CN110438204A - A method of optimizing loop-mediated isothermal amplification using carbon nanotube - Google Patents
A method of optimizing loop-mediated isothermal amplification using carbon nanotube Download PDFInfo
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 239000002041 carbon nanotube Substances 0.000 title claims abstract description 51
- 229910021393 carbon nanotube Inorganic materials 0.000 title claims abstract description 51
- 238000007397 LAMP assay Methods 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000003321 amplification Effects 0.000 claims abstract description 31
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 31
- 238000005457 optimization Methods 0.000 claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 17
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 17
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims description 33
- 239000000463 material Substances 0.000 claims description 20
- 238000011901 isothermal amplification Methods 0.000 claims description 11
- 230000001404 mediated effect Effects 0.000 claims description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 6
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 5
- 239000011535 reaction buffer Substances 0.000 claims description 5
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 claims 1
- 241000335053 Beta vulgaris Species 0.000 claims 1
- 239000003513 alkali Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 235000013305 food Nutrition 0.000 abstract description 3
- 206010058874 Viraemia Diseases 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 241000607142 Salmonella Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002071 nanotube Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000016776 visual perception Effects 0.000 description 1
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- 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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Abstract
The invention discloses a kind of methods using carbon nanotube optimization loop-mediated isothermal amplification, belong to field of biotechnology.Method provided by the present invention is to be added to carbon nanotube in loop-mediated isothermal amplification system to carry out nucleic acid amplification.This method can effectively improve the sensitivity of loop-mediated isothermal amplification, and be able to suppress the appearance of false positive results, reduce non-specific amplification, thus the detection efficiency improved.This method can have wide application prospect by fields such as application food safety detections.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of to optimize loop-mediated isothermal amplification using carbon nanotube
Method.
Background technique
Carbon nanotube is a kind of One-dimensional Quantum material with special construction, has many special mechanics, electricity and change
Performance is learned, was goed deep into recent years with carbon nanotube research, wide application prospect is also constantly shown.Yan Xiyun etc.
(CN200410102574.6), Zhang Zhizhou and Wang Mingchun (CN200610015471.5) are individually disclosed and are being optimized using nanotube
Application in PCR amplification.As another nucleic acid amplification technologies different from polymerase chain reaction action principle --- ring is situated between
In terms of the optimization for leading isothermal amplification, there is not yet the report of carbon nanotube application.
Loop-mediated isothermal amplification (Loop-mediated isothermal amplification, be abbreviated as LAMP)
It is a kind of technology for carrying out nucleic acid amplification under isothermal conditions, can be widely applied to the fields such as food safety detection.The technology
High sensitivity, expanding 15min-1h under isothermal conditions can produce 109~1010Amplicon again, if experimental situation is molten by gas
Glue stain is then easy to generate false positive results.In addition, due to being related to a plurality of primer during amplified reaction, between primer very
It is easy to happen non-specific binding and generates primer dimer, to consume the reaction substrate in reaction system, reduce reaction efficiency
And detection sensitivity, while it being easy to cause result false positive again, so that result is judged by accident.Therefore, how to optimize ring mediated isothermal expansion
Increase reaction so that its it is existing compared with high detection sensitivity, again inhibition non-specific amplification, be always that the technical field is urgently to be solved
Technical problem.
The present invention provides a kind of methods using carbon nanotube optimization loop-mediated isothermal amplification, can further mention
The sensitivity of high detection inhibits non-specific amplification, can be very good to solve to encounter in ring mediated isothermal amplification context of detection
Technical problem.
Summary of the invention
For encountered in existing ring mediated isothermal amplification detection technique due to high sensitivity, experimental situation easily by gas it is molten
The problem of non-specific binding occurs between glue stain and a plurality of primer and is easy to produce false positive and sensitivity decrease, the present invention
Purpose with provide it is a kind of using carbon nanotube optimization loop-mediated isothermal amplification method.The present invention to ring by mediating
Carbon nano-tube material is added in isothermal amplification reaction system to realize effect of optimization, provides a kind of utilization carbon nanotube optimization ring Jie
The method for leading isothermal amplification.It is detected using detection method of the invention, has the advantages that highly sensitive and high specific.
The object of the invention is realized by following scheme: a kind of side optimizing loop-mediated isothermal amplification using carbon nanotube
Method is realized by adding carbon nano-tube material into loop-mediated isothermal amplification system to the excellent of ring mediated isothermal amplification
Change, includes the following steps:
(1) amplification reaction system optimizes: carbon nanotube being added in loop-mediated isothermal amplification system, core is then carried out
Acid amplification；
(2) nucleic acid amplification product detects: can pass through electrophoresis detection, Turbidity measurement or color developing detection.
In the present invention, carbon nanotube preparation: carbon nanotube reagent can utilize prior art preparation.
The loop-mediated isothermal amplification system optimization, wherein dosage are as follows: in the nucleic acid amplification reaction system of 25 μ L
The middle 8.0 μ g/L of final concentration of 0.2- that carbon nanotube is added.
In the method for the present invention, the nucleic acid amplification reaction system includes each 0.2 μm of ol/L of outer primer F3 and B3, inner primer
FIP and BIP each 1.6 μm of ol/L, 8 U of Bst archaeal dna polymerase, 1 × polymerase buffer, Mg2+ 2-9 mmol/L, dNTP1.0-
8.0 μ g/ L of 1.6 mmol/L, glycine betaine 0-1.5 mol/L, carbon nanotube 0.2-.For example, 1 × Bst archaeal dna polymerase is anti-
It answers buffer that can select 1 × Thermopol reaction buffer, includes Tris-HCl (pH 8.8) 20 mmol/L, KCl 10
Mmol/L, (NH4)2SO4 10 mmol/L, 0.1% Triton X-100, MgSO4 2 mmol/L.1 × Bst archaeal dna polymerase
MgSO in reaction buffer4With the magnesium ion Mg in enzyme reaction system2+Do merging treatment.In the method for the present invention, the constant temperature
The response procedures of amplified reaction are 1. 60~65 DEG C of 10~90 min of incubation；2. 2 ~ 20 min are reacted in 80 DEG C of terminations.The present invention is not
Limitation realizes detection method by the way that other are suitable for response procedures.
In the method for the present invention, amplification detection method includes but is not limited to electrophoresis detection, Turbidity measurement or color developing detection
(including direct visual perception or by instrument carry out amplification curve judgement) etc..
Beneficial effect of the present invention includes: to have the advantages that high specificity, high sensitivity using detection method.With mesh
Preceding common loop-mediated isothermal amplification detection method is compared, and the present invention is by adding carbon into loop-mediated isothermal amplification system
Nano-tube material realizes the optimization to ring mediated isothermal amplification, and easy to operate, excellent is very suitable for food safety detection
It promotes the use of in equal fields.Above-mentioned each optimum condition can be subjected to any combination based on common sense in the field, belong to present invention protection model
It encloses.
Detailed description of the invention
Attached drawing 1 shows 1 carbon nanotube of the embodiment of the present invention to the effect of optimization of salmonella LAMP amplification reaction system；
Attached drawing 2 shows that 2 carbon nanotube of the embodiment of the present invention imitates the optimization of huichun viremia virus LAMP amplification reaction system
Fruit.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention,
Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below,
There are no special restrictions to content by the present invention.
Embodiment 1
A method of optimizing ring salmonella loop-mediated isothermal amplification (LAMP) system using carbon nanotube, by ring
Carbon nano-tube material is added in mediated isothermal amplification system to realize optimization, as follows:
(1) amplification reaction system optimizes: carbon nanotube being added in loop-mediated isothermal amplification system, core is then carried out
Acid amplification；
It prepares nucleic acid amplification reaction system (in addition to carbon nanotube), composition is as follows:
Wherein, primers F IP, BIP, F3 and B3 is salmonella specific nucleic acid amplimer, and template is come for salmonella DNA(
Derived from China General Microbiological Culture Collection Center CGMCC 1.1859, bacterial strain uses Beijing Tiangeng bioengineering public after cultivation
The bacterial nucleic acid extracts kit of department extracts genomic DNA), template quantity is 0(negative control) or 1 ng.
(2) processed optimization material carbon nanotube is added into the above system, 1.0 μ L are added in the system of every 25 μ L
Carbon nano-tube solution, final concentration of 0.2 μ g/L；Meanwhile the corresponding control experiment of optimization material is not added, then at 63 DEG C
Under the conditions of carry out 60 min of nucleic acid reaction；5 min are reacted in 80 DEG C of terminations.
(3) amplified production is subjected to agarose gel electrophoresis detection.
Amplification is as shown in Figure 1, M is molecular weight standard (DL2000), no addition optimization in sample 1-2 reaction system
Material carbon nanotube, 3-4 are added to optimization material quantum dot, wherein it is 0(i.e. feminine gender that 1 and 3 template quantities, which are the template quantity of 1ng, 2 and 4,
Control).It can be seen that template quantity is that the reaction system electrophoresis of 1 ng and 0 is presented in the processing of no addition carbon nanotube
Characteristic trapezoid-shaped strips show that there are non-specific amplification (i.e. false positives)；And in the processing of addition carbon nanotube, template
Amount is the trapezoid-shaped strips of the reaction system electrophoresis expression characteristics of 1 ng, is judged as positive, and the reaction system that template is 0 is (i.e.
Negative control) the not shown characteristic trapezoid-shaped strips of electrophoresis, it is judged as negative.The nucleic acid amplification is the results show that the present invention can be with
Non-specific amplification is eliminated, avoids false positive, effect of optimization is significant.
Embodiment 2
A method of optimizing huichun viremia virus loop-mediated isothermal amplification (LAMP) system using carbon nanotube, leads to
It crosses and adds carbon nano-tube material into loop-mediated isothermal amplification system to realize optimization, improve mediation etc. using carbon nanotube
The sensitivity of isothermal amplification reaction system, as follows:
(1) amplification reaction system optimizes: carbon nanotube being added in loop-mediated isothermal amplification system, core is then carried out
Acid amplification；
Nucleic acid reaction system is prepared, composition see the table below:
Wherein, primers F IP, BIP, F3 and B3 is huichun viremia virus specificity loop-mediated isothermal amplification (LAMP) primer, and template is
Huichun viremia virus RNA, and be serially diluted, carbon nanotube sample-adding amount is the final concentration of 8 μ g/ L of 1.0 μ L().
(2) processed optimization material carbon nanotube is added into the above system, 1.0 μ are added in the system of each 25 μ L
The carbon nano-tube solution (final concentration of 8.0 μ g/L) of L, meanwhile, the corresponding control experiment of optimization material is not added, then, 62
60 min of nucleic acid reaction is carried out under the conditions of DEG C, 10min is reacted in 80 DEG C of terminations.
(3) SYBR Green I is added in amplified production carry out color developing detection.
Amplification is as shown in Figure 2.The huichun viremia virus that sample is added in 1-5 reaction system from left to right in figure
The amount of RNA template is respectively 0(i.e. negative control), 100(i.e. stoste), 10-1、10-2With 10-3, first group be added without the present invention it is excellent
Change material carbon nanotube, second group of addition present invention optimizes material carbon nanotube.It can be seen from the figure that optimization material is not added
The system of carbon nanotube is 10 in template quantity0With 10-1Sample cell colour developing be bright green, be judged as positive, and template quantity be 0,
10-2With 10-3Sample cell colour developing be it is orange, be judged as negative, show not add the reaction system of optimization material carbon nanotube most
Low detectable dilution is 10-2Template；And the system template quantity that optimization material carbon nanotube is added is 100、10-1With 10-2
Sample cell colour developing be bright green, be judged as positive, and template quantity is 0 and 10-3Sample cell colour developing be it is orange, be judged as yin
Property, show that the minimum detectable dilution of reaction system of addition optimization material carbon nanotube is 10-2Template, it is more excellent than not being added
The sensitivity for changing the reaction system of material carbon nanotube improves 1 order of magnitude.The nucleic acid amplification is the results show that the present invention can mention
High reaction sensitivity, effect of optimization are significant.
Claims (3)
1. a kind of method using carbon nanotube optimization loop-mediated isothermal amplification, which is characterized in that by being mediated to ring
Carbon nano-tube material is added in isothermal amplification reaction system to realize optimization, is included the following steps:
(1) amplification reaction system optimizes: carbon nanotube being added in loop-mediated isothermal amplification system, core is then carried out
Acid amplification；
(2) nucleic acid amplification product detects: passing through electrophoresis detection, Turbidity measurement or color developing detection.
2. a kind of method using carbon nanotube optimization loop-mediated isothermal amplification according to claim 1, feature
It is, the 8.0 μ g/L of final concentration of 0.2- of carbon nanotube is added in the nucleic acid amplification reaction system of 25 μ L.
3. a kind of method using carbon nanotube optimization loop-mediated isothermal amplification according to claim 1 or 2, special
Sign is that the nucleic acid amplification reaction system includes each 0.2 μm of ol/L of outer primer F3 and B3, each 1.6 μ of inner primer FIP and BIP
8 U of mol/L, Bst archaeal dna polymerase, 1 × polymerase buffer, Mg2+ 2-9 mmol/L, dNTP1.0-1.6 mmol/L, beet
8.0 μ g/ L of alkali 0-1.5 mol/L, carbon nanotube 0.2-；Wherein, 1 × Bst DNA polymerase reaction buffer select 1 ×
Thermopol reaction buffer includes Tris-HCl (pH 8.8) 20 mmol/L, KCl 10 mmol/L, (NH4)2SO4
10 mmol/L, 0.1% Triton X-100, MgSO4 2 mmol/L；In 1 × Bst DNA polymerase reaction buffer
MgSO4With the magnesium ion Mg in enzyme reaction system2+Do merging treatment；The response procedures of isothermal amplification reactions are 1. to incubate for 60~65 DEG C
Educate 10~90 min；2. 2 ~ 20 min are reacted in 80 DEG C of terminations.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115725792A (en) * | 2022-09-20 | 2023-03-03 | 江苏省家禽科学研究所 | Universal nucleic acid detection kit for avian leukosis virus |
| CN116334255A (en) * | 2022-09-20 | 2023-06-27 | 江苏省家禽科学研究所 | Novel salmonella nucleic acid detection kit and non-diagnostic detection method thereof |
| CN116334254A (en) * | 2022-09-20 | 2023-06-27 | 江苏省家禽科学研究所 | Novel Pasteurella multocida nucleic acid detection kit and non-diagnostic detection method thereof |
-
2019
- 2019-08-20 CN CN201910767207.4A patent/CN110438204B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| ZHIZHOU ZHANG 等: "Aqueous suspension of carbon nanotubes enhances the specificity of long PCR", 《BIOTECHNIQUES》 * |
| 高翔: "副溶血弧菌LAMP检测法的建立及纳米材料应用", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115725792A (en) * | 2022-09-20 | 2023-03-03 | 江苏省家禽科学研究所 | Universal nucleic acid detection kit for avian leukosis virus |
| CN116334255A (en) * | 2022-09-20 | 2023-06-27 | 江苏省家禽科学研究所 | Novel salmonella nucleic acid detection kit and non-diagnostic detection method thereof |
| CN116334254A (en) * | 2022-09-20 | 2023-06-27 | 江苏省家禽科学研究所 | Novel Pasteurella multocida nucleic acid detection kit and non-diagnostic detection method thereof |
| CN116334254B (en) * | 2022-09-20 | 2024-01-16 | 江苏省家禽科学研究所 | Novel Pasteurella multocida nucleic acid detection kit and non-diagnostic detection method thereof |
| CN116334255B (en) * | 2022-09-20 | 2024-01-30 | 江苏省家禽科学研究所 | Novel salmonella nucleic acid detection kit and non-diagnostic detection method thereof |
| CN115725792B (en) * | 2022-09-20 | 2024-01-30 | 江苏省家禽科学研究所 | Universal avian leukosis virus nucleic acid detection kit |
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