CN109161582A - It is a kind of for the reagent and its kit of ring mediated isothermal amplification and application - Google Patents
It is a kind of for the reagent and its kit of ring mediated isothermal amplification and application Download PDFInfo
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- CN109161582A CN109161582A CN201810922940.4A CN201810922940A CN109161582A CN 109161582 A CN109161582 A CN 109161582A CN 201810922940 A CN201810922940 A CN 201810922940A CN 109161582 A CN109161582 A CN 109161582A
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Abstract
The present invention relates to a kind of reagents for ring mediated isothermal amplification comprising at least one of nano particle, dithiothreitol (DTT), peptide cow's serum, tetramethyl ammonium chloride, trehalose；The invention further relates to a kind of kit containing mentioned reagent and its application, the kits further include: 10 × constant temperature buffer, dNTP mixture, MgSO4, Bst archaeal dna polymerase, indicator and water, the final concentration of each component is as follows in the loop-mediated isothermal amplification system as made from the kit: nano particle 0-10mg/mL；Dithiothreitol (DTT) 0-10M；Peptide cow's serum 0-10g/mL；Tetramethyl ammonium chloride 0-100mM；Trehalose 0-50mM；DNTP mixture 1.0-3.0mM；MgSO44-10mM；Bst archaeal dna polymerase 0.1-0.5U/ μ L.The present invention ensures to detect quality while solution LAMP reagent high cost problem, kit of the present invention has detection effect identical with import reagent, its sensitivity, specificity and stability can meet clinical trial demand, but testing cost can be greatly lowered, and have a good application prospect.
Description
Technical field
The invention belongs to nucleic acid amplification technologies fields, and in particular to a kind of reagent for ring mediated isothermal amplification, and
The application of kit containing the reagent and the kit.
Background technique
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a kind of difference
In the beyond body nucleic acid gene magnification new technology of Standard PCR.LAMP is mainly set using two or three pairs of specific primers and with chain
Change active Bst archaeal dna polymerase, continuous rapid amplifying under constant temperature conditions.Compared with traditional round pcr, this technology tool
There are higher sensitivity, specificity and amplification efficiency, contrasuppressor ability is strong, low to nucleic acid-templated quality requirement, therefore can be with
The step of greatly simplifying nucleic acid extraction, this technology can amplify 10 in 1h9~1010Times target sequence copy, testing result
View mode is versatile and flexible, may is that electrophoresis, nephelometry (direct unaided visual or using professional equipment), dye method (pass through
The direct judging result of the variation of reaction system color), real-time fluorescence curves method (be similar to SYBR real-time PCR methodology), can satisfy
The requirement of various difference detection platforms, current application is increasingly extensive, has been applied to clinical diagnosis, environmental monitoring, food source peace
Congruent field.In terms of medicine, LAMP technology has been applied to the detection of bacterium class pathogenic microorganism, virus type pathogenic microorganism
Detection, the detection of fungal microbe, the rapid field detection of acute infectious disease, oncogene detection etc., the technology is instant
Application in terms of detection (POCT) has important value.
However, current LAMP reagent market is mainly monopolized by offshore company (such as Rong Yan company of Japan), it is on the one hand former
Because being: the technical monopoly of LAMP patent, another aspect assignable cause is: the price of imported L AMP reagent is too high, price
About 80 yuan of -100 yuan/person-portions, cost are much higher than regular-PCR method (3 yuan -10 yuan), significantly limit the technology pushing away at home
Extensively (domestic LAMP kit mostly is difficult to be received by client because of quality defect).Although more next currently in order to break up monopoly
More Research tendencies domesticizes in by high-cost import reagent, however does not obtain good effect.
Summary of the invention
The purpose of the present invention is to overcome the defects in the prior art, provides a kind of examination for ring mediated isothermal amplification
Agent, and the application of the kit containing the reagent and the kit, the present invention are solving the same of the high cost problem of LAMP reagent
When ensure detect quality, kit of the present invention have detection effect identical with import reagent, sensitivity, specificity
Clinical trial demand can be met with stability, but testing cost can be greatly lowered.
To achieve the above object, the present invention adopts the following technical scheme:
The first purpose of the invention is to provide a kind of reagents for ring mediated isothermal amplification comprising nano particle,
At least one of dithiothreitol (DTT) (DTT), peptide cow's serum (BSA), tetramethyl ammonium chloride (TMAC), trehalose.
In order to advanced optimize mentioned reagent, the technical measures that the present invention takes further include:
Further, the reagent includes nano particle；Alternatively, the reagent includes nano particle, dithiothreitol (DTT)
(DTT), peptide cow's serum (BSA)；Alternatively, the reagent include nano particle, dithiothreitol (DTT) (DTT), peptide cow's serum (BSA),
Tetramethyl ammonium chloride (TMAC).
Further, the material of the nano particle includes gold, silver, silica, polystyrene or graphene.More preferably
Are as follows: gold nano grain.
Further, the surface of the nanometer material particle is positively charged or neutral, a diameter of 0-1000nm；It is more excellent
It is selected as 1~200nm；More preferably 2~10nm.
Further, the diameter of the nanometer material particle is 5nm, concentration 0.1mg/mL.
A second object of the present invention is to provide a kind of examinations for ring mediated isothermal amplification containing any mentioned reagent
Agent box.
In order to advanced optimize mentioned reagent box, the technical measures that the present invention takes further include:
Further, the kit further include: 10 × constant temperature buffer, dNTP mixture (dNTP mix), MgSO4、
Bst archaeal dna polymerase, indicator.
Further, the final concentration of each component is as follows in loop-mediated isothermal amplification system: nano particle 0-10mg/
mL；Dithiothreitol (DTT) 0-10M；Peptide cow's serum 0-10g/mL；Tetramethyl ammonium chloride 0-100mM；Trehalose 0-50mM；DNTP mixing
Object 1.0-3.0mM；MgSO44-10mM；Bst archaeal dna polymerase 0.1-0.5U/ μ L.More preferably: nano particle 0-2mg/mL；
Dithiothreitol (DTT) 0-2M；Peptide cow's serum 0-2g/mL；Tetramethyl ammonium chloride 0-10mM；Trehalose 0-10mM；DNTP mixture 1.0-
2.0mM；MgSO45-8mM；Bst archaeal dna polymerase 0.2-0.4U/ μ L.
Further, the final concentration of each component is as follows in loop-mediated isothermal amplification system: dNTP mixture
1.5mM；MgSO46mM；Bst archaeal dna polymerase 0.32U/ μ L；Nano particle 0.012mg/mL；Dithiothreitol (DTT) 0.08M；Peptide ox
Serum 0.01g/mL；Tetramethyl ammonium chloride 2mM；Trehalose 3.2mM.
Further, the indicator be developing dye or fluorescence indicator, the developing dye include HNB,
Calcein, cresol red, phenol red, m-cresol purple, bromocresol purple, dimethyl diaminophenazine chloride, naphtholphthalein, thymol blue etc., the fluorescence indicator
Including SYBR Green, Ever Green, Pico Green, SYTO series etc..It is highly preferred that the indicator is dimethyl diaminophenazine chloride dye
Material.
Further, the original content of each component and its dosage are as follows: the concentration of dNTP mixture is 25mM, and dosage is 1.5 μ
L；MgSO4Concentration be 100mM, dosage be 1.5 μ L；The concentration of Bst archaeal dna polymerase is 8U/ μ L, and dosage is 1 μ L；Nanometer
The diameter of grain is 5nm, and concentration 0.1mg/mL, dosage is 3 μ L；The concentration of dithiothreitol (DTT) is 2M, and dosage is 1 μ L；Peptide ox blood
Clear concentration is 0.25g/mL, and dosage is 1 μ L；The concentration of tetramethyl ammonium chloride is 50mM, and dosage is 1 μ L；The concentration of trehalose
For 80mM, dosage is 1 μ L；Wherein, the reaction system of the reaction system is 25 μ L, also contains 10 × constant temperature buffer, 2.5 μ
L, 1 μ L of indicator and Primer composition and nucleic acid-templated, surplus is supplied by water.
Further, the amplification condition of loop-mediated isothermal amplification system are as follows: 60~68 DEG C of amplification 20-60min.It is more excellent
It is selected as: 65 DEG C of amplification 60min.
Third object of the present invention is to provide a kind of application of any mentioned reagent box in LAMP amplification.The kit
It is a kind of universal kit, can be used for any suitable LAMP amplified reaction, used primer is fixed according to different demands
System synthesis.
Further, the application is based on non-diagnostic and therapeutic purposes the detection for the mutation of CALR-2 type, in institute
State the Primer composition that the sequence as shown in SEQ ID NO:2~SEQ ID NO:7 is used in application.
Further, the target sequence of the Primer composition amplification is as shown in SEQ ID NO:1.
Further, in this application, the particular sequence of target sequence and Primer composition is as follows:
Further, in this application, in amplification reaction system, Primer composition and nucleic acid-templated concentration and dosage
It is as follows:
| Primer | Concentration | Dosage (μ L) | Final concentration in reaction system |
| Primers F 3 | 100μM | 0.05 | 0.2μM |
| Primers F 3 | 100μM | 0.05 | 0.2μM |
| Primer B3 | 100μM | 0.05 | 0.2μM |
| Primers F IP | 100μM | 0.4 | 1.6μM |
| Primer BIP | 100μM | 0.4 | 1.6μM |
| Primer LF | 100μM | 0.2 | 0.8μM |
| Primer LB | 100μM | 0.2 | 0.8μM |
| It is nucleic acid-templated | - | 1 | - |
Compared with prior art, the invention has the following advantages:
LAMP ring mediated isothermal amplification reagent and kit of the present invention, testing result are able to achieve visualization inspection
Survey, there is good comparativity with the import reagent box (Japanese Rong Yan etc.) of the marketization, be greatly reduced in reagent cost (detection at
Originally 2-3 member/person-portion can be reduced to) on the basis of, it can ensure that sensitivity, specificity, stability and the repeatability of reaction system,
It has a good application prospect.
Detailed description of the invention
Fig. 1 is the schematic diagram for carrying out specificity verification to 2 type mutant primer system of CALR gene with homemade LAMP reagent；
Wherein, B1: clinical CALR-1 is mutated positive gene group DNA；B2: clinical CALR-2 is mutated positive gene group DNA；B3: clinical
CALR wild type gene group DNA；B4:CALR-1 is mutated positive plasmid；B5:CALR-2 is mutated positive plasmid；B6:CALR wild type
Plasmid；B7: blank control.
Fig. 2 is the fluorescence detection comparative result figure of reagent of the present invention and Japan's Rong Yan reagent；
Fig. 3 is the Monitoring lower-cut value comparison diagram of reagent of the present invention and Japan's Rong Yan reagent；Wherein；Fig. 3's is upper
Sample representated by two partial amplification curves, is from left to right respectively as follows: 104copies/ml,103Copies/ml, other samples
Product are without any amplified signal, the Japanese Rong Yan reagent system of import；Representated by two amplification curves below the lower part of Fig. 3
Sample, be from left to right respectively as follows: 104copies/ml,103Copies/ml, other samples are without any amplified signal, self-control
LAMP reagent system.
Specific embodiment
The present invention provides a kind of reagents for ring mediated isothermal amplification comprising nano particle, dithiothreitol (DTT), peptide
At least one of cow's serum, tetramethyl ammonium chloride, trehalose；The invention further relates to a kind of kit containing mentioned reagent and
The application of the kit.
With reference to the accompanying drawings and examples, further description of the specific embodiments of the present invention.Following embodiment is only
For clearly illustrating technical solution of the present invention, and not intended to limit the protection scope of the present invention.
Embodiment 1- is used for the reagent of LAMP ring mediated isothermal amplification
Include following component: nano particle for the reagent of LAMP ring mediated isothermal amplification involved in the present embodiment
At least one of (0.1mg/mL), DTT (2M), BSA (0.25g/mL), TMAC (50mM), trehalose (80mM)；Wherein, it receives
The material of rice grain is gold, silver, silica, polystyrene or graphene, and surface is positively charged or neutral, diameter
For 0-1000nm.
Embodiment 2- is used for the kit of LAMP ring mediated isothermal amplification
Kit involved in the present embodiment include following ingredient: 10 × constant temperature buffer, dNTP mix (25mM),
MgSO4(100mM), Bst archaeal dna polymerase (8U/ μ L), neutral red dye, nano particle (0.1mg/mL), DTT (2M), BSA
(0.25g/mL), TMAC (50mM), trehalose (80mM)；Wherein, the material of nano particle is gold, silver, silica, polyphenyl second
Alkene or graphene, surface is positively charged or neutral, a diameter of 0-1000nm.Above-mentioned kit still alternatively wraps
Composition containing amplimer.
In the loop-mediated isothermal amplification system prepared by the kit, the final concentration of each ingredient is as follows: nano particle
0-10mg/mL；Dithiothreitol (DTT) 0-10M；Peptide cow's serum 0-10g/mL；Tetramethyl ammonium chloride 0-100mM；Trehalose 0-50mM；
DNTP mixture 1.0-3.0mM；MgSO44-10mM；Bst archaeal dna polymerase 0.1-0.5U/ μ L.
Embodiment 3- is used for the kit of LAMP ring mediated isothermal amplification
The present embodiment is the kit of another preferred versions, and it includes following ingredients: 10 × constant temperature buffer, dNTP mix
(25mM)、MgSO4(100mM), Bst archaeal dna polymerase (8U/ μ L), neutral red dye, nano particle (0.1mg/mL), DTT
(2M),BSA(0.25g/mL)；Wherein, the material of nano particle is gold nano grain, and surface is positively charged or neutral,
A diameter of 1~200nm.Above-mentioned kit still alternatively includes amplimer composition.
In the loop-mediated isothermal amplification system prepared by the kit, the final concentration of each ingredient is as follows: gold nano
Grain 0-2mg/mL；Dithiothreitol (DTT) 0-2M；Peptide cow's serum 0-2g/mL；DNTP mixture 1.0-3.0mM；MgSO44-10mM；
Bst archaeal dna polymerase 0.1-0.5U/ μ L.
Embodiment 4- is used for the kit of LAMP ring mediated isothermal amplification
The present embodiment is the kit of another preferred versions, and it includes following ingredients: 10 × constant temperature buffer, dNTP mix
(25mM)、MgSO4(100mM), Bst archaeal dna polymerase (8U/ μ L), neutral red dye, nano particle (0.1mg/mL), DTT
(2M),BSA(0.25g/mL),TMAC(50mM)；Wherein, the material of nano particle is nano SiO 2 particle, surface band
Positive charge or neutral, a diameter of 2~10nm.Above-mentioned kit still alternatively includes amplimer composition.
In the loop-mediated isothermal amplification system prepared by the kit, the final concentration of each ingredient is as follows: gold nano
Grain 0-2mg/mL；Dithiothreitol (DTT) 0-2M；Peptide cow's serum 0-2g/mL；Tetramethyl ammonium chloride 0-10mM；DNTP mixture 1.0-
2.0mM；MgSO45-8mM；Bst archaeal dna polymerase 0.2-0.4U/ μ L.
Embodiment 5- is used for the kit of LAMP ring mediated isothermal amplification
The present embodiment is the kit of another preferred versions, and it includes following ingredients: 10 × constant temperature buffer, dNTP mix
(25mM)、MgSO4(100mM), Bst archaeal dna polymerase (8U/ μ L), neutral red dye, nano particle (0.1mg/mL), TMAC
(50mM)；Wherein, the material of nano particle is graphene nano particle, and surface is positively charged or neutral, a diameter of
5nm.Above-mentioned kit still alternatively includes amplimer composition.
In the loop-mediated isothermal amplification system prepared by the kit, the final concentration of each ingredient is as follows: gold nano
Grain 0.06mg/mL；DNTP mixture 1.5mM；MgSO46mM；Bst archaeal dna polymerase 0.32U/ μ L.
Kit is applied to the quick detection of 2 type of CALR gene mutation (CALR-2) by Application Example 1-
The present embodiment carries out the quick detection of CALR-2 type mutation using kit as described in Example 2, to the reagent
The content of each component is specifically set in box.The detection method is specifically related to following steps:
1.CALR-2 type mutation-detection architecture target sequence information and design of primers；
CALR-2 type mutation-detection architecture target sequence utilizes routine side as shown in SEQ ID NO:1, according to the target sequence
Method design primer sequence, shown in table specific as follows:
2. the preparation of reaction system；
Corresponding reaction system is configured according to kit as described in example 2, reaction system total volume is 25 μ L, the reaction
The dosage of each component and its ingredient final concentration in reaction system are as shown in the table in system:
LAMP reaction, LAMP reaction condition are carried out using the reaction system that mentioned reagent box is prepared are as follows: 65 DEG C, 60min；On
Stating LAMP reaction equipment used is that regular-PCR instrument or constant-temperature metal bath etc. can be stablized and provide the equipment of 65 DEG C of constant temperature；Then
Visualization interpretation is carried out to identify whether sample is that the mutation of 2 type of CALR gene is positive, specially result judges: after reaction,
The color of reaction solution from it is original it is faint yellow become red (in reaction process, the PH of system is changing), that is, be judged to reacting sun
Property.
The TA cloned plasmids containing target sequence are constructed, the sample of gradient concentration is prepared, specific concentration is 101Copies/ml,
102Copies/ml, 103Copies/ml, 105Copies/ml, 107Copies/ml, 1010Copies/ml, with what is configured
LAMP reagent is detected, and is as a result prompted: being greater than 10 for concentration2The sample standard deviation of copies/ml can be by stable detection；To facing
The detection of the recombinant plasmid energy specificity of the genomic DNA and building of 2 type mutation patient whole blood's sample of bed CALR gene, to CALR
1 type of gene be mutated patient whole blood's sample genomic DNA and building recombinant plasmid or normal person wild type gene group DNA and
For the recombinant plasmid of building without nonspecific amplification, detection effect is good, and testing result is as shown in Figure 1.
It has been transferred to the 293T cell of CALR-2 type mutant plasmid, has been transferred to the 293T cell of wild plasmid, be 106It is a
Cell extracts DNA with genome DNA extraction kit, then dilutes in proportion, the sample of gradient mutational load concentration is made,
It is expanded with the LAMP reaction system established, discovery mutational load detection sensibility can reach 1%, real-time with sonde method
The optimum efficiency of PCR is consistent.
Application Example 2-kit of the present invention and Japan's Rong Yan kit test result compare
Test in the present embodiment can both be detected in PCR instrument with fluorescent dye determination, can also use the reality of Japanese import
When monitoring transmissometer (LA-500) detected.
(1) it on real time PCR instrument (AB7300), is detected with fluorescent dye determination；
The self-designed L AMP reagent (expansion in Application Example 1 as described above of the present invention is respectively adopted in the present embodiment
Increasing system), the Japanese Rong Yan reagent of import, while expanding the TA cloned plasmids of the segment of target containing CALR-2 of gradient concentration
(AB7300PCR instrument, 0.5min 1 circulation, 90 circulations, time-consuming 45min completes entire Determination altogether), can by such as Fig. 2
Know, the comparison of 1 concentration (every two lines be) successively from left to right are as follows: 108copies/ml,107copies/ml,
106copies/ml,105copies/ml,104copies/ml,103copies/ml.From the result of Fig. 2: the present invention is wanted
The LAMP reagent of protection and the Japanese Rong Yan reagent of import have the comparativity of height.
(2) on real-time monitoring transmissometer (LA-500), detection comparison is carried out with nephelometry method.
The Japan of reagent of the present invention (amplification system of Application Example 1), import is respectively adopted in the present embodiment
Rong Yan reagent, while expanding TA cloned plasmids (LA-500 transmissometer, the 60min completion of the dense segment of target containing CALR-2 of gradient
Entire Determination).As shown in figure 3, part and lower part are respectively indicated with CALR-2TA grams of Japan's Rong Yan reagent amplification thereon
Grand plasmid and CALR-2TA cloned plasmids are expanded with the homemade LAMP reagent of the present invention.TA clone matter used in the present embodiment experiment
Four gradient concentrations of grain sample are as follows: 104Copies/ml, 103Copies/ml, 102Copies/ml, 101copies/ml。
As shown in Figure 3: the Monitoring lower-cut of the homemade LAMP reagent of Japanese Rong Yan reagent sum can only achieve
103Copies/ml, and 102Copies/ml and the sample of following concentration can not detect.This shows self-control of the present invention
LAMP reagent and the Japanese Rong Yan reagent of import have the comparativity of height.
As can be seen from the above embodiments, LAMP loop-mediated isothermal amplification kit of the present invention, testing result are able to achieve
Visual retrieval has good comparativity with the import reagent box (Japanese Rong Yan etc.) of the marketization, substantially drops in reagent cost
On the basis of low, sensitivity, specificity, stability and the repeatability of reaction system can ensure that.
Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it any equivalent modifications to the practical progress and replaces
In generation, is also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by equal transformation and repair
Change, all should be contained within the scope of the invention.
SEQUENCE LISTING
<110>Guan Ming, Cao monarch
<120>a kind of for the reagent and its kit of ring mediated isothermal amplification and application
<130> IPI182623
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 221
<212> DNA
<213> Artificial Sequence
<220>
<223>target sequence
<400> 1
tcctggtcct gatgtcgggg gcgggcaggg ctggcagggg gcaaggccct gaggtgtgtg 60
ctctgcctgc aggcagcaga gaaacaaatg aaggacaaac aggacgagga gcagaggctt 120
aaggaggagg aagaagacaa gaaacgcaaa gaggaggagg aggcagagga caattgtcgg 180
aggatgatga ggacaaagat gaggatgagg aggatgagga g 221
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer F3
<400> 2
tcctggtcct gatgtcgg 18
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer B3
<400> 3
ctcctcatcc tcctcatcct 20
<210> 4
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer FIP
<400> 4
cctcgtcctg tttgtccttc atttgtttca aggccctgag gtgtgt 46
<210> 5
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer BIP
<400> 5
gaggcttaag gaggaggaag aagacacttt gtcctcatca tcctccgac 49
<210> 6
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer LF
<400> 6
tgcctgcagg cagagc 16
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>amplimer LB
<400> 7
gaggaggcag aggacaat 18
Claims (10)
1. a kind of reagent for ring mediated isothermal amplification, which is characterized in that including nano particle, dithiothreitol (DTT), peptide ox blood
Clearly, at least one of tetramethyl ammonium chloride, trehalose.
2. the reagent according to claim 1 for ring mediated isothermal amplification, which is characterized in that the material of the nano particle
Matter includes gold, silver, silica, polystyrene or graphene.
3. being used for the reagent of ring mediated isothermal amplification according to claims requirement 1, which is characterized in that the nanometer
The surface of material particle is positively charged or neutral, a diameter of 0-1000nm.
4. a kind of kit for ring mediated isothermal amplification, which is characterized in that containing described in any one of claims 1 to 3
The reagent for ring mediated isothermal amplification.
5. a kind of kit for ring mediated isothermal amplification according to claim 4, which is characterized in that further include: 10
× constant temperature buffer, dNTP mixture, MgSO4, Bst archaeal dna polymerase and indicator.
6. a kind of kit for ring mediated isothermal amplification according to claim 5, which is characterized in that mediated in ring etc.
The final concentration of each component is as follows in isothermal amplification reaction system: nano particle 0-10mg/mL；Dithiothreitol (DTT) 0-10M；Peptide cow's serum
0-10g/mL；Tetramethyl ammonium chloride 0-100mM；Trehalose 0-50mM；DNTP mixture 1.0-3.0mM；MgSO44-10mM；
Bst archaeal dna polymerase 0.1-0.5U/ μ L.
7. a kind of kit for ring mediated isothermal amplification according to claim 6, which is characterized in that mediated in ring etc.
The final concentration of each component is as follows in isothermal amplification reaction system: dNTP mixture 1.5mM；MgSO46mM；Bst archaeal dna polymerase
0.32U/μL；Nano particle 0.012mg/mL；Dithiothreitol (DTT) 0.08M；Peptide cow's serum 0.01g/mL；Tetramethyl ammonium chloride 2mM；
Trehalose 3.2mM.
8. a kind of kit for ring mediated isothermal amplification according to claim 6, which is characterized in that the ring mediates
The amplification condition of isothermal amplification system are as follows: 60~68 DEG C of amplification 20-60min.
9. a kind of application of kit as described in any one of claim 5~8 in LAMP amplification.
10. application according to claim 9, which is characterized in that the application is the detection being mutated for CALR-2 type,
The Primer composition of the sequence as shown in SEQ ID NO:2~SEQ ID NO:7 is used in the application.
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| CN201810922940.4A CN109161582A (en) | 2018-08-14 | 2018-08-14 | It is a kind of for the reagent and its kit of ring mediated isothermal amplification and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358811A (en) * | 2019-08-20 | 2019-10-22 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of optimization loop-mediated isothermal amplification |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110453013A (en) * | 2019-08-19 | 2019-11-15 | 上海纳米技术及应用国家工程研究中心有限公司 | A method of aquatic animal epidemic disease detection efficiency is promoted using graphene oxide |
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| CN110358811B (en) * | 2019-08-20 | 2024-02-13 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for optimizing loop-mediated isothermal amplification reaction |
| CN112342318A (en) * | 2020-12-09 | 2021-02-09 | 陕西师范大学 | Primer pair, reaction freeze-drying tube and kit for detecting novel coronavirus SARS-CoV2 |
| CN112442555A (en) * | 2020-12-09 | 2021-03-05 | 陕西师范大学 | Visual LAMP detection system for preventing aerosol pollution and preparation method, use method and application thereof |
| CN112442555B (en) * | 2020-12-09 | 2023-05-26 | 陕西师范大学 | Visual LAMP detection system for preventing aerosol pollution and preparation method, using method and application thereof |
| CN112342318B (en) * | 2020-12-09 | 2023-05-30 | 陕西师范大学 | Primer pair, reaction freeze-drying tube and kit for detecting novel coronavirus SARS-CoV2 |
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