CN109136337A - It is a kind of to be denaturalized the target nucleic acids amplification method and its dedicated kit and application that bubble mediates - Google Patents
It is a kind of to be denaturalized the target nucleic acids amplification method and its dedicated kit and application that bubble mediates Download PDFInfo
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Abstract
The invention discloses a kind of target nucleic acids amplification method that denaturation bubble mediates and its dedicated kit and applications.The kit includes one or more pairs of Oligonucleolide primers for expanding target nucleic acids, one or more archaeal dna polymerases, reaction buffer, dNTPs and the preparation for assisting unwinding.The preparation of the auxiliary unwinding includes single strand binding protein and/or polyethylene glycol.Be experimentally confirmed: kit of the invention has higher sensitivity, can be realized 10‑14The detection of M target improves 1000 times of SEA technology of sensitivity, is suitble to the Gene Detectings such as multiple pathogenic microorganisms and species identification, and have stability good, the advantages of can storing for a long time.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of denaturation bubble mediate target nucleic acids amplification method and its specially
With kit and application.
Background technique
Nucleic acid can be divided into DNA (DNA) and ribonucleic acid (RNA), be the fundamental of all life forms.
DNA carries hereditary information and is responsible for the basic unit amino acid of coding protein.RNA is in the coding of gene, decoding, adjusting and table
It is played an important role in reaching.Therefore, nucleic acid is used as important biomarker for biological study and medical diagnosis, and core
The appearance of sour amplification technique provides important theoretical foundation for the detection of pathogenic microorganism and meat derived components, establishes a kind of letter
Single, easily operated and sensitive quick nucleic acid detection method is the main target in field of biological detection.
Polymerase chain amplification reacts (Polymerase Chain Reaction, PCR) technology as the core occurred earliest
Sour amplification technique, is so far still widely used.The technology using long-fragment nucleic acid as target, draw by design upstream and downstream two
Object, high annealing unlock double-strand, low temperature bond primer, and when thermophilic extends in the presence of polymerase, through iterative cycles, realize target
The exponential amplification of standard film section.Traditional round pcr amplification needs agarose gel electrophoresis to be verified, and develops later
Real-Time Fluorescent Quantitative PCR Technique realizes fluorescence signal output experimental result by the way that fluorescent dye is added.But since three steps expand
Increasing causes round pcr always to be unable to do without accurate temperature control instrument, limits the research and development and base's application of the technology.
Since early 1990s, various isothermal amplification techniques are developed to mediate for the substitute of PCR, such as ring
Temperature amplification (Loop Mediated Isothermal Amplification, LAMP), the nucleic acid isothermal amplification for relying on unwindase
The technologies such as technology (Helicase-dependent Isothermal Deoxyribonucleic Acid, HDA).LAMP technology
It is known because high sensitivity, specificity are good, but the technology is easy to pollute, and design of primers is difficult, cannot achieve height
It is mutated the detection of species target.And two enzymes are needed in the reaction system of HAD technology, dual-enzyme system easily causes non-specific expansion
Increase, influences the judgement of experimental result.These disadvantages limit the popularization and use of these technologies to a certain extent.
Nineteen fifty-three Watson and Crick propose DNA double helical structure.In physiological conditions, DNA is to be in dynamic equilibrium
In, due to fluctuation, can be broken between some base hydrogen bonds, the denaturation that double-stranded DNA can be spontaneous, and double-spiral structure is caused to be sent out
Changing, the phenomenon are referred to as the respiration of DNA, and the single-stranded regions unlocked are called DNA denaturation bubble.
Chain exchange amplification technique (Strand Exchange Amplification, SEA) mediated based on denaturation bubble is one
Kind is based on the isothermal amplification that denaturation bubble mediates caused by DNA respiration.Target nucleic acid is being detected based on SEA technology
When, it is only necessary to two primers of upstream and downstream are designed, which can be denaturalized bubble structure by intrusion, extend juxtaposition under the action of polymerase
Original complementary strand is changed to form exponential amplification.SEA technology can detecte DNA, and the one-step method inspection of RNA also may be implemented
It surveys, has many advantages, such as simple, quick, high specificity, chain exchange amplified reaction can be completed under isothermal conditions, got rid of to large size
The dependence of complex instrument is suitble to the field quick detection of medical institutions, the disorder in screening of grass-roots unit, the inspection of food pathogenic microorganisms
It surveys and species identification, inspection and quarantine mechanism, handles large-scale emergency event, the occasions such as military field quickly detects.
But the chain switching technology that the denaturation bubble reported at present mediates, minimum detection limit is higher, for low concentration target
Detection capability it is weaker.
Summary of the invention
The technical problem to be solved by the present invention is to the chain switching technologies (SEA) how to be mediated based on denaturation bubble to realize low concentration
The quick detection of target nucleic acids.
In order to solve the above-mentioned technical problem, present invention firstly provides a kind of target nucleic acids amplification sides that denaturation bubble mediates
Method.
The target nucleic acids amplification method that denaturation bubble provided by the invention mediates includes steeping nucleic acid samples to be detected in denaturation
With the step of carrying out selective amplification under the action of archaeal dna polymerase；
The reaction system of the amplification includes one or more pairs of Oligonucleolide primers for expanding the target nucleic acids, one
Kind or a variety of archaeal dna polymerases, reaction buffer, dNTPs and the preparation for assisting unwinding；
The preparation of the auxiliary unwinding includes single strand binding protein and/or polyethylene glycol.
In the above method, the amplification is isothermal duplication.The reaction temperature of the isothermal duplication can be 20-75 DEG C.
Further, the reaction temperature of the isothermal duplication can be 35-65 DEG C.
Further, the reaction temperature of the isothermal duplication can be 55-65 DEG C；Preferably 59 DEG C, 60 DEG C, 61 DEG C or 62
℃。
In the above method, the target nucleic acids can be single strand dna or single strand RNA molecule, or double-stranded DNA
Molecule.
The length of the target nucleic acids can be 20-60bp.
Further, the length of the target nucleic acids can be 35-50bp.
Further, the length of the target nucleic acids can be 38-50bp.In the present invention, the length of the target nucleic acids
Degree is specially 38bp, 41bp, 43bp or 50bp.
In the above method, the Oligonucleolide primers, the archaeal dna polymerase, the dNTPs in the reaction system,
The proportion of the single strand binding protein and the polyethylene glycol is 10-6Mol:(1-30) × 106U:(0.1-10) × 10-3Mol:
(0.1-100)×103μ g:(0.001-0.2) L.
In the above method, the Oligonucleolide primers are a pair of of Oligonucleolide primers, wherein a primer and the target
The end 5' of nucleic acid hybridizes, and another primer hybridizes with the end 3' of the target nucleic acids.
Further, model of the melting temperature of the Oligonucleolide primers in reaction temperature ± 5 DEG C of the isothermal duplication
In enclosing.
The length of the Oligonucleolide primers can be 15-30bp.
The G/C content of the Oligonucleolide primers can be 40-60%.
Concentration of the Oligonucleolide primers in the reaction system can be 10-7-10-5M, concretely 10-6M。
In the present invention, the target nucleic acids refer to the partial sequence in template, and the both ends of the partial sequence are respectively with two
Primer sequence complementation is the end primer 5' to the nucleic acid moiety between another end primer 5'.
In the above method, the archaeal dna polymerase can be selected from the Klenow segment and Bst polymerization of e. coli dna polymerase I
Enzyme large fragment can also be the polymerase mutant or isomery with Bst polymerase Large fragment with 80% or 80% or more homology
Enzyme also with other compounds or nucleic acid or protein in conjunction with and can have the enzyme of hot start effect for Bst polymerase Large fragment
Compound.The archaeal dna polymerase lacks 5' to 3' exonuclease activity, has strand-displacement activity.
When target nucleic acids are RNA molecule, the archaeal dna polymerase also has reverse transcription living other than with polymerase activity
Property.
Further, concentration of the archaeal dna polymerase in the reaction system can be 1-30U/ μ L.
Further, concentration of the archaeal dna polymerase in the reaction system can be 8-24U/ μ L, concretely
8U/ μ L, 16U/ μ L or 24U/ μ L.
In the above method, the single strand binding protein includes but is not limited to bacteriophage T4 32SSB, E.Coli SSB, phagocytosis
Body T7 2.5SSB and bacteriophage phi 29SSB or their derivative.
Further, the single strand binding protein is the high thermal stability single strand binding protein from Escherichia coli
(ET SSB).The high thermal stability single strand binding protein still has greater activity in range of reaction temperature, combinable
Single-stranded DNA or RNA, can make the nucleic acid double chain unlocked due to respiration is more stable to deposit with single stranded form
To increase the joint efficiency of primer and target.In addition to this, high thermal stability single strand binding protein can also improve
The activity of archaeal dna polymerase, to accelerate reaction speed.
Concentration of the single strand binding protein in the reaction system can be 0.1-100 μ g/mL.
Further, concentration of the single strand binding protein in the reaction system can be 1.0-50 μ g/mL, specifically
It can be 5 μ g/mL, 12.5 μ g/mL, 25 μ g/mL or 50 μ g/mL, preferably 5 μ g/mL.
In the above method, the polyethylene glycol is selected from polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 2000 and poly- second
Glycol 4000.
Further, the polyethylene glycol is polyethylene glycol 200 (PEG 200).The polyethylene glycol 200 can be accelerated to hybridize
Process increases the joint efficiency of complementary double-stranded DNA, to accelerate reaction speed.
Volume fraction of the polyethylene glycol in the reaction system can be 0.1-20%.
Further, volume fraction of the polyethylene glycol in the reaction system can be 1.0-10%, specifically may be used
It is 1.0%, 2.5%, 5% or 10%, preferably 2.5%.
In the above method, concentration of the dNTPs in the reaction system can be 0.1-10mM.
Further, concentration of the dNTPs in the reaction system can be 0.5-1.5mM, concretely 0.5mM,
0.8mM or 1.0mM, preferably 0.8mM.
In the above method, the reaction buffer can be Isothermal reaction buffer, be formulated as follows:
20mMTris-HCl, 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4, 0.1%Triton X-100；pH8.8@25℃.
In the above method, the reaction system can for it is following 1) or 2):
It is described 1) in, the reaction system is by the Oligonucleolide primers, the single strand binding protein, the polyethylene glycol
200, the archaeal dna polymerase, the dNTPs, the reaction buffer, template (nucleic acid samples to be detected) and water composition；
It is described 2) in, the reaction system is made of Buffer solution A, Buffer B solution and water；
The Buffer solution A is by the reaction buffer, the dNTPs, the Oligonucleolide primers and the poly- second
Glycol composition；
The Buffer B solution is made of the reaction buffer, the archaeal dna polymerase and the single strand binding protein.
Further, according to described in Fluorometric assay when amplified production, it is described 1) in reaction system or it is described 2) in
Buffer solution A further includes fluorescent dye；According to described in colorimetric determination when amplified production, it is described 1) in reaction system
With it is described 2) in reaction system further include acid-base indicator.
Further, it is described 1) in reaction system it is specific as follows: it is template (nucleic acid samples to be detected) 1 μ L, a pair of few
(final concentration in the reaction system is 10 to nucleotide primer-6M)、dNTPs(10mM)0.8μL、Isothermal reaction
Buffer reaction buffer (1 ×), 0.25 μ L of Evagreen (20 ×), Bst 2.0WarmStartTMDNA polymerase
(8U/ μ L) 0.1 μ L, ET SSB (final concentration of 5 μ g/mL in the reaction system), 200 0.25 μ L of PEG, add water to supply system
Total volume is 10 μ L.
It is described 2) in reaction system be by Buffer A and Buffer B mix, add water supply system total volume be 25 μ
L.The Buffer A formula is specific as follows: Isothermal reaction buffer (10 ×) 1.75 μ L, dNTPs (10mM)
2 μ L, (final concentration in the reaction system is 10 to a pair of of Oligonucleolide primers-6M)、PEG 2000.625μL、Evagreen(20
×)0.625μL；The Buffer B formula is specific as follows: Isothermal reaction buffer (10 ×) 0.75 μ L, ET
SSB (final concentration of 5 μ g/mL in the reaction system), Bst 2.0WarmStartTM DNA polymerase(8U/μL)0.25
μL。
The above method further includes the steps that detecting amplified production；The method of the detection amplified production is fluorescence detection or electricity
Swimming detection or colorimetric determination.
In the above method, the target nucleic acids are the pathogen come in biological sample, can be by expanding the target core
Acid detects the pathogen.
In the above method, the target nucleic acids are located at chromosome or mitochondria or ribosomes, and the method also includes detections
The step of target nucleic acid sequence variation situation.The method of the detection target nucleic acid sequence variation can be in the prior art
Conventional method.The sequence variations can be single nucleotide polymorphism.
In the above method, the nucleic acid samples to be detected can be the genomic DNA of the biological sample extracted using kit
And RNA, 5min then can also be heated under the conditions of 95 DEG C, then be centrifuged receipts for biological sample directly to be smashed to pieces to rear mixed pyrolysis liquid
The supernatant containing target nucleic acids of collection.
Method of the invention steeps the Exchange reaction of chain (SEA) opening double-strand and being extended based on denaturation.Draw in constant temperature process
Object can be denaturalized bubble structure by intrusion, extend juxtaposition under the action of polymerase and change original complementary strand to form exponential expansion
Increase.In addition, archaeal dna polymerase target chain length about 100bp and it is following when have stronger reverse transcriptase activity.And Exchange reaction of chain
Target between design detection overall length about 35-50bp, design two primers of upstream and downstream are opened moment in conjunction with target in double-strand, are led to
The effect for crossing archaeal dna polymerase expands, it can be achieved that the one-step method of DNA or RNA detects under isothermal conditions.
In order to solve the above-mentioned technical problem, the present invention also provides target nucleic acids amplification kits.
Target nucleic acids amplification kit provided by the invention includes above-mentioned Oligonucleolide primers, above-mentioned archaeal dna polymerase, above-mentioned
The preparation of reaction buffer, above-mentioned dNTPs and above-mentioned auxiliary unwinding.
Further, the kit includes Buffer solution A and Buffer B solution；
The Buffer solution A includes the reaction buffer, the dNTPs, Oligonucleolide primers and described poly-
Ethylene glycol；
The Buffer B solution includes the reaction buffer, the archaeal dna polymerase and the single strand binding protein.
It further include fluorescent dye in the kit according to described in Fluorometric assay when amplified production.
It further include acid-base indicator in the kit according to described in colorimetric determination when amplified production.
Further, the kit by the Oligonucleolide primers, the archaeal dna polymerase, the reaction buffer,
The dNTPs, the polyethylene glycol, the single strand binding protein, the fluorescent dye and water composition or the kit by
Buffer solution A, Buffer B solution and water composition.The Buffer solution A by the reaction buffer, the dNTPs,
The Oligonucleolide primers, the polyethylene glycol and fluorescent dye composition；The Buffer B solution is slow by the reaction
Fliud flushing, the archaeal dna polymerase and single strand binding protein composition.
In order to solve the above-mentioned technical problem, the present invention also provides the above method or the new applications of mentioned reagent box.
The present invention provides the application of the above method or mentioned reagent box in the pathogenic microorganism examination.
The present invention also provides the application of the above method or mentioned reagent box in species identification.
The present invention also provides the application of the above method or mentioned reagent box in genetic test.
The genetic test includes but is not limited to defective gene detection, drug resistant gene detection, disease detection, disease risks
The detection fields such as prediction or gene physical examination.
The present invention provides a kind of target nucleic acids amplification methods and its used kit that denaturation bubble mediates.It is demonstrate,proved by experiment
Bright: kit of the invention has higher sensitivity, can be realized 10-14The detection of M target improves 1000 times of SEA technology
Sensitivity, be suitble to the genetic tests such as multiple pathogenic microorganisms and species identification field, and have stability it is good, can deposit for a long time
The advantages of storage.
Detailed description of the invention
Fig. 1 is Exchange reaction of chain schematic diagram.Exchange reaction of chain only needs two primers of upstream and downstream, and a kind of archaeal dna polymerase is waiting
The detection of nucleic acid rapid amplifying is realized under the conditions of temperature.The denaturation bubble that " breathing " effect of nucleic acid double chain itself generates is so that nucleic acid is presented
Single-chain state, up or down trip primer at this time extend in the presence of polymerase to the end 3', shape after a circulation in conjunction with target
At the short chain target DNA of 35-70bp, continues to generate a large amount of reaction product as target circulation, realize signal amplification.
Fig. 2 is the fluorogram that ET SSB greatly improves reaction efficiency.Wherein-▆-is represented contains in 10 μ L reaction systems
Amplification curve when the ET SSB of final concentration of 50 μ g/mL ,-●-represent and contain final concentration of 25 μ g/ in 10 μ L reaction systems
Amplification curve when the ET SSB of mL ,-▲-represent the ET SSB for containing final concentration of 12.5 μ g/mL in 10 μ L reaction systems
When amplification curve, amplification when the 10 μ L reaction systems that-▼-represent contain the ET SSB of final concentration of 5.0 μ g/mL is bent
Line ,-◆-represent amplification curve when 10 μ L reaction systems do not add ET SSB；10 μ L are represented using water as target
Contain the amplification curve when ETSSB of final concentration of 50 μ g/mL in reaction system,10 μ L are represented using water as the anti-of target
The amplification curve when ET SSB for containing final concentration of 25 μ g/mL in system is answered,10 μ L are represented using water as the anti-of target
Amplification curve when answering in system containing final concentration of 12.5 μ g/mLET SSB ,-★-represent 10 μ L using water as the anti-of target
Amplification curve when system being answered to contain the ET SSB of final concentration of 5 μ g/mL,10 μ L are represented using water as the reactant of target
System does not add amplification curve when ET SSB.
Fig. 3 is the influence result figure of 200 pairs of PEG amplification tests of various concentration.Wherein-▆-represents 10 μ L reactants
Contain amplification curve when final concentration of 1.0% PEG 200 in system ,-●-represent in 10 μ L reaction systems and contain final concentration
For 2.5% PEG 200 when amplification curve ,-▲-represent in 10 μ L reaction systems contain final concentration of 5.0% PEG
Amplification curve when 200, amplification when-▼-is represented in 10 μ L reaction systems containing final concentration of 10.0% PEG 200 are bent
Line ,-◆-represent amplification curve when 10 μ L reaction systems do not add PEG 200；10 μ L are represented using water as target
Contain amplification curve when final concentration of 1.0% PEG 200 in reaction system,10 μ L are represented using water as the anti-of target
Amplification curve when answering in system containing final concentration of 2.5% PEG 200,10 μ L are represented using water as the reaction of target
Contain amplification curve when final concentration of 5.0% PEG 200 in system ,-★-represents 10 μ L using water as the reactant of target
Contain the amplification curve of final concentration of 10.0% PEG200 in system,10 μ L are represented using water as in the reaction system of target
Amplification curve when PEG 200 is not added.
Fig. 4 is influence result figure of the Bst DNA polymerase to amplification test of various concentration.Wherein-▆-generation
Amplification curve when in 10 μ L reaction system of table containing 8U Bst DNA polymerase ,-●-represent 10 μ L reaction systems
In containing 16U Bst DNA polymerase when amplification curve ,-▲-represent in 10 μ L reaction systems containing 24U Bst
Amplification curve when DNA polymerase ,-▼-, which is represented, contains 24U Bst DNA polymerase in 10 μ L reaction systems
But amplification curve when without target nucleic acids.
Fig. 5 is influence result figure of the dNTPs to amplification test of different volumes.Wherein-▆-represents 10 μ L reaction systems
In using water as target when amplification curve ,-● and-amplification when representing in 10 μ L reaction systems containing 0.8 μ L dNTPs is bent
Line, amplification curve when-▲-represents in 10 μ L dNTPs reaction systems containing 1 μ L dNTPs ,-▼-represent 10 μ L reaction
Amplification curve when in system containing 0.5 μ L dNTPs.
Fig. 6 is that kit of the present invention detects staphylococcus aureus feasibility verification result figure.In-▼-generation, is wherein schemed in A
Table is using water as the blank control of target.Its excess-three fluorescence curve is three repeatability verifyings of same target concentration.Scheme B be with
Scheme the corresponding PAGE figure of A, wherein swimming lane M is the Marker of 20bp, and corresponding three positives of swimming lane 1-3 repeat, and swimming lane 4 is blank pair
According to.Scheming C is corresponding with figure A than chromatic graph, and wherein corresponding three positives of a-c repeat, and d corresponds to blank control.
Fig. 7 is the SEA amplified reaction figure for the target nucleic acids that two kinds of distinct methods obtain.Wherein-●-represent by kit
Amplification curve when the duck genomic DNA and RNA mixture of extraction are as target ,-▆-representative are obtained by simple cleavage
Amplification curve when duck genomic DNA and RNA mixture are as target ,-▲-represent the blank control using water as target.
Fig. 8 is the senile experiment of SEA reaction system of the present invention.Wherein-▆-representative has just been taken out from -20 DEG C of refrigerators
React Mix amplification curve ,-●-represent room temperature storage 1 month reaction mix amplification curve ,-▲-represent room temperature storage
The amplification curve of 4 months reaction mix is deposited ,-▼-represents the amplification curve of room temperature storage 8 months reaction mix ,-◆-
Represent the blank control using water as target.
Fig. 9 is that the multigelation of SEA reaction system of the present invention is tested.Wherein 1 positive targets when representing multigelation one time
Amplification curve, the amplification curve of 2 positive targets when representing multigelation twice, 3 positive targets when representing multigelation three times
Amplification curve, the amplification curve of 4 positive targets when representing multigelation four times, 5 positive targets when representing multigelation five times
Amplification curve, the amplification curve of 6 positive targets when representing multigelation six times, 7 when representing multigelation one time using water as target
Target amplification curve, 8 when representing multigelation twice using water as the amplification curve of target, 9 when representing multigelation three times with water
It is represented multigelation five times using water as the amplification curve of target when being represented multigelation four times for the amplification curve of target, 10,11
Shi Yishui is the amplification curve of target, 12 when representing multigelation six times using water as the amplification curve of target.
Figure 10 is that the detection of SEA reaction system of the invention limits.Wherein-▆-represents target (staphylococcus aureus
PCR product) final concentration of 10-11Amplification curve when M ,-●-represent target (staphylococcus aureus PCR product) final concentration
It is 10-12Amplification curve when M ,-▲-represent target (staphylococcus aureus PCR product) final concentration of 10-13Expansion when M
Increase curve ,-▼-represents target (staphylococcus aureus PCR product) final concentration of 10-14Amplification curve when M ,-◆-
Represent target (staphylococcus aureus PCR product) final concentration of 10-15Amplification curve when M,It represents using water as target
Blank control.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
The genomic DNA of biological sample and RNA (genomic DNA and RNA mixture) pass through DNA/ in following embodiments
RNA Isolation Kit, which is extracted, to be obtained, and DNA/RNA Isolation Kit is TIANGEN Biotech (Beijing) Co., Ltd.
Product, article No. DP422.
The solvent of Isothermal reaction buffer reaction buffer is water, solute and its dense in following embodiments
Degree is as follows respectively: 20mM Tris-HCl, 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4, 0.1%Triton X-100；
pH8.8@25℃。
Embodiment 1: single strand binding protein is improving the application in SEA reaction efficiency
Since single strand binding protein (SSB) can speed up the functioning efficiency of archaeal dna polymerase, stablize the structure of single stranded DNA, pole
It is big to promote amplified reaction.High thermal stability single strand binding protein (ET SSB) conduct that the present embodiment will be extracted from Escherichia coli
It assists the preparation of unwinding to carry out isothermal duplication, and optimizes and obtain ET SSB optium concentration.Specific step is as follows:
1, the target nucleic acids of isothermal duplication: with Bursaphelenchus xylophilus (Bursaphelenchus xylophilus) 28S rRNA base
Because (sequence 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3') (SEQ ID No.1) is target core
Acid.
2, the reaction system (SEA reaction system) of isothermal duplication is as follows:
Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2)): the end in SEA reaction system
Concentration is 10-6M；
Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3)): in SEA reaction system
Final concentration of 10-6M；
DNTPs (10mM): 0.8 μ L；
Isothermal reaction buffer reaction buffer (1 ×)；
Evagreen (20 ×) (Bridgen, D013-1): 0.25 μ L；
Bst 2.0WarmStartTMDNA polymerase (8U/ μ L) (New England Biolabs, M0537L):
0.1μL；
Genomic DNA and RNA Mix of B.Xylophilus (Bursaphelenchus xylophilus genomic DNA and RNA mixture)
(10-10M): 1 μ L；
In addition to the above ingredient, ET SSB solution (500 μ g/mL) (New England of different volumes is added into system
Biolabs, #M2401S), making the final concentration of ET SSB in the reaction system is respectively 0,5 μ g/mL, 12.5 μ g/mL, 25 μ g/
ML, 50 μ g/mL, remaining supplies system total volume to 10 μ L with water.
Simultaneously in the SEA reaction system containing various concentration ET SSB using be not added target nucleic acids (NTC) as pair
According to remaining supplies system total volume to 10 μ L with water.
3, Bole CFX96 the reaction condition of isothermal duplication: is utilized at 62 DEG CTMReal-time fluorescence quantitative PCR instrument carries out isothermal
Amplification, and fluorescence signal is scanned per minute, as a result such as Fig. 2.
It can be seen from the figure that the ET SSB that various concentration is added has apparent facilitation to SEA Exchange reaction of chain.
It comparing with ET SSB is not added, the reaction time can be greatly shortened in the ET SSB that debita spissitudo is added in SEA reaction system,
Improve reaction rate.Wherein, when final concentration of 5 μ g/mL of the ET SSB in SEA reaction system, it is earliest to play peak time.
The optimization of embodiment 2:SEA reaction system
One, Polyethylene glycol optimization experiment
It is 201610101384.5 in number of patent application since polyethylene glycol can increase the joint efficiency of complementary nucleic acid chain
On the basis of, isothermal duplication is carried out using polyethylene glycol as the preparation of auxiliary unwinding, further to carry out to Polyethylene glycol
Optimization.Specific step is as follows:
1, the target nucleic acids of isothermal duplication: with Bursaphelenchus xylophilus (Bursaphelenchus xylophilus) 28S rRNA base
Because (sequence 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3') (SEQ ID No.1) is target core
Acid.
2, the reaction system of isothermal duplication is as follows:
Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2)): the end in SEA reaction system
Concentration is 10-6M；
Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3)): in SEA reaction system
Final concentration of 10-6M；
DNTPs (10mM): 0.8 μ L；
Isothermal reaction buffer reaction buffer (1 ×)；
Evagreen (20 ×): 0.25 μ L；
ET SSB (500 μ g/mL): 0.1 μ L；
Bst 2.0WarmStartTMDNA polymerase (8U/ μ L): 0.1 μ L；
Genomic DNA and RNA Mix of B.Xylophilus (Bursaphelenchus xylophilus genomic DNA and RNA mixture)
(10-10M): 1 μ L；
In addition to the above ingredient, the polyethylene glycol 200 (PEG 200, Sigma of various concentration is added into system
Aldrioh, 1002300576P3015-250G), make its volume fraction in SEA reaction system be respectively 1%, 2.5%,
5% and 10%.
Simultaneously target nucleic acids are not added in SEA reaction system and make containing volume fraction for 10% PEG 200 (NTC)
For control.
3, Bole CFX96 the reaction condition of isothermal duplication: is utilized at 62 DEG CTMReal-time fluorescence quantitative PCR instrument carries out isothermal
Amplification, and fluorescence signal is scanned per minute, as a result such as Fig. 3.
As can be seen from the figure: the PEG 200 that various concentration is added there is apparent promotion to make SEA Exchange reaction of chain
With.It compares with PEG 200 is not added, reaction can be greatly shortened in the PEG 200 that debita spissitudo is added in SEA reaction system
Time improves reaction rate.Wherein, when volume fraction of the PEG 200 in SEA reaction system is 2.5%, peak time is played most
It is early.
Two, Bst DNA polymerase concentration optimization is tested
Isothermal duplication is carried out using the Bst DNA polymerase of various concentration, studies Bst DNA polymerase couple
The influence of amplified reaction.Specific step is as follows:
1, the target nucleic acids of isothermal duplication: with Bursaphelenchus xylophilus (Bursaphelenchus xylophilus) 28S rRNA base
Because (sequence 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3') (SEQ ID No.1) is target core
Acid.
2, the reaction system of isothermal duplication is as follows:
Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2)): the end in SEA reaction system
Concentration is 10-6M；
Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3)): in SEA reaction system
Final concentration of 10-6M；
DNTPs (10mM): 0.8 μ L；
Isothermal reaction buffer reaction buffer (1 ×)；
Evagreen (20 ×): 0.25 μ L；
ET SSB (500 μ g/mL): 0.1 μ L；
PEG 200 (100%): 0.25 μ L；
Genomic DNA and RNA Mix of B.Xylophilus (Bursaphelenchus xylophilus genomic DNA and RNA mixture)
(10-10M): 1 μ L；
In addition to the above ingredient, the Bst that 0.1 μ L concentration is 8U/ μ L, 16U/ μ L and 24U/ μ L is separately added into system
2.0WarmStartTMDNA polymerase, remaining supplies system total volume to 10 μ L with water.
Simultaneously target nucleic acids are not added in SEA reaction system and make containing 24U Bst DNA polymerase (NTC)
For control.
3, Bole CFX96 the reaction condition of isothermal duplication: is utilized at 62 DEG CTMReal-time fluorescence quantitative PCR instrument carries out isothermal
Amplification, and fluorescence signal is scanned per minute, as a result such as Fig. 4.
As can be seen from the figure: under the Bst DNA polymerase of 3 kinds of various concentrations, without obvious between positive findings
Difference.
Three, dNTPs concentration optimization is tested
Isothermal duplication is carried out using the dNTPs of different volumes, studies influence of the dNTPs to amplified reaction.Specific steps are such as
Under:
1, the target nucleic acids of isothermal duplication: with Listeria monocytogenes (Listeria monocytogenes) 16S rRNA base
Because of (sequence is 5'-GTCATTGGAAACTGGAAGACTGGAGTGCAGAAGAGGAGAGTGG-3'(SEQ ID No.4)) it is target
Mark nucleic acid.
2, the reaction system of isothermal duplication is as follows:
Primer P1 (sequence is 5'-GTCATTGGAAACTGGAAGACTG-3'(SEQ ID NO.5)): in SEA reaction system
In final concentration of 10-6M；
Primer P2 (sequence is 5'-CCACTCTCCTCTTCTGCAC-3'(SEQ ID NO.6)): in SEA reaction system
Final concentration of 10-6M；
Isothermal reaction buffer reaction buffer (1 ×)；
Evagreen (20 ×): 0.25 μ L；
ET SSB (500 μ g/mL): 0.1 μ L；
PEG 200:0.25 μ L；
Bst 2.0WarmStartTMDNA polymerase (8U/ μ L): 0.1 μ L；
Genomic DNA and RNA Mix of L.monocytogenes (Listeria monocytogenes genomic DNA and RNA
Mixture) (10-10M): 1 μ L；
In addition to the above ingredient, the dNTPs (10mM) of 0.5 μ L, 0.8 μ L and 1.0 μ L is added into system respectively, make its
Final concentration in system is respectively 0.5mM, 0.8mM and 1.0mM, remaining supplies system total volume to 10 μ L with water.
Using water as target nucleic acids (NTC) and contain the dNTPs of 1.0 μ L as compareing simultaneously.
3, Bole CFX96 the reaction condition of isothermal duplication: is utilized at 62 DEG CTMReal-time fluorescence quantitative PCR instrument carries out isothermal
Amplification, and fluorescence signal is scanned per minute, as a result such as Fig. 5.
As can be seen from the figure: the dNTPs of various concentration shows different facilitations to SEA reaction, reaction is added
When the dNTPs volume of system is 0.8 μ L (the final concentration of 0.8mM of dNTPs in the reaction system), positive findings play peak time more
Short, reaction efficiency is higher.
Embodiment 3: the preparation and its feasibility verifying of the kit for target nucleic acids detection
One, the preparation for the kit of target nucleic acids detection
Kit for target nucleic acids detection of the invention includes Buffer A and Buffer B.Buffer A and
The formula difference of Buffer B is as follows:
Buffer A formula is as follows:
Isothermal reaction buffer (10 ×): 1.75 μ L；
DNTPs (10mM): 2 μ L；
Primer P1: final concentration of 10 in overall reaction system-6M；
Primer P2: final concentration of 10 in overall reaction system-6M；
PEG 200:0.625 μ L；
Evagreen (20 ×): 0.625 μ L；
Buffer B formula is as follows:
Isothermal reaction buffer (10 ×): 0.75 μ L；
ET SSB (500 μ g/mL): 0.25 μ L；
Bst 2.0WarmStartTMDNA polymerase (8U/ μ L): 0.25 μ L.
When needing to detect target nucleic acids, Buffer A and Buffer B are mixed, core to be detected is then added
Sour sample realizes SEA augmentation detection under isothermy.In practical application, the total volume of reaction system is 25 μ L, and water is added to supply always
Volume is 25 μ L.SEA amplification can also be carried out according to 10 μ L systems in Examples 1 and 2.
When carrying out result detection using colorimetric method, Evagreen fluorescent dye is not added in Buffer A, in reaction system
In be additionally added final concentration of 10% acid-base indicator, realize reaction monitoring.
Two, the feasibility detection for the kit of target nucleic acids detection
1, the target nucleic acids of isothermal duplication: with staphylococcus aureus (Staphylococcus aureus) 16S rRNA
(sequence is 5'-GGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGC-3' (SEQ ID to gene
It No.7)) is target nucleic acids.
The primer sequence for expanding staphylococcus aureus target nucleic acids is as follows: 5'-
GGTTCAAAAGTGAAAGACGGTCTTG-3'(SEQ ID No.8) and 5'-GCGCGGATCCATCTATAAGTGAC-3'(SEQ
ID No.9)。
2, the reaction system of isothermal duplication: by Buffer A, Buffer B and 1 μ L staphylococcus aureus gene group DNA
With RNA mixture (10-10M) it is uniformly mixed so as to obtain SEA reaction system.Three repetitions of experimental setup.
Simultaneously using water as target nucleic acids (NTC) as blank control.
3, Bole CFX96 the reaction condition of isothermal duplication: is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument carries out isothermal
Amplification.
4, result detects
(1) fluorescence method realizes detection
Bole CFX96 is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument is per minute scanned fluorescence signal, as a result
Such as Fig. 6 A.
As can be seen from the figure: three repeating samples play peak simultaneously in one minute error range, and blank control does not have fluorescence
Value variation.System after illustrating optimization is reproducible, and stability is strong.
(2) PAGE glue electrophoresis realizes detection
Amplified production is carried out to the PAGE electrophoresis of 10-18%, first 180V voltage 1min, then 135V voltage 55min.As a result such as
Fig. 6 B.
It can be seen from the figure that swimming lane 1-3 has apparent product band in 41bp, it is consistent with design length, it was demonstrated that have production
Object.Swimming lane 4 only has unreacted primer band in 20bp or so without spawn band.
(3) colorimetric method of acid-base indicator instruction realizes detection
It will indicate that the acid-base indicator of acid-base property (is purchased from Qingdao Nai De Bioisystech Co., Ltd, article No. is
K3005) be added reaction system in, make its final concentration of 10%.Observing response system before the reaction after color change situation.If
Reaction system before the reaction after from yellow become red, then show that sample to be tested contains target nucleic acids, if reaction system is being reacted
There is no color changes for front and back, then show sample to be tested without containing target nucleic acids.As a result such as Fig. 6 C.
It can be seen from the figure that color from yellow becomes red to a-c after the completion of reaction, blank control does not have color change
Change.
Embodiment 4: the performance detection of kit of the present invention
One, the detection effect for the target nucleic acids that distinct methods obtain compares
Isothermal duplication is carried out using the duck target that two kinds of different modes obtain, the specific steps are as follows:
1, the acquisition of target nucleic acids
1) kit extracts: extracting mallard (Anas using TIAN DNA/RNA Isolation Kit
Platyrhynchos) duck genomic DNA and RNA obtain Duck genome DNA and RNA mixture (pork 1).
2) it directly smashs cracking to pieces: after mallard (Anas platyrhynchos) duck is directly smashed to pieces, being mixed with PBS,
Then 5min is heated at 95 DEG C, then is centrifuged with compact centrifuge, and Duck genome DNA and RNA mixture (pork 2) are obtained.
2, the detection of target nucleic acids
With mallard chondriogen (sequence 5'-CGCATAACCCTCCTAGTCCAAGCCGGACGGACTCGTATC
CC-3'(SEQ ID No.10)) it is target nucleic acids.
The primer sequence for expanding mallard chondriogen target nucleic acids is as follows: 5'-CGCATAACCCTCCTAGTCCAAG-
3'(SEQ ID No.11) and 5'-CCCTCTGCTCAGGCAGGC-3'(SEQ ID No.12).
Duck genome DNA and the RNA mixture (pork 1) and Duck genome DNA and RNA obtained respectively with step 1 mixes
Object (pork 2) is template, and the detection of target nucleic acids is carried out using the kit in embodiment 3.
Two kinds of Duck genome DNA and RNA mixtures are separately added into 2.5 μ L.
Simultaneously using water as target nucleic acids (NTC) as blank control.
Bole CFX96 is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument is per minute scanned fluorescence signal, as a result
Such as Fig. 7.
It can be seen from the figure that Duck genome DNA and the RNA mixture (pork 1) that kit extracts are in 17min fluorescence
Value has started peak, directly smash to pieces Duck genome DNA and the RNA mixture (pork 2) of the cracking fluorescent value at 24min occur it is bright
Aobvious variation, illustrates that amplification system of the invention can either quickly detect Genomic targets, while also can detecte without complexity
The sample of pre-treatment provides theoretical foundation for on-site test.
Two, the senile experiment of kit of the present invention
Isothermal duplication, tool are carried out to duck target using the bufferA and bufferB that store different time at different conditions
Steps are as follows for body: Duck genome DNA and the RNA mixture (pork 1) obtained with step 1 is template, using in embodiment 3
The detection of kit progress target nucleic acids.It is divided into according to the resting period of bufferA and bufferB with storage condition difference as follows
Each group:
- 20 DEG C: the bufferA and bufferB that take out from -20 DEG C of refrigerators carry out the detection of target nucleic acids；
A month: the detection of target nucleic acids is carried out with room temperature storage 1 month bufferA and bufferB；
Four months: the detection of target nucleic acids is carried out with room temperature storage 4 months bufferA and bufferB；
Eight months: the detection of target nucleic acids is carried out with room temperature storage 8 months bufferA and bufferB；
Simultaneously using water as target nucleic acids (NTC) as blank control.
Bole CFX96 is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument is per minute scanned fluorescence signal, as a result
Such as Fig. 8.
As can be seen from the figure: room temperature storage 4 months reaction solutions still have preferable reaction efficiency, room temperature storage 8
The reaction time long 13min of reaction solution of a month reaction solution compared with room temperature storage 4 months, but detectability does not subtract
It is weak.Illustrate that stabilization of kit of the present invention is good, it can be with long-time storage and not vulnerable.
Three, the multigelation experiment of kit of the present invention
Isothermal duplication is carried out to duck target using naturally frozen and the SEA reaction system for melting different numbers, it is specific to walk
Rapid as follows: Duck genome DNA and the RNA mixture (pork 1) obtained with step 1 is template, using the reagent in embodiment 3
The detection of box progress target nucleic acids.The same batch bufferA and bufferB SEA reaction system being mixed to get is dispensed into six
In a Eppendorf pipe, following each group is divided into according to SEA reaction system naturally frozen and the difference for melting number:
1 time (multigelation 1 time) of naturally frozen and thawing；
2 times (multigelation 2 times) of naturally frozen and thawing；
3 times (multigelation 3 times) of naturally frozen and thawing；
4 times (multigelation 4 times) of naturally frozen and thawing；
5 times (multigelation 5 times) of naturally frozen and thawing；
6 times (multigelation 6 times) of naturally frozen and thawing.
Above-mentioned six groups are respectively target as blank control using water simultaneously.
Bole CFX96 is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument is per minute scanned fluorescence signal, as a result
Such as Fig. 9.
It can be seen from the figure that buffer solution at multigelation 6 times except rise peak time relatively rearward in addition to, still can be with
Reach good detection effect.
Embodiment 5: the DNA of kit of the present invention detects limit
The present embodiment carries out isothermal duplication by template of the target nucleic acids of various concentration, the specific steps are as follows: with different dense
(staphylococcus aureus PCR product is the genomic DNA with staphylococcus aureus to the staphylococcus aureus PCR product of degree
For template, the PCR product that PCR amplification obtains is carried out using primer shown in SEQ ID No.13 and SEQ ID No.14) template,
The detection of target nucleic acids is carried out using the kit in embodiment 3, and it is dense that equal volume difference is added into each reaction tube respectively
The staphylococcus aureus PCR product of degree carries out isothermal duplication.In the reaction system according to staphylococcus aureus PCR product
Final concentration difference is divided into following each group: staphylococcus aureus PCR product final concentration of 10-11M；Staphylococcus aureus PCR is produced
Object final concentration of 10-12M；Staphylococcus aureus PCR product final concentration of 10-13M；Staphylococcus aureus PCR product is dense eventually
Degree is 10-14M；Staphylococcus aureus PCR product final concentration of 10-15M.It is simultaneously target as blank control using water.
Bole CFX96 is utilized at 61 DEG CTMReal-time fluorescence quantitative PCR instrument is per minute scanned fluorescence signal, as a result
Such as Figure 10.
It can be seen from the figure that the detection limit of SEA reaction system of the invention reaches 10-14M is with number of patent application
Comparing in 201610101384.5 without the SEA reaction system of optimization, sensitivity improves 1000 times, is more conducive to low
The detection of concentration target provides advantage for field quick detection.
Sequence table
<110>University Of Qingdao
<120>a kind of target nucleic acids amplification method that denaturation bubble mediates and its dedicated kit and application
<160>14
<170>PatentIn version 3.5
<210>1
<211>38
<212>DNA
<213>Bursaphelenchus xylophilus
<400>1
agccttctgg gcgcgtgatt ggtgtttgtg cattgccg 38
<210>2
<211>16
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>2
agccttctgg gcgcgt 16
<210>3
<211>19
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>3
cggcaatgca caaacacca 19
<210>4
<211>43
<212>DNA
<213>Listeria monocytogenes
<400>4
gtcattggaa actggaagac tggagtgcag aagaggagag tgg 43
<210>5
<211>22
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>5
gtcattggaa actggaagac tg 22
<210>6
<211>19
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>6
ccactctcct cttctgcac 19
<210>7
<211>50
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<213>Staphylococcus aureus
<400>7
ggttcaaaag tgaaagacgg tcttgctgtc acttatagat ggatccgcgc 50
<210>8
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<213>artificial sequence (Artificial Sequence)
<400>8
ggttcaaaag tgaaagacgg tcttg 25
<210>9
<211>23
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<213>artificial sequence (Artificial Sequence)
<400>9
gcgcggatcc atctataagt gac 23
<210>10
<211>41
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<213>Anas platyrhynchos
<400>10
cgcataaccc tcctagtcca agccggacgg actcgtatcc c 41
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<213>artificial sequence (Artificial Sequence)
<400>11
cgcataaccc tcctagtcca ag 22
<210>12
<211>18
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<213>artificial sequence (Artificial Sequence)
<400>12
ccctctgctc aggcaggc 18
<210>13
<211>26
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<213>artificial sequence (Artificial Sequence)
<400>13
cgtggataac ctacctataa gactgg 26
<210>14
<211>26
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<213>artificial sequence (Artificial Sequence)
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ggtaccgtca agatgtgcac agttac 26
Claims (36)
1. a kind of target nucleic acids amplification method that denaturation bubble mediates, including nucleic acid samples to be detected are polymerize in denaturation bubble and DNA
The step of selective amplification is carried out under the action of enzyme；
The reaction system of the amplification includes one or more pairs of for expanding Oligonucleolide primers, the Yi Zhonghuo of the target nucleic acids
A variety of archaeal dna polymerases, reaction buffer, dNTPs and the preparation for assisting unwinding；
The preparation of the auxiliary unwinding includes single strand binding protein and/or polyethylene glycol.
2. according to the method described in claim 1, it is characterized by: the amplification is isothermal duplication.
3. according to the method described in claim 2, it is characterized by: the reaction temperature of the isothermal duplication is 20-75 DEG C.
4. according to the method described in claim 3, it is characterized by: the reaction temperature of the isothermal duplication is 37-65 DEG C.
5. according to the method described in claim 4, it is characterized by: the reaction temperature of the isothermal duplication is 55-65 DEG C.
6. according to the method described in claim 1, it is characterized by: the target nucleic acids are DNA molecular or RNA molecule.
7. method according to claim 1 or 6, it is characterised in that: the length of the target nucleic acids is 20-60bp.
8. according to the method described in claim 7, it is characterized by: the length of the target nucleic acids is 35-50bp.
9. according to the method described in claim 1, it is characterized by: the Oligonucleolide primers are a pair of of Oligonucleolide primers,
Wherein a primer hybridizes with the end 5' of the target nucleic acids, and another primer hybridizes with the end 3' of the target nucleic acids.
10. -5 or 9 any method according to claim 1, it is characterised in that: the melting temperature of the Oligonucleolide primers
In the range of reaction temperature ± 5 DEG C of the isothermal duplication.
11. method described according to claim 1 or 9 or 10, it is characterised in that: the length of the Oligonucleolide primers is 15-
30bp。
12. according to claim 1 or any method of 9-11, it is characterised in that: the G/C content of the Oligonucleolide primers
For 40-60%.
13. according to the method described in claim 1, it is characterized by: the archaeal dna polymerase is selected from e. coli dna polymerase I
Klenow segment and Bst polymerase Large fragment.
14. according to the method described in claim 1, it is characterized by: the archaeal dna polymerase is to have with Bst polymerase Large fragment
There are the polymerase mutant or isomerase of 80% or 80% or more homology.
15. according to the method described in claim 1, it is characterized by: the archaeal dna polymerase is Bst polymerase Large fragment and its
His compound or nucleic acid or protein combine and the compound of the enzyme with hot start effect.
16. according to claim 1 or any method of 13-15, it is characterised in that: the archaeal dna polymerase lacks 5 ' to 3 '
Exonuclease activity.
17. according to claim 1 or any method of 13-16, it is characterised in that: the archaeal dna polymerase has strand displacement
Activity.
18. according to the method described in claim 1, it is characterized by: the single strand binding protein be selected from bacteriophage T4 32SSB,
E.Coli SSB, phage t7 2.5SSB and bacteriophage phi 29SSB or their derivative.
19. according to the method for claim 18, it is characterised in that: the single strand binding protein is from Escherichia coli
High thermal stability single strand binding protein.
20. according to the method described in claim 1, it is characterized by: the polyethylene glycol is selected from polyethylene glycol 200, poly- second two
Alcohol 400, polyethylene glycol 2000 and Macrogol 4000.
21. according to the method for claim 20, it is characterised in that: the polyethylene glycol is polyethylene glycol 200.
22. -21 any method according to claim 1, it is characterised in that: the oligonucleotides in the reaction system
Primer, the archaeal dna polymerase, the dNTPs, the single strand binding protein and the polyethylene glycol proportion be 10-6Mol:(1-
30)×106U:(0.1-10) × 10-3Mol:(0.1-100) × 103μ g:(0.001-0.2) L.
23. according to claim 1 or 18 or 19 or 22 any methods, it is characterised in that: the single strand binding protein exists
Concentration in the reaction system is 0.1-100 μ g/mL.
24. according to claim 1 or any method of 20-22, it is characterised in that: the polyethylene glycol is in the reactant
Volume fraction in system is 0.1-20%.
25. according to claim 1 or any method of 13-17 or 22, it is characterised in that: the archaeal dna polymerase is described
Concentration in reaction system is 1-30U/ μ L.
26. according to claim 1 or method described in 22, it is characterised in that: concentration of the dNTPs in the reaction system
For 0.1-10mM.
27. according to the method described in claim 1, it is characterized by: further including fluorescent dye or soda acid in the reaction system
Indicator.
28. according to the method described in claim 1, it is characterized by: the step of the method also includes detection amplified productions；Institute
The method for stating detection amplified production is fluorescence detection or electrophoresis detection or colorimetric determination.
29. according to the method described in claim 1, it is characterized by: the target nucleic acids carry out the pathogen in biological sample,
The pathogen can be detected by expanding the target nucleic acids.
30. the method according to claim 1, it is characterised in that: the target nucleic acids are located at chromosome or mitochondria or ribosomes,
The method also includes detecting the target nucleic acid sequence variation situation.
31. method according to claim 30, it is characterised in that: the sequence variations are single nucleotide polymorphism.
32. target nucleic acids amplification kit comprising any Oligonucleolide primers, claim in claim 1-31
Any archaeal dna polymerase in 1-31, any reaction buffer in claim 1-31 are appointed in claim 1-31
The preparation of any auxiliary unwinding in dNTPs described in one and claim 1-31.
33. kit according to claim 32, it is characterised in that: the kit include Buffer solution A and
Buffer B solution；
The Buffer solution A includes the reaction buffer, the dNTPs, the Oligonucleolide primers and the poly- second two
Alcohol；
The Buffer B solution includes the reaction buffer, the archaeal dna polymerase and the single strand binding protein.
34. the kit according to claim 32 or 33, it is characterised in that: the kit further includes fluorescent dye or acid
Neutralization indicator.
35. any any kit of the method or claim 32-34 of claim 1-31 is following 1) -3) appoint
A kind of application in:
1) the pathogenic microorganism examination；
2) species identification；
3) genetic test.
36. application according to claim 35, it is characterised in that: the genetic test includes that defective gene detects, is resistance to
Medicine genetic test, disease detection, disease risks prediction and gene physical examination.
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| PCT/CN2019/101293 WO2020048307A1 (en) | 2018-09-05 | 2019-08-19 | Degenerated vacuole-mediated target nucleic acid amplification method, special kit and use thereof |
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| WO2021147910A1 (en) | 2020-01-21 | 2021-07-29 | Qingdao Navid Biotechnology Co., Ltd. | Methods and kits for amplification and detection of nucleic acids |
| CN113215224A (en) * | 2020-01-21 | 2021-08-06 | 青岛大学 | Method and kit for amplifying and detecting nucleic acid |
| CN113528624A (en) * | 2020-04-17 | 2021-10-22 | 青岛大学 | Method and kit for amplifying and detecting nucleic acid |
| WO2024067192A1 (en) * | 2022-09-30 | 2024-04-04 | 华南农业大学 | Linear displacement isothermal amplification method and use thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020048307A1 (en) * | 2018-09-05 | 2020-03-12 | 青岛耐德生物技术有限公司 | Degenerated vacuole-mediated target nucleic acid amplification method, special kit and use thereof |
| WO2021147910A1 (en) | 2020-01-21 | 2021-07-29 | Qingdao Navid Biotechnology Co., Ltd. | Methods and kits for amplification and detection of nucleic acids |
| CN113215224A (en) * | 2020-01-21 | 2021-08-06 | 青岛大学 | Method and kit for amplifying and detecting nucleic acid |
| CN113528624A (en) * | 2020-04-17 | 2021-10-22 | 青岛大学 | Method and kit for amplifying and detecting nucleic acid |
| WO2024067192A1 (en) * | 2022-09-30 | 2024-04-04 | 华南农业大学 | Linear displacement isothermal amplification method and use thereof |
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| WO2020048307A1 (en) | 2020-03-12 |
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