O。
(3) amplification reaction system for preparing 20 μ L, mixes on the oscillator, is put into ABI7500 real-time fluorescence quantitative PCR instrument
In, setting reaction temperature is 52 DEG C, and the reaction time is 90 minutes, detects first order fluorescence signal per minute.
Testing result is as shown in fig. 6, detection method of the present invention can effectively detect human source gene GAPDH as the result is shown
MRNA, target gene can be detected in 1ng total serum IgE.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art
Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention
Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle
Within the scope of.
Sequence table
<110>the biological Co., Ltd of Shenzhen's match wise man
<120>a kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 174
<212> DNA
<213>people (human)
<400> 1
cgggtgatgc ttttcctaga ttattctctg gtaaatcaaa gaagtgggtt tatggaggtc 60
ctcttgtgtc ccctccccgc agaggtgtgg tggctgtggc atggtgccaa gccgggagaa 120
gctgagtcat gggtagttgg aaaaggacat ttccaccgca aaatggcccc tctg 174
<210> 2
<211> 22
<212> DNA
<213>people (human)
<400> 2
cgggtgatgc ttttcctaga tt 22
<210> 3
<211> 19
<212> DNA
<213>people (human)
<400> 3
cagaggggcc attttgcgg 19
<210> 4
<211> 22
<212> DNA
<213>people (human)
<400> 4
ctgagtcatg ggtagttgga aa 22
<210> 5
<211> 186
<212> DNA
<213>clostridium difficile (Clostridium difficile)
<400> 5
cacaagtggt agaagaaagg attgaagaag ctaaaagctt aacttctgac tctattaatt 60
atataaagaa tgaatttaaa ctaatagaat ctatttctga tgcactatac gatttaaaac 120
aacagaatga attagaagag tctcatttta tatcttttga ggatatatcg gagactgatg 180
aaggct 186
<210> 6
<211> 24
<212> DNA
<213>clostridium difficile (Clostridium difficile)
<400> 6
cacaagtggt agaagaaagg attg 24
<210> 7
<211> 21
<212> DNA
<213>clostridium difficile (Clostridium difficile)
<400> 7
agccttcatc agtctccgat a 21
<210> 8
<211> 23
<212> DNA
<213>clostridium difficile (Clostridium difficile)
<400> 8
aagagtctca ttttatatct ttt 23
<210> 9
<211> 193
<212> DNA
<213>people (human)
<400> 9
acccattcat tgaccttgat tacatggttt acatgttcca gtacgactcc acccatggaa 60
agtacaaggg tgaggttaag gcagaaggcg gcaaactggt cattgatggt catgcaatca 120
cagtctatag cgagagggac ccagccaaca ttaagtgggg tgatgcaggt gctacttatg 180
ttgtggagtc tac 193
<210> 10
<211> 20
<212> DNA
<213>people (human)
<400> 10
acccattcat tgaccttgat 20
<210> 11
<211> 20
<212> DNA
<213>people (human)
<400> 11
gtagactcca caacataagt 20
<210> 12
<211> 22
<212> DNA
<213>people (human)
<400> 12
tggagtcgta ctggaacatg ta 22
Claims (10)
1. a kind of isothermal duplication nucleic acid detection method based on unwindase and nicking enzyme, which comprises the steps of:
S1. according to target nucleic acid sequence design specificity amplification primer and with the Taqman probe of nicking enzyme recognition site；
S2. by sample nucleic acid, enzyme treated to obtain single-stranded target nucleic acid-templated；
S3. it by the nucleic acid-templated specific binding of single-stranded target obtained by Taqman probe obtained by S1 and S2, obtains specific binding and produces
Object specifically binds product, and real-time detection fluorescence signal using nicking enzyme digestion.
2. isothermal duplication nucleic acid detection method according to claim 1, which is characterized in that enzyme described in step S2 is uncoiling
One of enzyme, strand displacement enzyme, recombinase are a variety of.
3. isothermal duplication nucleic acid detection method according to claim 2, which is characterized in that enzyme described in step S2 untwists for UvrD
Enzyme and strand displacement archaeal dna polymerase.
4. isothermal duplication nucleic acid detection method according to claim 1, which is characterized in that enzyme described in step S2 is reverse transcriptase
And RNaseH.
5. the isothermal duplication nucleic acid detection method according to Claims 2 or 3, which is characterized in that pass through in the step S2 by
Specificity amplification primer and the nucleic acid-templated combination of single-stranded target make under the enzyme alternating action described in polymerase and step S2 single-stranded
Target nucleic acid template constantly expands.
6. detection method according to claim 1, which is characterized in that the condition of the detection is 50~60 DEG C of reaction temperature,
Detection time 90min.
7. a kind of isothermal duplication kit for detecting nucleic acid based on unwindase and nicking enzyme, which is characterized in that dense including following end
The each component of degree: 8~12 μM of specificity amplification primers, 8~12 μM of Taqman probes, 180~220ng/ μ L enzyme, 4~6 μ g/ μ L
32 albumen of T4 gene, 5~10U/ μ L polymerase, 8~12U/ μ L nicking enzyme, 8~12mM dNTP.
8. isothermal duplication kit for detecting nucleic acid according to claim 7, which is characterized in that the isothermal duplication detection of nucleic acids
Kit further includes 5 × buffer；5 × the buffer includes each component of following final concentration: 25mM Tris-HCl,
PH7.8,125mM NaCl, 50mM (NH4)2SO4, 250mM MgCl2, 2.5mg/mL BSA, 1M glycine betaine, 0.5%
Tween20。
9. isothermal duplication kit for detecting nucleic acid according to claim 7, which is characterized in that the polymerase is Escherichia coli
Any one of the Klenow segment of DNA polymerase i, T7 archaeal dna polymerase, BST polymerase.
10. isothermal duplication kit for detecting nucleic acid according to claim 7, which is characterized in that the nicking enzyme is
Nb.BbvCI、Nb.BsmI、Nb.BsrDI、Nb.BssSI、Nb.BtsI、Nt.AlwI、Nt.BbvCI、Nt.BsmAI、
Any one of Nt.BspQI, Nt.BstNBI, Nt.CviPII.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810922821.9A CN109097448A (en) | 2018-08-14 | 2018-08-14 | A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810922821.9A CN109097448A (en) | 2018-08-14 | 2018-08-14 | A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109097448A true CN109097448A (en) | 2018-12-28 |
Family
ID=64849559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810922821.9A Pending CN109097448A (en) | 2018-08-14 | 2018-08-14 | A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097448A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628925A (en) * | 2019-11-04 | 2019-12-31 | 湖南融健基因生物科技有限公司 | Primer, kit and method for detecting clostridium difficile |
| CN111218529A (en) * | 2020-03-13 | 2020-06-02 | 湖南融健基因生物科技有限公司 | Primer composition, kit and method for detecting novel coronavirus |
| WO2020181947A1 (en) * | 2019-03-13 | 2020-09-17 | 江苏宏微特斯医药科技有限公司 | Method for detecting one or more target nucleic acid sequences to be tested by single tube and kit thereof |
| CN112852927A (en) * | 2021-02-02 | 2021-05-28 | 中国科学院合肥物质科学研究院 | Isothermal amplification system and method based on fluorescence self-inhibition probe |
| CN113736858A (en) * | 2020-05-28 | 2021-12-03 | 香港科技大学 | Real-time monitoring method of nucleic acid amplicon mediated by circular oligonucleotide probe |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810989A (en) * | 2005-08-31 | 2006-08-02 | 中国科学院广州分院 | Constant temperature reaction process for detecting DNA and RNA with sequence specificity |
| CN101215600A (en) * | 2007-01-04 | 2008-07-09 | 中国科学院广州生物医药与健康研究院 | Detection probe and detection method for nucleic acid aim sequence |
| CN103108961A (en) * | 2010-08-17 | 2013-05-15 | 凯杰有限公司 | Helicase dependent isothermal amplification using nicking enzymes |
| CN103305612A (en) * | 2013-06-04 | 2013-09-18 | 西安交通大学 | Lead ion detection kit based on constant-temperature cascade nucleic acid amplification and detection method of lead ion detection kit |
| CN103436608A (en) * | 2013-08-08 | 2013-12-11 | 中国科学院广州生物医药与健康研究院 | Rapid detection method based on nucleic acid aptamers and kit |
-
2018
- 2018-08-14 CN CN201810922821.9A patent/CN109097448A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810989A (en) * | 2005-08-31 | 2006-08-02 | 中国科学院广州分院 | Constant temperature reaction process for detecting DNA and RNA with sequence specificity |
| CN101215600A (en) * | 2007-01-04 | 2008-07-09 | 中国科学院广州生物医药与健康研究院 | Detection probe and detection method for nucleic acid aim sequence |
| CN103108961A (en) * | 2010-08-17 | 2013-05-15 | 凯杰有限公司 | Helicase dependent isothermal amplification using nicking enzymes |
| CN103305612A (en) * | 2013-06-04 | 2013-09-18 | 西安交通大学 | Lead ion detection kit based on constant-temperature cascade nucleic acid amplification and detection method of lead ion detection kit |
| CN103436608A (en) * | 2013-08-08 | 2013-12-11 | 中国科学院广州生物医药与健康研究院 | Rapid detection method based on nucleic acid aptamers and kit |
Non-Patent Citations (1)
| Title |
|---|
| YANHONG TONG: "Development of isothermal TaqMan assays for detection of biothreat organisms", 《BIOTECHNIQUES》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020181947A1 (en) * | 2019-03-13 | 2020-09-17 | 江苏宏微特斯医药科技有限公司 | Method for detecting one or more target nucleic acid sequences to be tested by single tube and kit thereof |
| CN110628925A (en) * | 2019-11-04 | 2019-12-31 | 湖南融健基因生物科技有限公司 | Primer, kit and method for detecting clostridium difficile |
| CN110628925B (en) * | 2019-11-04 | 2023-02-24 | 湖南融健生物科技有限公司 | Primer, kit and method for detecting clostridium difficile |
| CN111218529A (en) * | 2020-03-13 | 2020-06-02 | 湖南融健基因生物科技有限公司 | Primer composition, kit and method for detecting novel coronavirus |
| CN111218529B (en) * | 2020-03-13 | 2023-06-23 | 湖南融健生物科技有限公司 | Primer composition, kit and method for detecting novel coronaviruses |
| CN113736858A (en) * | 2020-05-28 | 2021-12-03 | 香港科技大学 | Real-time monitoring method of nucleic acid amplicon mediated by circular oligonucleotide probe |
| CN113736858B (en) * | 2020-05-28 | 2024-05-10 | 香港科技大学 | Real-time monitoring method for nucleic acid amplicon mediated by cyclic oligonucleotide probe |
| CN112852927A (en) * | 2021-02-02 | 2021-05-28 | 中国科学院合肥物质科学研究院 | Isothermal amplification system and method based on fluorescence self-inhibition probe |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2018201671B2 (en) | Compositions and methods for quantifying a nucleic acid sequence in a sample | |
| Zhong et al. | Isothermal amplification technologies for the detection of foodborne pathogens | |
| CN109097448A (en) | A kind of isothermal duplication nucleic acid detection method and kit based on unwindase and nicking enzyme | |
| CN105087825B (en) | Room temperature isothermal quickly detects method, reagent and the primer and probe of Ebola virus | |
| US9845495B2 (en) | Method and kit for detecting target nucleic acid | |
| CN103571962B (en) | Multi-enzyme cleavage site mediated nucleic acid isothermal amplification detecting method | |
| Zhang et al. | Sensitive and visual detection of Cronobacter spp. in powdered infant formula by saltatory rolling circle amplification method | |
| CN106987643A (en) | A kind of Taq DNA polymerase activity detection methods | |
| CN113481283A (en) | Method for isothermal amplification of nucleic acid target sequences | |
| CN104975098A (en) | Method for rapid fluorescence detection of polynucleotide target objects simultaneously at room temperature and constant temperature | |
| JP2023518217A (en) | Loop primer and loop de loop method for detecting target nucleic acid | |
| JP2020533974A5 (en) | ||
| US20230031670A1 (en) | Method and kit for detection of polynucleotide | |
| Li et al. | One-pot, ultrasensitive, and multiplex detection of SARS-CoV-2 genes utilizing self-priming hairpin-mediated isothermal amplification | |
| Zeng et al. | Strand displacement amplification for multiplex detection of nucleic acids | |
| JP6468555B2 (en) | Detection kit and detection method | |
| Shi et al. | Isothermal amplification detection of nucleic acids by a double-nicked beacon | |
| Milton et al. | Isothermal Nucleic Acid Amplification Techniques: New Horizons in Microbial Food Safety Applications | |
| JP2017521083A (en) | Sequence for detecting Listeria monocytogenes and use of this sequence | |
| CN116694816A (en) | Isothermal multiplex detection reagent and detection method for SARS-CoV-2, influenza A virus and influenza B virus nucleic acid | |
| Kubota | Engineering a simple, rapid, inexpensive, nucleic acid based system for strain selective detection of the bacterial wilt pathogen Ralstonia solanacearum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190301 Address after: 510320 Research and Development Area A, No. 1 Spiral Fourth Road, Guangzhou International Biological Island, Guangdong Province, Third Floor Applicant after: GUANGZHOU SAGENE BIOTECH Co.,Ltd. Address before: 518067 International Plaza 604-23, West of Nanhai Avenue, Merchants Street, Nanshan District, Shenzhen City, Guangdong Province Applicant before: SHENZHEN SAIZHE BIOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191217 Address after: No. 502, building 10, Shenzhen Biomedical Innovation Industrial Park, No. 14, Jinhui Road, Kengzi street, Pingshan District, Shenzhen City, Guangdong Province Applicant after: Shenzhen Baina Xinzhi Life Science Co.,Ltd. Applicant after: GUANGZHOU SAGENE BIOTECH Co.,Ltd. Address before: 510320 Research and Development Area A, No. 1 Spiral Fourth Road, Guangzhou International Biological Island, Guangdong Province, Third Floor Applicant before: GUANGZHOU SAGENE BIOTECH Co.,Ltd. |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181228 |