CN104293945B - Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid - Google Patents
Method for carrying out nucleic acid isothermal amplification by using paper-based microfluid Download PDFInfo
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- CN104293945B CN104293945B CN201410528335.0A CN201410528335A CN104293945B CN 104293945 B CN104293945 B CN 104293945B CN 201410528335 A CN201410528335 A CN 201410528335A CN 104293945 B CN104293945 B CN 104293945B
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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
Abstract
The invention discloses a method for carrying out nucleic acid isothermal amplification by using paper-based microfluid. The method comprises the following steps: (1) preparing a sample DNA solution; (2) coating a primer on a reaction zone of paper-based microfluid, wherein the microfluid comprises a reaction layer made of filter paper and at least one sampling layer; sampling holes are formed at the centers of the sampling layers; a plurality of reaction holes are evenly distributed around the sampling holes; each orifice is coated by a hydrophobic material; the sampling layers are stacked above the reaction layer; the reaction layer is provided with a sample zone, a plurality of reaction zones and reaction channels; the sample zone is drawn by the hydrophobic material and corresponds to the sampling hole; the plurality of reaction zones and reaction channels correspond to the reaction holes; the reaction zones are the same as the reaction holes in diameter; the diameter of the sample zone is smaller than those of the sampling holes; and the reaction zones are communicated with the sample zones through the reaction channels of which the widths are gradually reduced from the reaction zones to the sample zones; (3) dropwise adding the reaction liquid into the sampling holes, heating the microfluid to reach amplification temperature; and (4) observing the color change of the reaction zones, so as to obtain the detection result. The method is low in cost and accurate in result.
Description
Technical field
The present invention relates to micro-fluidic technologies field, more particularly to one kind carry out nucleic acid isothermal amplification using papery microfluid
Method and implement this method device.
Background technology
Nucleic acid amplification technologies with pcr as representative are commonly used always in scientific research and clinical position.Based on molecule
Biological fast development, successively have developed many new nucleic acid isothermal amplification methods, for example: Nucleic acid sequence amplification
Method (nasba), from sequence replicating (3sr), ring mediated isothermal amplification (lamp), strand displacement amplification method (sda) etc..But it is traditional
Pcr amplification needs expensive special amplification instrument, and operator are also required to the training of specialty, and testing cost is high, due to amplification
Equipment instrument is larger, heavier-weight is so that detection site can not flexibly move.
All the time, a platform can simple, quick, the accurate and effective multi-channel detection that carries out be medical science, life
The common expectation of all circles scholars such as change, environment.Microfluidic chip technology (microfluidics technology) side of having
Just, be easy to carry about with one, efficiently, the feature of low consumption (including time, specimen, reagent), analysis miniaturization and experiment flux, and lead to
Cross with reference to sensing or sample pre-treatments module, increased analysis detection effectiveness, decrease cross-contamination, be Site Detection or
In time detection (point-of-care, poc) provides a kind of method of quick diagnosis.Constant temperature microfluid nucleic acid amplification chip skill
Art is that one kind of a kind of novelty bind nucleic acid isothermal duplication and micro-fluidic chip is used for nucleic acid rapid amplifying and result naked eyes are sentenced
Fixed new technique.But microfluidic device typically adopts quasiconductor or integrated circuit processing pattern to realize, and process is loaded down with trivial details, technique
Complexity, cost is still far from reaching disposable target so far.
Content of the invention
The purpose of the present invention is for the deficiencies in the prior art, provides one kind to carry out nucleic acid isothermal expansion using papery microfluid
The method increasing, the method is easy to operate, do not need special nucleic acid amplification equipment, expands low cost, can carry out multinomial inspection simultaneously
Survey.
The object of the present invention is achieved like this: a kind of method carrying out nucleic acid isothermal amplification using papery microfluid, bag
Include following steps:
(1) prepare testing sample dna solution；
(2) according to detection project, corresponding primer is coated on the reaction zone of papery microfluid, described papery microfluid
Including the conversion zone being made up of filter paper and at least one of which sample-adding layer, described sample-adding layer overlap is placed on the top of conversion zone, described
Sample-adding layer center is provided with well, and described sample-adding layer is evenly equipped with multiple reacting holes, described well and reaction around well
It is coated with hydrophobic material on the aperture in hole；Described conversion zone is provided with the sample corresponding with well being drawn by hydrophobic material
Product area multiple reaction zones corresponding with reacting hole and reaction channel, described reaction zone diameter is identical with the diameter of reacting hole, institute
The diameter stating sample area is less than the diameter of well, and each reaction zone is connected with sample area by a reaction channel, described anti-
The width answering passage tapers into from reaction zone to sample area；
(3) testing sample dna solution prepared by step 1 is mixed into amplification reaction system according to detection project, Deca exists
In the well of papery microfluid, heating papery microfluid makes its temperature reach the temperature required for nucleic acid amplification and keep permanent
Temperature；
(4) after the completion of amplified reaction, observe the color change of papery microfluid reaction zone, you can obtain testing result.
Using technique scheme,
Hydrophobic material is coated with the aperture of the well of papery microfluid and reacting hole, conversion zone is provided with by hydrophobic
The sample area corresponding with well multiple reaction zones corresponding with reacting hole that material draws and reaction channel are so that treat
Survey sample solution and the other local of filter paper layer can not be diffused into by hydrophobic material after well is added drop-wise to sample area, thus
The detection of disparity items is separated, a miniflow physical ability carries out multinomial different detection simultaneously.
Preferably, being coated primer in described step 2 on the reaction zone of papery microfluid is to be drawn by biotinylation
Thing and Avidin microgel are coupled and combine, and drying at room temperature is deposited on reaction zone.Can be according to the difference of detection object, in advance
Micro fluid reaction area is coated corresponding primer, realizes testing goal.
Preferably, the sample-adding layer of the papery microfluid in described step 2 is more than two-layer, sample-adding layer from top to bottom
The diameter of well successively reduces, and the diameter of described sample area is less than the diameter of the well of orlop sample-adding layer.Well
Diameter successively reduces so that the central authorities of microfluid form a hole by outer reduced diameter inside, decreases sample and is filtered
The loss that paper absorption brings.
Preferably, the center of circle of all reacting holes of the papery microfluid in described step 2, on same circumference, owns
The center of circle of reaction zone also on same circumference so that microfluidic device is beautiful and clean, result be easy to observe statistics.
Preferably, the hydrophobic material in the papery microfluid in described step 2 is paraffin or dimethyl siloxane
Or su-8 photoresistance glue.
Preferably, the method heating papery microfluid in described step 3 is to put into papery microfluid to mix up in advance
It is not necessary to miscellaneous equipment in the artificial incubator of temperature, simple to operate.
Preferably, the papery microfluid in described step 2 also includes the zone of heating below conversion zone, described plus
Thermosphere is made up of filter paper, and its lower surface is provided with resistance wire, can connect resistance, makes ply of paper temperature reach nucleic acid after turn-on current
Amplified reaction is temperature required.
Preferably, the method heating papery microfluid in described step 3 is the external variable resistance of resistance wire, connects electricity
Stream, adjusts variable resistance so that the temperature of papery microfluid reaches the temperature required for amplification, can according to amplification temperature not
Reach different temperatures with needing regulation variable resistance.
Preferably, the resistance wire of the zone of heating of papery microfluid in described step 2 is by metal powder spray precipitation
Form, resistance wire is fine, small volume, can combine closely with filter paper layer, and be beneficial to be uniformly distributed so that ply of paper temperature is uniform.
Preferably, the sample-adding layer of papery microfluid in described step 2, conversion zone and zone of heating are glued by layers of two-sided
Patch superposition is fixed, and the layers of two-sided between wherein said sample-adding layer, between sample-adding layer and conversion zone is provided with and is positioned above
The well of sample-adding layer and the corresponding hole of reacting hole so that whole microfluidic structures consolidate, each layer can not shift and avoid causing
Experimental result is inaccurate.
The invention has the beneficial effects as follows: the method carrying out nucleic acid isothermal amplification using papery microfluid of the present invention, it is not required to
Want expensive nucleic acid amplification equipment, the papery microfluid for isothermal duplication nucleic acid is made up of filter paper, liquid passes through blood capillary
Effect and liquid wick act on carry out horizontal and vertical flowing between paper fiber so that testing sample solution can be coated on
The primer of reaction zone reacts and realizes amplification purpose.This papery microfluid is lightweight, easy to carry, can be as needed by paper
Matter microfluid takes different places to and carries out Site Detection, and this papery microfluid is with low cost, and multiple reacting holes allow to same
When detection same target different detection projects, can single use；This papery microfluid can be connect from outside by conductive layer
Enter power supply so that ply of paper temperature reaches the needs of amplification temperature it is also possible to must not zone of heating directly only incite somebody to action by sample-adding layer and reaction
The microfluid that layer assembling obtains is put into and is carried out amplified reaction in the artificial incubator mix up temperature, simplifies the preparation of this microfluid
Process, reduce cost.The small volume of sample needed for this amplification method, analyze speed are fast, low cost, can carry out as needed
Scene is detected immediately, has good application prospect.
Brief description
Fig. 1 is the papery microfluid assembling schematic diagram of the present invention.
Fig. 2 is the sample-adding layer structural representation of the papery microfluid of the present invention.
Fig. 3 is the conversion zone structural representation of the papery microfluid of the present invention.
Fig. 4 is the zone of heating structural representation of the papery microfluid of the present invention.
Specific embodiment
Embodiment 1 carries out the ring mediated isothermal amplification detection of seven kinds of bacterium using papery microfluid simultaneously
Detect in testing sample, whether to contain following seven kinds of bacterium using papery microfluid: Pseudomonas aeruginosa, pair are molten simultaneously
Courageous and upright vibrio, streptococcus pneumoniae, staphylococcus aureuses, colon bacillus, Klebsiella Pneumoniae, Acinetobacter bauamnnii, adopt
With ring mediated isothermal amplification (loop-mediated isothermal amplification, lamp), grasp in accordance with the following steps
Make:
First, extract the dna solution for standby of sample, using water-boiling method rapid extraction nucleic acid.
2nd, the lamp primer of seven kinds of bacterium is deposited on seven reaction zones 22 of papery microfluid respectively, different bacterium detections
Gene and use primer as shown in table 1: be coupled by biotinylated primer and Avidin microgel and combine, drying at room temperature
It is deposited on reaction zone 22, surplus next one reaction zone 22 is not coated primer as negative control.
The detection gene of 1 seven kinds of bacterium of table and corresponding primer
As shown in figures 1-4, papery microfluid by least one of which sample-adding layer 1, conversion zone 2 and layers of two-sided 4 from top to bottom according to
The secondary papery microfluid stacking formation isothermal duplication nucleic acid, selects two-layer sample-adding layer 1 here.
From top to bottom: ground floor sample-adding layer 1 is made up of filter paper, its center is provided with the well 11 of a diameter of 10mm, around
Well 11 is laid with the reacting hole 12 of 8 a diameter of 5mm, and reacting hole 12 is apart from well 11 apart from for 5mm, well 11
It is coated with hydrophobic material paraffin or dimethyl siloxane (polydimethylsiloxane, pdms) with the aperture of reacting hole 12
Or su-8 photoresistance glue (su-8photoresist) makes in the hole liquid can not pass through.
Second layer sample-adding layer 1 is identical with the first Rotating fields, also is provided with well 11 and reacting hole 12, and layer 2-only is loaded
The diameter of the well 11 of layer 1 is less than the diameter of upper strata sample-adding layer 1, preferably the diameter of the well 11 of second layer sample-adding layer 1
For 9mm.
Ground floor sample-adding layer 1 and second layer sample-adding layer 2 pass through layers of two-sided 4 bonding, and this layer of layers of two-sided 4 is provided with and the
The well 11 of one layer of sample-adding layer 1 and the corresponding hole of reacting hole 12.
Pass through layers of two-sided 4 bonding conversion zone 2 below second layer sample-adding layer 1, this layer of layers of two-sided 4 is provided with and the
The well 11 of two layers of sample-adding layer 1 and the corresponding hole of reacting hole 12.
Conversion zone 2 is made up of filter paper, and conversion zone 2 is provided with hydrophobic material paraffin or dimethyl siloxane or su-8
The sample area 21 corresponding with well 11 and 8 reaction zones 22 corresponding with reacting hole 12 and reaction channel that photoresistance tempera painting goes out
23, a diameter of 6～10mm in sample area, reaction zone diameter is 5mm, and a reaction channel 23 and sample area are passed through in each reaction zone 22
21 connections, the width of reaction channel 23 tapers into sample area 21 from reaction zone 22.
3rd, the testing sample dna solution preparing step one and other reagent are mixed into lamp reaction system: cumulative volume is
25 μ l, comprising: 1.6 μm of fip, 1.6 μm of bip, 0.4 μm of f3,0.4 μm of b3,1.0mm dntps, 0.8mmbetaine,
6mmmgso4, 1 × bstdna polymerase buffer (20mm tris-hcl, 10mm (nh4)2so4, 10mmkcl, 2mmmgso4,
0.1%tritonx-100, ph8.8), 8ubstdna polymerase, 1 μ l3mmhnb, 1 μ ldna template.By the reactant mixing
It is that Deca is dropped on sample area 21 in the well 11 of papery microfluid, the metal indicator hnb of addition makes reactant
Be color be pansy, so that the sample area 21 of papery microfluid is changed into violet after reaction system is added drop-wise on sample area 21
Color.Liquid is acted on by capillary action and liquid wick and carries out horizontal and vertical flowing between paper fiber, by reaction
Passage 23 reaches reaction zone 22 so that testing sample solution can react with the lamp primer being coated on reaction zone realizes expansion
Gaining.Covering one layer of heat resistant plastice film after sample-adding in papery microfluid prevents from volatilizing, and papery microfluid is placed in temperature is
React 60min in the artificial incubator of 65 DEG C of constant temperature, the temperature of artificial incubator is adjusted to 80 DEG C of inactivation 2min.Take out papery micro-
Fluid, the color change in observing response area 22, observe the color finding negative control reaction zone still for pansy, the false list of Aerugo
Born of the same parents bacterium, streptococcus pneumoniae, the color of the reaction zone 22 of these three bacterium of Acinetobacter bauamnnii become for sky blue, and testing sample is described
In contain these three bacterium；These four bacterium of Klebsiella Pneumoniae, colon bacillus, vibrio parahaemolyticus, staphylococcus aureuses
Reaction zone 22 color still be pansy, illustrate to be not detected by these four bacterium in testing sample.
Embodiment 2 carries out the ring mediated isothermal amplification detection of seven kinds of bacterium using the papery microfluid of Resistant heating simultaneously
Other same as Example 1, except for the difference that: it is pasted with zone of heating 3 in conversion zone 2 below by layers of two-sided 4, should
Any pattern and hole are not had on layers of two-sided 4.Zone of heating 3 is made up of filter paper, and its lower surface is provided with resistance wire 31, preferably resistance
Silk 31 is formed by metal powder spray precipitation, and resistance wire 31 is connected with power supply and switch by wire with after variable resistance 5 series connection.
The size of electric current is changed by the regulation of variable resistance 5, thus playing the effect of the temperature adjusting zone of heating 3.The gold using
Belonging to indicator is not hnb but orange-yellow calcein.
The testing sample Deca of metal indicator calcein will be mixed with well 11, in papery microfluid during use
Upper one layer of heat resistant plastice film of covering prevents from volatilizing, and switches on power, adjusts variable resistance 5, make papery microfluid temperature reach 65 DEG C,
Reaction 60min；Adjust variable resistance 5, the temperature of microfluid is adjusted to 80 DEG C of inactivation 2min, closes power supply.
The color change in observing response area 22, observes the color finding negative control reaction zone still for orange-yellow, Aerugo vacation
Zymomonas mobiliss, streptococcus pneumoniae, the color of the reaction zone 22 of these three bacterium of Acinetobacter bauamnnii become for grass green, illustrate to treat test sample
These three bacterium are contained in product；Klebsiella Pneumoniae, colon bacillus, vibrio parahaemolyticus, staphylococcus aureuses these four
The color of the reaction zone 22 of bacterium is still orange-yellow, illustrates to be not detected by these four bacterium in testing sample.
Claims (10)
1. a kind of carry out the method for nucleic acid isothermal amplification it is characterised in that comprising the following steps using papery microfluid:
(1) prepare testing sample dna solution；
(2) according to detection project, corresponding primer is coated on the reaction zone (22) of papery microfluid, described papery microfluid
Including the conversion zone (2) being made up of filter paper and at least one of which sample-adding layer (1), described sample-adding layer (1) overlap is placed on conversion zone (2)
Top, described sample-adding layer (1) center is provided with well (11), described sample-adding layer (1) is evenly equipped with around well (11) many
Individual reacting hole (12), the aperture of described well (11) and reacting hole (12) is coated with hydrophobic material, described hydrophobic material
For paraffin or dimethyl siloxane or su-8 photoresistance glue；Described conversion zone (2) be provided with drawn by hydrophobic material with plus
The corresponding sample area (21) in sample hole (11) multiple reaction zones (22) corresponding with reacting hole (12) and reaction channel (23),
Described reaction zone (22) diameter is identical with the diameter of reacting hole (12), and the diameter of described sample area (21) is less than well (11)
Diameter, each reaction zone (22) are connected with sample area (21) by a reaction channel (23), the width of described reaction channel (23)
Degree tapers into sample area (21) from reaction zone (22)；
(3) testing sample dna solution prepared by step 1 is mixed into amplification reaction system according to detection project, Deca is in paper
In the well of matter microfluid, heating papery microfluid makes its temperature reach the temperature required for nucleic acid amplification and keep constant temperature.
2. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 1
Being coated primer in step 2 on the reaction zone (22) of papery microfluid is by biotinylated primer and Avidin microgel
It is coupled and combines, drying at room temperature is deposited on reaction zone (22).
3. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 1
The sample-adding layer (1) of the papery microfluid in step 2 is more than two-layer, the diameter of the well (11) of sample-adding layer (1) from top to bottom
Successively reduce, the diameter of described sample area (21) is less than the diameter of the well (11) of orlop sample-adding layer (1).
4. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 1
The center of circle of all reacting holes (12) of the papery microfluid in step 2 on same circumference, the center of circle of all reaction zones (22)
On same circumference.
5. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 1
The method heating papery microfluid in step 3 is to put into papery microfluid in the artificial incubator mixing up temperature in advance.
6. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 1
Papery microfluid in step 2 also includes the zone of heating (3) below conversion zone (2), and described zone of heating (3) is by filter paper system
Become, its lower surface is provided with resistance wire (31).
7. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 6
The method heating papery microfluid in step 3 is the external variable resistance of resistance wire (31), and turn-on current adjusts variable resistance, makes
The temperature obtaining papery microfluid reaches the temperature required for amplification.
8. carry out the method for nucleic acid isothermal amplification using papery microfluid it is characterised in that described as described in claim 6
The resistance wire (31) of the zone of heating (3) of the papery microfluid in step 2 is to be formed by metal powder spray precipitation.
9. the described method carrying out nucleic acid isothermal amplification using papery microfluid as arbitrary in Claims 1 to 5, its feature exists
In, the sample-adding layer (1) of the papery microfluid in described step 2, conversion zone (2) are pasted superposition by layers of two-sided (4) and are fixed, its
Described in layers of two-sided (4) between sample-adding layer (1), between sample-adding layer (1) and conversion zone (2) be provided with and be positioned above
The well (11) of sample-adding layer (1) and the corresponding hole of reacting hole (12).
10. the described method carrying out nucleic acid isothermal amplification using papery microfluid as arbitrary in claim 6～8, its feature exists
In the sample-adding layer (1) of the papery microfluid in described step 2, conversion zone (2) and zone of heating (3) are pasted by layers of two-sided (4)
Superposition is fixed, the layers of two-sided (4) between wherein said sample-adding layer (1), between sample-adding layer (1) and conversion zone (2) be provided with and
The well (11) of the sample-adding layer (1) being positioned above and the corresponding hole of reacting hole (12).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105273997B (en) * | 2015-10-21 | 2017-10-20 | 西安交通大学 | Paper substrate nucleic acid extraction, the amplification detecting system and detection method integrated with detection |
| CN107513582A (en) * | 2017-10-23 | 2017-12-26 | 蔡慧娜 | Authentication chip, kit and the authentication method of positive blood culture |
| CN107988046B (en) * | 2018-01-23 | 2021-02-19 | 吉林大学 | LAMP-based self-suction type multi-channel pathogen detection micro-fluidic chip |
| EP3880839A4 (en) * | 2018-11-16 | 2022-08-03 | Precigenome, LLC | Integrated microfluidic system for droplet generation, nucleic acid amplification, and detection |
| CN109709316A (en) * | 2018-12-27 | 2019-05-03 | 天津昌和生物医药技术有限公司 | A kind of more target item miniflow test cards and preparation method |
| CN111088155A (en) * | 2019-12-30 | 2020-05-01 | 航天神舟生物科技集团有限公司 | PCR detection microfluidic paper chip and preparation method and application thereof |
| CN111575354A (en) * | 2020-05-25 | 2020-08-25 | 广州新诚生物科技有限公司 | Nucleic acid detection equipment and use method thereof |
| CN111575167A (en) * | 2020-05-25 | 2020-08-25 | 广州新诚生物科技有限公司 | Nucleic acid detection equipment and use method thereof |
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