CN106979940A - A kind of device and method for carrying out nucleic acid amplification detection - Google Patents
A kind of device and method for carrying out nucleic acid amplification detection Download PDFInfo
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- CN106979940A CN106979940A CN201710248640.8A CN201710248640A CN106979940A CN 106979940 A CN106979940 A CN 106979940A CN 201710248640 A CN201710248640 A CN 201710248640A CN 106979940 A CN106979940 A CN 106979940A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The present invention provides a kind of device for carrying out nucleic acid amplification detection, including detecting system and chip, chip is placed in detecting system, detecting system includes light excitation device and signal collecting device, and the light sent by light excitation device is irradiated on chip, transmitting light is produced in detection zone, through being filtrated to get fluorescence signal, signal collecting device gathers the fluorescence signal to be detected, the chip can be integrated on wearable device.The present invention also provides a kind of method that application said apparatus carries out nucleic acid amplification detection.Nucleic acid can quickly be detected using the device of the present invention, even can be with the content of quantitative nucleic acid, and required external equipment condition requires low, it can also be widely used in poverty-stricken area, the detection such as poverty-stricken area incident serious infectious diseases such as HIV, HBV, Ebola and Zika is significant, also has important meaning to some sudden illness Pathogen tests such as Mers, influenza etc..
Description
Technical field
The present invention relates to technical field of biological, and in particular to nucleic acid amplification detection technique and body-worn medical equipment skill
Art.
Background technology
Wearable device（wearable device）It is that directly can wear or be integrated directly into the clothes of user
Or the equipment in accessory.The equipment can realize that quick data are obtained with technologies such as integrated multimedia, sensor and radio communications
Take, ultrafast data transfer, break away from traditional handheld device and obtain seamless network access experience.With comfortable wearing even
It is noninductive, do not disturb normal life, outward appearance to be adapted to use occasion and the advantages of environment using process.Wearable market rule global at present
Mould is about 3,000,000,000 ~ 5,000,000,000 dollars, and the following great market for being expected to grow into 30,000,000,000 ~ 50,000,000,000 dollars for 2 to three years, particularly
With the popularization of 4G and mobile terminal, wearable device has huge development potentiality.Current most widely used wearable device
Mainly glasses, wrist-watch, bracelet and necklace etc., the function of realization are mainly health monitoring, such as bracelet monitor heart rate；Safety is protected
Barrier, such as bluetooth short-range communication realizes that wallet is thief-proof；And Entertainment, such as Kinect somatosensory game machine.With support
The continuous progress of technology, wearable product is towards lighter, more hidden, more efficiently direction transformation, and fashion type and functional form will
The two main lines of development as wearable product.With the development of hardware technology and Internet technology, the work(of wearable product
Energy can be more comprehensive, and with huge market potential and wide development space.
Medical control class wearable device is current topmost developing direction.Using integrated various sensors to wearable
In equipment, the index of the parameter of certain in human body can be monitored in real time for a long time.Due to flexible material such as dimethyl silicone polymer
（PDMS）, polymethyl methacrylate（PMMA）Can fully it be contacted with human body skin Deng in the presence of Van der Waals force, and
With the preferable transparency, heattransfer effect and electrochemistry resistance, by the way that the shape of bending, and skin can be made after project treatment
Skin is more closely contacted, and is generally used for integrated sensor and is made wearable device.Flexible current sensor is integrated into stealthy eye
In mirror, for detecting the glucose content in tear；Biology sensor is integrated into mouth guard and be can be used in detection saliva
Lactic acid content；It also have developed in the recent period and biology sensor be integrated into temporary tatt, application on human skin is measured in the way of non-intrusive
Or the sweat composition in interstitial liquid.By the sensor integration based on polyester piece into flexible material, available for record human body
Electroencephalogram（EEG）, electrocardiogram（ECG）And electromyogram（EMG）, and result is sent into computer end by way of wireless data transmission
The detected status at end, in real time analysis human body.Although the application field of current wearable device is very extensive, do not have so far
Wearable device can be used for nucleic acid amplification and detection.
Current nucleic acid amplification technologies have many kinds, and most common is exactly PCR（Polymerase Chain
Reaction, PCR）.Party's hair is the method for denaturation, extension, renaturation by nucleic acid under high temperature, but is the need for temperature and follows
The equipment of ring, required condition is higher, and some isothermal amplification technologies by cleavage base sequence, it is necessary to which a steady temperature is
Nucleic acid amplification can be achieved, is applied to field of nucleic acid amplification for wearable device and provides possibility.Recombinase polymeric enzymatic amplification technology
（Recombinase Polymerase Amplification, RPA）It is a kind of isothermal amplification technology, amplification temperature is 31 ~ 42
C, close to body temperature, and can within more than ten minutes by nucleic acid amplification to detectable level, be that a kind of Rapid nucleic acid is permanent
Warm amplification technique.
The content of the invention
The technical problem that the present invention first has to solve is to provide a kind of device for carrying out nucleic acid amplification detection, can be to nucleic acid
Quickly detected, and required external equipment condition requires low, can be detected whenever and wherever possible.
The present invention solves the technical scheme that is used of above-mentioned technical problem：A kind of device for carrying out nucleic acid amplification detection,
Including detecting system and chip, chip is placed in detecting system, and detecting system includes light excitation device and signal collecting device, by
The light that light excitation device is sent, is irradiated on chip, transmitting light is produced in detection zone, through being filtrated to get fluorescence signal, signal
Collecting device gathers the fluorescence signal to be detected, the chip can be integrated on wearable device.
While using above-mentioned technical proposal, the present invention can also be using further technical scheme once：
Detecting system includes being provided with driving equipment, light excitation device, signal collecting device, detection device and inspection in housing, housing
Chamber is surveyed, driving equipment includes power supply and transformer, and driving equipment is that light excitation device and signal collecting device are powered, and light is excited
Equipment includes fan, radiator and LED light source, and detection device includes exciting piece, two to point mirror and a sheet emitting, signal collecting device
For industrial camera, piece is excited to be placed in the front end of LED light source, sheet emitting is placed in the camera lens front end of industrial camera, two to point mirror and a core
Piece is placed in detection chambers, and light excitation device is located at the side of detection chambers and LED light source is towards the direction of detection chambers, industrial
Camera is located at the top of detection chambers and camera lens is towards the direction of detection chambers, and chip is placed in the bottom of detection chambers, two to points
Mirror is placed in detection chambers into 45 degree of angles, and one is facing to light excitation device, and another side is towards industrial camera.
Chip includes the reaction chamber for being arranged on the hexagon at middle part, the two ends of the reaction chamber be respectively equipped with inlet and
Liquid outlet, the upper and lower surface of reaction chamber is respectively equipped with top layer and bottom as sealing, when chip is placed in detection chambers, its
Lower section is provided with heating plate, and heating plate is powered by driving equipment.
Chip is the high molecular polymer chip with biocompatibility, and such as PDMS, the top layer of chip includes passage knot
Structure, the bottom of chip is the thick PDMS films of 0.5mm, and the thickness of PDMS passages is 0.5-1.5mm, inlet and liquid outlet aperture
For 1-4mm.
The channel design of chip top-layer is single-pass configuration or multi-channel structure, and the cumulative volume of passage is not less than 50 μ L.
The power of LED light source is 5-15W.
The band-pass wavelength for exciting piece is 435-480nm.
The band-pass wavelength of sheet emitting is 500-560nm.
The chip can be integrated on watch, worn at human body wrist.
Another technical problem to be solved by this invention is to provide a kind of method for carrying out nucleic acid amplification detection, this method
Using above-mentioned device, and comprise the following steps：
（1）Measure human body temperature：The water filling into chip, is fixed on human skin by wearable device by chip, passes through heat
Fluid temperature in galvanic couple measurement chip, the feasibility that checking is expanded with body temperature；
（2）Build the standard curve of detection of nucleic acids：
（2.1）Since original concentration it is nucleic acid-templated dilute in proportion, obtain the template concentrations of gradient dilution；
（2.2）According to the method in RPA kit specifications, the good mixture needed for reaction of configuration；
（2.3）The template and magnesium acetate for adding various concentrations in the mixture are fully mixed；
（2.4）Reactant after mixing is squeezed into chip from inlet, detecting system is put into, photographs to record fluorescence intensity；
（2.5）Chip is worn on human skin, expanded by body temperature, after reaction certain time, core is taken out
Piece is put into detecting system and photographed to record；
Repeat step（2.1）-（2.5）At least three times,
（2.6）ImageJ analyzes the green fluorescence intensity of detection zone, and it is bent that the fluorescence intensity after being terminated according to reaction sets up standard
Line；
（3）Concentration of specimens is detected：According to step（2.1）-（2.5）Detection sample simultaneously brings testing result in standard curve into, obtains
Nucleic acid concentration into sample.
The beneficial effects of the invention are as follows：The present invention devises the wearable device directly contacted with human body skin, will contain
In the chip insertion wrist-watch of RPA reagents, the temperature needed for RPA amplifications is provided using the body temperature at wrist, passes through the optics carried
The fluorescence signal produced after system detectio reaction.Nucleic acid can quickly be detected using the device of the present invention, or even can
With the content of quantitative nucleic acid, and required external equipment condition requires low, can also be widely used in poverty-stricken area, to poverty
The detections such as area incident serious infectious diseases such as HIV, HBV, Ebola and Zika are significant, sudden to some
Disease pathogen detection such as Mers, influenza also have important meaning.Because the time required for detection is short, as long as 20 minutes left sides
The right side, the personnel for being also applied for entry and exit do infectious disease detection.This method possesses in terms of Rapid nucleic acid detection, Prevention of Infectious Diseases
Wide business potential.
Brief description of the drawings
Fig. 1 is the structure chart of chip.
Fig. 2 is the decomposing state schematic diagram of chip.
Fig. 3 is the structure chart of detection means.
Fig. 4 is real-time fluorescence amplification figure.
Fig. 5 is nucleic acid concentration examination criteria curve.
Label in figure：1- chips, 2- top layers, 2-1- inlets, 2-2- liquid outlets, 2-3- reaction chambers, 3- bottoms, 4- adds
Backing, 5- light excitation devices, 5-1- fans, 5-2- radiators, 5-3-led light sources, 6- power supplys, 7- driving equipments, 8- industry phases
Machine, 9- detecting systems, 9-1- sheet emittings, the lanes of 9-2- bis- point mirror, 9-3- excites piece, No. bis- baffle plates of 10-, 11- detection chambers.
Embodiment
Technical scheme is described in further detail with reference to embodiments, embodiment is only used for explaining this hair
Bright embodiment or process, is not limiting the scope of the present invention.
Embodiment 1, a kind of device for carrying out nucleic acid amplification detection, referring to the drawings 1-3.
The present invention provides a kind of device for carrying out nucleic acid amplification detection, and the device can be worn in human body using wearable device
Skin surface, so as to experience human body temperature, carries out nucleic acid amplification detection, specifically, the inventive system comprises detecting system 9
With chip 1, chip 1 can be placed in wearable device, such as wrist strap or watch, so as to directly be contacted with human body skin.
The concrete structure of chip 1 is：Chip 1 includes the reaction chamber 2-3 for being arranged on the hexagon at middle part, the reaction chamber
2-3 two ends are respectively equipped with inlet 2-1 and liquid outlet 2-2, and reaction chamber 2-3 upper and lower surface is respectively equipped with top layer 2 and bottom
Layer 3 is as sealing, and when chip 1 is placed in the detection chambers of detecting system 9, below provided with heating plate 4, heating plate 4 is by examining
Driving equipment 7 in examining system 9 is powered.
Chip 1 in the present invention is the high molecular polymer chip with biocompatibility, such as PDMS, the top of chip 1
Channel design is included in layer 2, channel design can be single-pass configuration or multi-channel structure, it is only necessary to ensure the cumulative volume of passage
Not less than 50 μ L.As 1 PDMS of chip, the bottom 3 of chip 1 is the thick PDMS films of 0.5mm, and the thickness of PDMS passages is 0.5-
1.5mm, inlet 2-1 and liquid outlet 2-2 aperture are 1-4mm.
Detecting system 9 includes light excitation device 5 and signal collecting device, and the light sent by light excitation device 5 is irradiated to core
On piece 1, transmitting light is produced in detection zone, through being filtrated to get fluorescence signal, signal collecting device gather the fluorescence signal so as to
Detected.
Detecting system 9 includes housing, and housing is interior to be set provided with driving equipment 7, light excitation device 5, signal collecting device, detection
Standby and detection chambers 11, driving equipment 7 is that light excitation device and signal collecting device are powered, and driving equipment 7 includes power supply 6 and become
The Power convert of power supply 6 is different voltage by depressor, transformer, to adapt to the demand of different power devices in detecting system.
Light excitation device 5 includes fan 5-1, radiator 5-2 and LED light source 5-3, and fan 5-1 rated voltage is 12V,
LED light source 5-3 power is 11W, and rated voltage is 11V, and required voltage is provided by power supply 6, and by the transformation in drive module 7
Device is converted to corresponding voltage.
Detection device includes exciting piece 9-3, two to a point mirror 9-2 and sheet emitting 9-1, and signal collecting device is industrial camera 8,
Piece 9-3 is excited to be placed in LED light source 5-3 front end, sheet emitting 9-1 is placed in the camera lens front end of industrial camera 8 and is adjacent to industrial camera 8
Camera lens, two are placed in detection chambers 11 to point mirror 9-2 and chip 1, and the centre wavelength for exciting piece 9-3 is 475 nm, with a width of
40 nm, the distance away from LED light source 5-3 is 2.3 mm；Light excitation device 5 is located at the right side of detection chambers 11 and LED light source 5-3
The direction of the launch towards the direction of detection chambers, industrial camera 8 is located at the top of detection chambers 11 and camera lens is towards detection chambers
11 direction, chip is placed in the bottom of detection chambers, and detection chambers 11 are 55 × 55 mm square chambers, and two exist to a point mirror
Placed in detection chambers into 45 degree of angles, one is facing to light excitation device, and another side is towards industrial camera, and No. two baffle plates 10 are one
The PMMA plates of block black, for covering interference light, away from piece 9-3 distance is excited for 2.3 mm, prevent scattered light from producing background and doing
Disturb.
Embodiment 2, builds standard curve, referring to the drawings 5.
The present embodiment using HIV-1 DNA plasmids be detection template, inject the mixture into chip, chip by wrist-watch with
Human body is close to, and using body temperature amplification of nucleic acid, and detects, sets up the standard curve of various concentrations.Specifically include following steps：
（1）From original concentration 109 Copies/mL HIV-1 DNA plasmid templates start by 1：10 dilution proportion, obtains ladder
Spend the template concentrations of dilution.
（2）RPA kits are bought from twist, according to the method on supporting specification, 29.5 μ L Rehydration
Buffer, 2.1 μ L Primer-F, 2.1 μ L Primer-R, 0.6 μ L Probe and 3.2 μ L water, have configured mixture.
（3）The μ L of 10 copies/mL HIV-1 DNA profilings 10 are added in the mixture, and 2.5 μ L magnesium acetates are fully mixed
It is even, obtain reactant.
（4）Reactant is squeezed into chip 4 on ice, is put into detecting system, CCD industrial cameras 8 photograph to record detection
The fluorescence intensity of region initial reaction.
（5）Chip is embedded in wrist-watch, is worn at the wrist of human body, after reacting 24 minutes, chip is taken out and is placed in inspection
Photographed to record in examining system.
（6）Will（2）In template change template concentrations into respectively for 105 - 102Copies/m, repeats (2)-(5).
（7）Repeat (2)-(6) three times.
（8）ImageJ analysis detection zones 2-3 green fluorescence intensity, the fluorescence intensity after being terminated according to reaction, which is set up, to be marked
Directrix curve.
（9）Negative control group replaces nucleic acid-templated using RNA-free water, repeats (4)-(5).Standard curve such as Fig. 4.
Embodiment 3, nucleic acid amplification detection, referring to the drawings 4.
1）50 μ L water are squeezed into PDMS chips 1, are placed on 4 on heating plate, insertion thermocouple surveys the temperature of passage reclaimed water
Degree, adjusts heating-up temperature, the temperature of water is reached 33 ~ 34 C, imitates body temperature.
2）By HIV-1 DNA plasmid concentrations from 109Copies/mL starts by 1:10 start dilution, obtain different dense
Spend gradient.
2）According to the method for RPA reagent specifications, 29.5 μ L Rehydration buffer, 2.1 μ LPrimer-F,
2.1 μ L Primer-R, 0.6 μ L Probe and 3.2 μ L water, have configured mixture.
3）The 10 copies/mL μ L of HIV-1 DNA profilings 10, the μ L of magnesium acetate 2.5 are added, fully mixes, must react
Thing.
4）Reactant is squeezed into chip 1 on ice, is put into detecting system, CCD industrial cameras 8 photograph to record detection
The fluorescence intensity of region initial reaction.
5）Heating plate 4 is begun to warm up, and industrial camera 8 recorded an amplification figure picture every three minutes, and 30 points are counted on altogether
Clock.
6）Concentration is 105~102Copies/mL HIV-1 DNA are used as amplification template, repeat step 6）~8）.
7）Repeat 5）~9）Three times.
8）ImageJ analysis detection zones 2-3 fluorescence intensity, sets up real-time fluorescence amplification curve.Testing result such as Fig. 3.
Claims (10)
1. a kind of device for carrying out nucleic acid amplification detection, it is characterised in that including detecting system and chip, chip is placed in detection system
In system, detecting system includes light excitation device and signal collecting device, and the light sent by light excitation device is irradiated on chip,
Transmitting light is produced in detection zone, through being filtrated to get fluorescence signal, signal collecting device gathers the fluorescence signal to be examined
Survey, the chip can be integrated on wearable device.
2. a kind of device for carrying out nucleic acid amplification detection according to claim 1, it is characterised in that detecting system includes shell
Driving equipment, light excitation device, signal collecting device, detection device and detection chambers are provided with body, housing, driving equipment includes
Power supply and transformer, driving equipment are that light excitation device and signal collecting device are powered, and light excitation device includes fan, radiator
And LED light source, detection device is including exciting piece, two to point mirror and a sheet emitting, and signal collecting device is industrial camera, excites piece to put
In the front end of LED light source, sheet emitting is placed in the camera lens front end of industrial camera, and two are placed in detection chambers to point mirror and chip, light
Excitation device is located at the side of detection chambers and LED light source is towards the direction of detection chambers, and industrial camera is located at detection chambers
Top and camera lens is towards the direction of detection chambers, chip is placed in the bottom of detection chambers, two to point mirror in detection chambers into 45
Spend angle to place, one is facing to light excitation device, and another side is towards industrial camera.
3. a kind of device for carrying out nucleic acid amplification detection according to claim 2, it is characterised in that chip includes being arranged on
The reaction chamber of the hexagon at middle part, the two ends of the reaction chamber are respectively equipped with above and below inlet and liquid outlet, reaction chamber
Two sides is respectively equipped with top layer and bottom as sealing, when chip is placed in detection chambers, below provided with heating plate, heating plate
Powered by driving equipment.
4. a kind of device for carrying out nucleic acid amplification detection according to claim 3, it is characterised in that chip is that have biology
The high molecular polymer chip of compatibility, the top layer of chip includes channel design, when chip is PDMS chips, the bottom of chip
It is PDMS films, the thickness of PDMS passages is 0.5-1.5mm, the aperture of inlet and liquid outlet is 1-4mm.
5. a kind of device of carry out nucleic acid amplification detection according to any one of claim 3, it is characterised in that chip
The channel design of top layer is single-pass configuration or multi-channel structure, and the cumulative volume of passage is not less than 50 μ L.
6. a kind of device for carrying out nucleic acid amplification detection according to claim 2, it is characterised in that the power of LED light source
For 5-15W.
7. a kind of device for carrying out nucleic acid amplification detection according to claim 2, it is characterised in that excite the band logical ripple of piece
A length of 435-480nm.
8. a kind of device for carrying out nucleic acid amplification detection according to claim 2, it is characterised in that the band logical ripple of sheet emitting
A length of 500-560nm.
9. a kind of device of carry out nucleic acid amplification detection according to any one of claim 1-8, it is characterised in that the core
Piece can be integrated on watch, worn at human body wrist.
10. a kind of method for carrying out nucleic acid amplification detection, it is characterised in that any one of this method application claim 1-9 institutes
The device stated, and comprise the following steps：
（1）Measure human body temperature：The water filling into chip, is fixed on human skin by wearable device by chip, passes through heat
Fluid temperature in galvanic couple measurement chip, the feasibility that checking is expanded with body temperature；
（2）Build the standard curve of detection of nucleic acids：
（2.1）Since original concentration it is nucleic acid-templated dilute in proportion, obtain the template concentrations of gradient dilution；
（2.2）According to the method in RPA kit specifications, the good mixture needed for reaction of configuration；
（2.3）The template and magnesium acetate for adding various concentrations in the mixture are fully mixed；
（2.4）Reactant after mixing is squeezed into chip from inlet, detecting system is put into, photographs to record fluorescence intensity；
（2.5）Chip is worn on human skin, expanded by body temperature, after reaction certain time, core is taken out
Piece is put into detecting system and photographed to record；
Repeat step（2.1）-（2.5）At least three times,
（2.6）ImageJ analyzes the green fluorescence intensity of detection zone, and it is bent that the fluorescence intensity after being terminated according to reaction sets up standard
Line；
（3）Concentration of specimens is detected：According to step（2.1）-（2.5）Detection sample simultaneously brings testing result in standard curve into, obtains
Nucleic acid concentration into sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317223A (en) * | 2020-09-30 | 2022-04-12 | 富佳生技股份有限公司 | Nucleic acid detecting cassette and nucleic acid detecting apparatus |
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