CN109207563A - A kind of antipollution method of LAMP - Google Patents
A kind of antipollution method of LAMP Download PDFInfo
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- CN109207563A CN109207563A CN201710530484.4A CN201710530484A CN109207563A CN 109207563 A CN109207563 A CN 109207563A CN 201710530484 A CN201710530484 A CN 201710530484A CN 109207563 A CN109207563 A CN 109207563A
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims abstract description 17
- 239000000203 mixture Substances 0.000 claims abstract description 10
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- 238000001514 detection method Methods 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 5
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 5
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Classifications
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- 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
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- 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
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The invention discloses a kind of antipollution methods of LAMP, specifically comprise the following steps: the identification sequence that two inner primers of (1) design LAMP contain restriction endonuclease；(2) by the outer primer of inner primer and LAMP in step (1) and amplification target hybridization, and polymerase and restriction endonuclease is added, mixture is incubated for a period of time, the pollutant in elimination reaction pipe；(3) mixture is incubated for a period of time under the high temperature conditions, inactivates the restriction endonuclease in reaction tube；(4) mixture is placed in preference temperature to react, finally obtains a large amount of amplified production.The characteristics of DNA double chain is cut using restriction endonuclease, the last LAMP product of the identification sequence containing restriction endonuclease entered in LAMP reaction tube because of pollution is cut, prevent primer from being combined with pollution products, to avoid pollution.
Description
Technical field
The invention belongs to nucleic acid detection technique fields, and in particular to a kind of antipollution method of LAMP.
Background technique
Ring mediated isothermal amplification method (LAMP) is a kind of novel constant temperature nucleic acid amplification method of exploitation in 2000, feature
It is 6 regions design, 2 pairs of specific primers for target gene, using a kind of strand displacement archaeal dna polymerase at (63 DEG C of isothermy
Left and right) 30~60min of heat preservation, nucleic acid amplification reaction can be completed.Compared with Standard PCR, thermal denaturation, the temperature of template are not needed
The processes such as circulation, electrophoresis and ultraviolet visualization.LAMP is a kind of completely new nucleic acid amplification method, has simple, quick, high specificity
The characteristics of.The technology can match in excellence or beauty even better than round pcr in the indexs such as sensitivity, specificity and detection range.
But the excessively high sensitivity of LAMP technology causes it easily to pollute, and generates false positive results, severe jamming LAMP inspection
The accuracy of survey.When LAMP is expanded most important pollution sources be containing amplified production identical with target sequence, high concentration
LAMP product easily splashes during experimental implementation or is formed aerosol, to air, reagent, pipettor and operator
Gloves clothing pollute.Currently, it is anti-pollution that researcher both domestic and external proposes many kinds of LAMP in order to solve this problem
Technology, wherein physical isolation method, ultraviolet irradiation method etc. are commonly used in current LAMP experiment.Have become LAMP experimental implementation
Specification process.UNG-dUTP system is invented after occurring the problem of a kind of LAMP pollution causes false positive results for preventing
The method of product pollution.This method has the advantages that pollution sources, UNG (Uracil-DNAGlycosylase, uracil can be eliminated
DNA glycosylase) it handles and can be carried out in the same reaction tube with LAMP, there is simplicity.Its technical principle is: in LAMP
DUTP is added in reaction system to replace dTTP or dUTP and dTTP is added simultaneously according to a certain percentage, makes in PCR reaction product
A large amount of dUTP is mixed, upper primary LAMP product (dU- can be eliminated with UNG processing PCR reaction system before PCR again
DNA residual contamination).UNG is under the conditions of certain temperature, N- glycosyl bond between cleavable uracil base and sugared phosphoric acid backbone,
The dUTP in last PCR product double-strand is removed, preventing archaeal dna polymerase is that template extends, and UNG is not to or not dU-DNA
The amplification of template containing dUTP has no effect.
Although physical isolation method, ultraviolet irradiation method etc. has been proved in current LAMP experiment can be effectively controlled expansion
Increase production the diffusion and pollution of object, but still can not decontaminate completely.And UNG-dUTP system needs to uncap again enzyme, this can lead
Pollution sources are caused to be again introduced into reaction system.
Summary of the invention
For there is technical issues that in ring mediated isothermal amplification method existing in the prior art occur, it is of the invention
Be designed to provide a kind of loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification,
LAMP) antipollution method.
The technical scheme adopted by the invention is as follows:
A kind of antipollution method of LAMP, which is characterized in that specifically comprise the following steps:
(1) the identification sequence that two inner primers of LAMP contain restriction endonuclease is designed；
(2) by the outer primer of inner primer and LAMP in step (1) and amplification target hybridization, and polymerase and limitation is added
Property endonuclease, mixture is incubated for a period of time, the pollutant in elimination reaction pipe；
(3) mixture is incubated for a period of time under the high temperature conditions, inactivates the restriction endonuclease in reaction tube；
(4) mixture is placed in preference temperature to react, finally obtains a large amount of amplified production.
Different reaction methods can be used according to different restriction enzymes for above-mentioned steps (3) and step (4), that is, have each
Kind of situation, some need elder generation low-temperature treatment high-temperature inactivation again, or some do not need low-temperature treatment, and room temperature sample-adding is just eliminated dirty on the way
Dye, direct high-temperature inactivation is just.LAMP reaction is carried out again or directly, is just eliminated during being warming up to reaction temperature dirty
Dye inactivates restriction endonuclease.
Further, in the step (1) the upstream inner primer of two inner primers of LAMP and downstream inner primer sequence
In enzyme recognition site sequence all containing one section of restriction enzyme.
All contain one in the upstream inner primer of two inner primers of LAMP and the sequence of downstream inner primer in the step (1)
I (Bpm I) restriction enzyme site sequence of section 6nt restriction enzyme Gsu, wherein the restriction enzyme site sequence in the inner primer of upstream is 5-
CTGGAG-3, the restriction enzyme site sequence in the inner primer of downstream is 5-CTGGAG-3.
Further, the amplifying target nucleic acid sequence in the step (2) is vibrio parahemolyticus DNA.
Further, restriction enzyme cuts off LAMP Product Sequence in the step (3), makes primer and cleaved products
Between at the reaction temperatures can not in conjunction with extend.
Further, in the step (3) restriction enzyme cleavage site inner primer terminal position.
Further, the identification sequence of step (3) restriction enzyme is different from cleavage site, and cleavage site is being known
After 3 ' ends of other sequence.
Further, the incubation temperature in the step (3) is lower than the reaction temperature of LAMP.
Further, the normal temperature condition in the step (3) is 20-45 DEG C, incubation time 0-20min.
Further, the normal temperature condition in the step (3) is 30-40 DEG C, incubation time 5-10min.
Further, the hot conditions in the step (4) will be lower than the deactivation temperature of LAMP reaction enzymes.
Further, the hot conditions in the step (4) are 60-90 DEG C, incubation time 0-20min.
Further, the hot conditions in the step (4) are 70-80 DEG C, incubation time 5-10min.
Further, restriction enzyme is inactivated at 30-70 DEG C in the step (4).
Further, restriction enzyme is inactivated at 65 DEG C in the step (4).
Further, it is just inactivated during being warming up to LAMP reaction temperature if obtaining low temperature restriction endonuclease,
The step (3) is omitted.
Further, the preference temperature in the step (5) is LAMP reaction temperature.
Further, the optimum temperature in the step (5) is 59-72 DEG C.
Further, the optimum temperature in the step (5) is 60-65 DEG C.
LAMP amplification method can be replaced, such as strand displacement amplification reaction (Strand displacement
Amplification, SDA), rolling circle amplification (Rolling circleamplification, RCA), ring mediated isothermal amplification skill
Art (Loop-mediated isothermal amplification, LAMP), the amplified reaction for relying on unwindase
(Helicase-dependent amplification, HDA) and the strand displacement mediated based on denaturation bubble expand (denaturation
Bubble-mediated strand exchange amplification) etc. or other isothermal nucleic acid amplification methods in appoint
It anticipates one kind.In the identification sequence of the suitable position addition restriction endonuclease of the primer of its design.
The invention has the benefit that
The identification sequence of one section of restriction endonuclease is added between two sections of sequences that the present invention passes through the inner primer in LAMP
Column, using restriction endonuclease to DNA double chain cut the characteristics of, by enters because of pollution in LAMP reaction tube contain it is limited
The last LAMP product of the identification sequence of property endonuclease processed is cut, and prevents primer from being tied with pollution products
It closes, to avoid pollution.High-temperature process inactivates restriction endonuclease, therefore will not be to subsequent normal LAMP product
Generation effect.The present invention can carry out in same reaction tube with LAMP, not need process of uncapping again, simple and efficient.
(1) easy to operate, a pipe is completed, and is not required to uncap again.
(2) high-efficient, 103 pollution can be completely eliminated, under the conditions of high density pollution object, being handled can also be postponed
20min。
(3) normal LAMP reaction is not influenced.To its sensitivity, detection limit is without influence.
Detailed description of the invention
Fig. 1 is LAMP constant-temperature amplification schematic diagram；
Fig. 2 is LAMP experimental specificity primer and normal primer comparison diagram；
Fig. 3 is LAMP product 10-4To 10-8Whether there is or not I enzyme comparison diagrams of Gsu；
Fig. 4 is that whether there is or not the LAMP production concentration gradient comparison curve graphs of I enzymatic treatment of Gsu；
Fig. 5 for no I enzymatic treatment of Gsu normal LAMP concentration gradient electrophoretogram；
Fig. 6 is that whether there is or not the LAMP electrophoretograms of I enzymatic treatment of Gsu.
Specific embodiment
With reference to the accompanying drawing and gene order table further illustrates technical solution of the present invention.
The application provides corresponding sequence table: the correlation in sequence table is labeled as the primer of LAMP amplification vibrio parahemolyticus
The position and direction schematic diagram of design.
LAMP constant-temperature amplification principle
As shown in Fig. 1 and sequence table: upstream primer is respectively " GCGCAAGGTTACAACATCAC "
“TGATACTCACGCCTTGTTCGA”“GCACGGTTTCGTGAACGCG”
Downstream primer is respectively " TTGGACATCAACCGCTCATCGT " " CTCTGAGTGTGCAGCGTC "
“TGTGTTCTGGAATGTCACGC”
The identification sequence of restriction enzyme in FIP and BIP is " CTGGAG "
Restriction enzyme is separately added among the F2 and F1C of interior primers F IP and among the B2 and B1C of inner primer BIP
The identification sequence R1 of Gsu I, carries out LAMP reaction with this primer.Therefore the product obtained contains restriction enzyme Gsu I
Identification sequence R1.By this product after the processing of restriction enzyme Gsu I, LAMP product can be cut into unique DNA chain, this
The new LAMP primer of sample will be unable to it is in combination reacted, thus achieve the purpose that eliminate pollution.
As shown in Fig. 1 and sequence table, the method for the invention carries out under normal temperature conditions first, restriction endonuclease
Pollutant product contained in reaction tube is cut in inner primer region, pollutant is cut into a section and is contained
The small fragment of part inner primer can not carry out LAMP amplification again, and to target nucleic acid sequence and primer without effect.Then it is being suitable for
Restriction endonuclease is inactivated under the conditions of temperature, and polymerase is unaffected.Finally under preference temperature, LAMP is being carried out just
Paradoxical reaction.Therefore this method can eliminate the Aerosol Pollution of LAMP.
Embodiment 1
65 DEG C of 10ul system reaction temperature, 60min
LAMP-FIP is being just: 1.6*10-6M LAMP-BIP is being just: 1.6*10-6M
LAMP-FIP2:1.6*10-6M LAMP-BIP2:1.6*10-6M
LAMP-B3:2.0*10-7M LAMP-F3:2.0*10-7M
Bst 2.0WarmStart archaeal dna polymerase (8units/ul): 0.2ul
1 × Thermopol Buffer (20mM Tris-HCl, 10mM KCl, 10mM (NH4)2SO4, 2mM MgSO4,
25 DEG C of 8.8@of 0.1%Triton X-100, pH)
+MgSO4: 2mM
1.6mM dNTPs
0.25×Eva green
Remaining deficiency H2O filling-in
Experimentation: four groups of reactions are respectively normal LAMP inner primer, target (0.01 × vibrio parahemolyticus bacterium solution),
LAMP inner primer, the target (0.01 × vibrio parahemolyticus bacterium solution) specifically designed, normal LAMP inner primer, without target are special
The LAMP inner primer of different design, without target, other compositions are identical, react at a temperature of 65 degree, experimental result such as Fig. 1.
In the LAMP experimental specificity primer and normal primer comparison diagram of Fig. 2:
Normal primer: normal LAMP inner primer, 0.01 × vibrio parahemolyticus bacterium solution target；
Specific primer: LAMP inner primer, the 0.01 × vibrio parahemolyticus bacterium solution target specifically designed；
Normal primer NTC: normal LAMP inner primer, without target；
Specific primer NTC: the LAMP1-FIP primer that specifically designs, without target；
As shown in Fig. 2, normal LAMP inner primer and the LAMP inner primer specifically designed rise in the case where there is target
Peak and rise a peak time be not much different.And peak is not played in the case where no target.The result illustrates to draw in the LAMP specifically designed
Object can replace normal LAMP inner primer, and not influence detection of the LAMP reaction to vibrio parahemolyticus.
Embodiment 2
37 DEG C of low temperature of 10ul system reaction temperature, 10min；70 DEG C of high temperature, 10min；65 DEG C, 60min
LAMP-FIP2:1.8*10-6M LAMP-BIP2:1.8*10-6M
LAMP-B3:2.0*10-7M LAMP-F3:2.0*10-7M
Bst 2.0WarmStart archaeal dna polymerase (8units/ul): 0.2ul
1×FastDigest Buffer
1.6mM dNTPs
0.25×Eva green
Target: I endonuclease recognition sequence LAMP product of Gsu is had
Remaining deficiency H2O filling-in
Experimentation: two groups of experiments, I restriction endonuclease (5units/ul) of addition 0.1ul Gsu, another is not added
I restriction endonuclease of Gsu.
Every group is done target dilution 104——108Target concentration gradient again.Other conditions are identical.The following Fig. 3 of experimental result
It is shown:
10-4: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 104Times
10-5: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 105Times
10-6: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 106Times
10-7: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 107Times
10-8: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 108Times
10- 4,Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 104Times
10- 5,Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 105Times
10- 6,Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 106Times
10- 7,Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 107Times
10- 8,Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 108Times
Conclusion: target concentration is in dilution 107Times when, through I inscribe enzymatic treatment of Gsu, LAMP experiment will not play a peak.
Embodiment 3
37 DEG C of 10ul system reaction temperature, 10min；70 DEG C, 10min；65 DEG C, 60min
LAMP-FIP2:1.8*10-6M LAMP-BIP2:1.8*10-6M
LAMP-B3:2.0*10-7M LAMP-F3:2.0*10-7M
Bst 2.0WarmStart archaeal dna polymerase (8units/ul): 0.2ul
1×FastDigest Buffer
1.6mM dNTPs
0.25×Eva green
Target: I endonuclease recognition sequence LAMP product of Gsu is had
Remaining deficiency H2O filling-in
Experimentation: two groups of experiments, I restriction endonuclease (5units/ul) of addition 0.1ul Gsu, another is not added
I restriction endonuclease of Gsu.
Every group is done dilution 107Times --- 1010Target concentration gradient again.Other conditions are identical.The following Fig. 4 institute of experimental result
Show:
10-7: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 107Times
10-8: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 108Times
10-9: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 109Times
10-10: I restriction endonuclease of Gsu is not added, target concentration: LAMP product dilution 1010Times
10-7Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 107Times
10-8Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 108Times
10-9Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 109Times
10-10Gsu I: addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 1010Times
As shown in the normal LAMP concentration gradient of Fig. 5 and detection limit:
Take experiment in do not add I restriction endonuclease of Gsu target 1 μ L of reaction product, add water supply 10 μ L add again 2 μ L6 ×
Loading buffer runs glue 25min result such as Fig. 5 under conditions of 2% agarose gel electrophoresis, voltage 110V.
M:2000bp DNA Marker；
1: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 107Times
2: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 108Times
3: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 109Times
4: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 1010Times
Gsu as shown in Figure 6 has to have no contrast
It takes in experiment and dilutes 107Times --- 109Times target 1 μ L of reaction product, add water supply 10 μ L add again 2 μ L6 ×
Loading buffer runs glue 25min result such as Fig. 6 under conditions of 2% agarose gel electrophoresis, voltage 110V.
M:2000bp DNA Marker；
1: the product of addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 107Times
2: the product of addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 108Times
3: the product of addition I restriction endonuclease of Gsu, target concentration: LAMP product dilution 109Times
4: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 107Times
5: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 108Times
6: not adding the product of I restriction endonuclease of Gsu, target concentration: LAMP product dilution 109Times
From Fig. 4, Fig. 5, Fig. 6, we are it can be concluded that the detection without the normal LAMP of I inscribe enzymatic treatment of Gsu is limited to
LAMP product dilution 1010Times, in dilution 107Times --- 109Without the normal of I inscribe enzymatic treatment of Gsu in target concentration again
LAMP plays peak, and the LAMP through I inscribe enzymatic treatment of Gsu does not play peak.Therefore the LAMP experiment through I inscribe enzymatic treatment of Gsu is anti-
Pollution can achieve 103Magnitude.
The above is not limitation of the present invention, it should be pointed out that: those skilled in the art are come
It says, under the premise of not departing from essential scope of the present invention, several variations, modifications, additions or substitutions can also be made, these improvement
It also should be regarded as protection scope of the present invention with retouching.
Sequence table
V.parahaemolyticus thermolabile hemolysin (TL) gene, complete cds
GenBank: M36437.1
GenBank listens Graphics
>gi|155177|gb|M36437.1|VIBHEMOX V.parahaemolyticus thermolabile hemolysin
(TL) gene, complete cds
TCGACTGTCTGGAGTATTTACTCAGAATTAGAGGGTTTAGTGAGAAAAATTCTCATCTAAACAATGTTATAGC
CAAGTATTTTTTCAATGTGCTTGGGTCAATAACCATTACAAGAAGAGTGAATGATGAAAAAAACAATCACACTATTA
ACTGCATTACTCCCGCTTGCTTCTGCAGTTGCCGAAGAGCCAACCTTATCACCAGAAATGGTTTCAGCGTCTGAAGT
GATCAGCACGCAAGAAAACCAAACCTATACCTATGTTCGCTGTTGGTATCGCACCAGCTACTCGAAAGATGATCCAG
CGACCGATTGGGAATGGGCAAAAAACGAAGATGGTAGCTACTTCACCATTGACGGCTACTGGTGGAGCTCCGTTTCA
TTTAAAAACATGTTCTACACCAACACGTCGCAAAACGTTATCCGTCAGCGTTGTGAAGCAACATTAGATTTGGCGAA
CGAGAACGCAGACATTACGTTCTTCGCCGCTGACAATCGCTTCTCATACAACCACACGATCTGGAGCAACGACGCAG
CAATGCAGCCAGATCAAATCAACAAAGTGGTTGCACTCGGTGACAGCTTGTCTGATACAGGCAACATCTTTAACGCA
TCACAATGGCGCTTCCCTAACCCGAACAGCTGGTTCTTAGGTCACTTCTCCAACGGTTTTGTGTGGACAGAATACAT
TGCCAAAGCGAAGAACCTTCCGCTCTACAACTGGGCAGTTGGCGGCGCGGCTGGTGAGAACCAATACATCGCGCTAA
CAGGGGTTGGTGATCAAGTTTCTTCGTACTTAACCTACGCAAAACTGGCGAAGAACTACAAACCAGCAAACACCTTG
TTTACGCTTGAGTTTGGTTTGAATGACTTCATGAACTACAACCGTGGCGTTCCAGAAGTGAAAGCGGATTATGCAGA
AGCACTGATTCGTTTGACGGACGCAGGTGCGAAGAACTTCATGTTGATGACACTGCCAGATGCGACGAAAGCGCCTC
AGTTTAAGTACTCAACACAAGAAGAGATCGACAAAATTCGTGCGAAAGTGCTTGAGATGAACGAGTTCATCAAGGCA
CAAGCGATGTACTACAAAGCGCAAGGTTACAACATCACGTTGTTTGATACTCACGCCTTGTTCGAGACGCTAACTTC
TGCGCCCGAAGAGCACGGTTTCGTGAACGCGAGCGATCCTTGTTTGGACATCAACCGCTCATCGTCTGTCGATTACA
TGTACACCCACGCATTGCGCTCTGAGTGTGCAGCGTCTGGTGCTGAGAAGTTTGTGTTCTGGAATGTCACGCATCCA
ACAACAGCAACTCACCGCTATGTTGCAGAGAAAATGCTAGAAAGTAGCAACAACTTAGCCGAGTACCGTTTCTAACC
GGACACGGCTTCTGAGTTGAAACCTTATCTTCGTACACACGTTGATAACGAACACATCGTGGCCATTTTTATCGAAG
GAACGTTGTGGTCACAGCAGTCACAACGCTAAACAAGTTACAGTGGCGCGACGTCGGTTCCCCCTAAAGACTGGTAA
AGCGTAACCTGAGTGAGAAACTGGTTGTAGCGGTTCTCCAACAAAGAAGC
F3:GCGCAAGGTTACAACATCAC(20) nt
B3:GCGTGACATTCCAGAACACA(20) nt
FIP:CGCGTTCACGAAACCGTGC CTGGAG TGATACTCACGCCTTGTTCGA(19+6+21) nt
BIP:TTGGACATCAACCGCTCATCGT CTGGAG GACGCTGCACACTCAGAG(22+6+18) nt
Claims (9)
1. a kind of antipollution method of LAMP, which is characterized in that specifically comprise the following steps:
(1) the identification sequence that two inner primers of LAMP contain restriction endonuclease is designed；
(2) by the outer primer of inner primer and LAMP in step (1) and amplification target hybridization, and polymerase and restricted core is added
Mixture is incubated for a period of time, the pollutant in elimination reaction pipe by sour restriction endonuclease；
(3) mixture is incubated for a period of time under the high temperature conditions, inactivates the restriction endonuclease in reaction tube；
(4) mixture is placed in preference temperature to react, finally obtains a large amount of amplified production.
2. a kind of antipollution method of LAMP according to claim 1, which is characterized in that the two of LAMP in the step (1)
Enzyme recognition site sequence in the upstream inner primer of inner primer and the sequence of downstream inner primer all containing one section of restriction enzyme
Column.
3. a kind of antipollution method of LAMP according to claim 1, which is characterized in that in restricted in the step (3)
LAMP Product Sequence is cut off in enzyme cutting, and making at the reaction temperatures can not be in conjunction with extension between primer and cleaved products.
4. a kind of antipollution method of LAMP according to claim 1, which is characterized in that step (3) restriction enzyme
The identification sequence of enzyme is different from cleavage site, and cleavage site is after 3 ' ends of identification sequence.
5. a kind of antipollution method of LAMP according to claim 1, which is characterized in that the incubation temperature in the step (3)
Degree is lower than the reaction temperature of LAMP.
6. a kind of antipollution method of LAMP according to claim 1, which is characterized in that the high temperature item in the step (4)
Part will be lower than the deactivation temperature of LAMP reaction enzymes.
7. a kind of antipollution method of LAMP according to claim 1, which is characterized in that if obtaining in low temperature restriction nuclease
Enzyme cutting just inactivates during being warming up to LAMP reaction temperature, and the step (3) is omitted.
8. a kind of antipollution method of LAMP according to claim 1, which is characterized in that the suitable temperature in the step (5)
Degree is LAMP reaction temperature.
9. a kind of antipollution method of LAMP according to claim 1, which is characterized in that the LAMP is anti-pollution to be can be replaced
It replaces amplified reaction, rolling circle amplification, loop-mediated isothermal amplification technique, the amplified reaction for relying on unwindase and is mediated based on denaturation bubble
Strand displacement amplification or other isothermal nucleic acid amplification methods in any one.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109112224A (en) * | 2018-08-24 | 2019-01-01 | 暨南大学 | Primer and its kit and method based on digital LAMP technology detection vibrio parahaemolytious |
| CN112301102A (en) * | 2019-07-31 | 2021-02-02 | 华南师范大学 | High-specificity loop-mediated isothermal amplification method and application thereof |
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| US5516292A (en) * | 1991-05-02 | 1996-05-14 | The Research Foundation Of State University Of New York | Primer directed nucleic acid amplification including the addition of specific template DNA inactivating enzyme |
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| CN112301102A (en) * | 2019-07-31 | 2021-02-02 | 华南师范大学 | High-specificity loop-mediated isothermal amplification method and application thereof |
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