CN107478629A - A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method - Google Patents
A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method Download PDFInfo
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- CN107478629A CN107478629A CN201710783914.3A CN201710783914A CN107478629A CN 107478629 A CN107478629 A CN 107478629A CN 201710783914 A CN201710783914 A CN 201710783914A CN 107478629 A CN107478629 A CN 107478629A
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Classifications
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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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- 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 invention discloses a kind of large area digital pcr droplet fluorescence high pass amount detecting device, it includes fluorescence excitation light path and fluorescence detection optical path;Along fluorescence excitation light path, line focus device and objective apparatus are incident on the digital pcr chip for being placed in objective table the exciting light of light supply apparatus transmitting successively;Along fluorescence detection optical path, fluorescence caused by digital pcr chip stimulated luminescence is transmitted to image acquiring sensor after the imaging of objective apparatus, focusing arrangement and imaging device successively;Wherein, objective table is additionally provided with temperature control device, and temperature control device controls the circulating temperature that digital pcr chip reacts in real time.The invention discloses a kind of large area digital pcr droplet fluorescence high-flux detection method, can obtain the accuracy that higher detection efficiency and large area high flux detect.The present invention can realize the real-time digital qPCR detections of high flux, high sensitivity, high specific, significant for improving digital pcr detecting instrument performance, precision and flux.
Description
Technical field
The present invention relates to biomedical inspection field field of nucleic acid detection, more particularly to one kind can realize large area numeral
PCR droplets fluorescence high pass amount detecting device and method.
Background technology
In fields such as the analysis of early diagnosis of tumor examination, blood disease parting and carrying capacity, noninvasive pre-natal diagnosis, it is required for pair
The nucleic acid of super low concentration carries out the detection of high speed high sensitivity high specific, and conventional PCR increasingly can not meet demand.For
This function is realized, people have developed a kind of digital pcr technology on the basis of PCR, and detection sensitivity is improved into 1~2 quantity
Level.
Droplet type digital pcr use " dividing and rule " (divide and conquer) inspection policies, by sample,
PCR reagent mixture, using oil as continuous phase, using microflow control technique, sample mixture is formed one by one as dispersed phase
Suspending drops are in continuous phase.And then a standard PCR amplification is distributed into a large amount of droplets (nanoliter to picoliters), Mei Gewei
The target molecule (DNA profiling) of 0 or 1 copy is only included in drop.Independent PCR amplifications are carried out to each droplet, amplification terminates
Afterwards, detect the fluorescence of each droplet, the droplet of positive signal counted, so as to obtain the absolute copy number of sample template and
Nucleic acid concentration.The size and number of droplet determine the detection sensitivity and lower limit of digital pcr, existing digital pcr droplet number
Between 20,000~10,000,000.
Existing commercial droplet type digital pcr technical operation is as follows:Prepared in special droplet and drop is prepared on chip;It is logical
Cross pipettor droplet is transferred in centrifuge tube, temperature cycles amplification is carried out using the PCR instrument of routine;After the completion of amplification, utilize
Streaming technology carries out fluorescence excitation and detection, result of calculation to each droplet successively.Problems be present in such detection mode:
(1) flow cytometer detection speed is slower, and detection speed takes long in 200~500/s, detection process, and flux is low;
(2) end-point method detection can only be carried out to single droplet, it is impossible to real-time qPCR, reduce specificity and sensitivity;
(3) light path hardware is complicated, it is difficult to realizes multiple digital pcr detection (more than 4 weights);
(4) transfer process of drop will cause droplet to lose twice, and the outside contamination that may be attracted;
(5) the heat block thermal inertia of standard PCR amplification instrument is big, thermal cycle times length (2 hours or so).
To solve above-mentioned five problems, applicant has been presented for integral type digital pcr chip, and has applied for patent of invention
(application number 201510961967.0).Referring to accompanying drawing 1, integral type digital pcr chip includes body 1, continuous phase disposed thereon
It is hand-hole 11, sample hand-hole 12, individual layer droplet tiling cavity 15, vacuum access aperture 16, micro- for generating "+the font " of droplet
Structure 13, and for connecting the fluid path 14 of continuous phase hand-hole 11 and "+font " micro-structural 13, for connecting sample hand-hole
12 and "+font " micro-structural 13 the first fluid path 17, for connecting "+font " micro-structural 13 and individual layer droplet tiling cavity 15
Second fluid path 18.All micro-structurals are made on a piece of polymeric substrates by MEMS technology, it is then poly- with another piece
Compound flat board is mutually bonded and forms the micro sprue system of closing, in continuous phase hand-hole 11, sample hand-hole 12, vacuum access aperture
Perforate at 16 3, for injecting sample and applying negative pressure.
Reference picture 2, its general principle are:After continuous phase hand-hole 11 injects oil, sample hand-hole 12 injects sample,
The applying vacuum negative pressure of vacuum access aperture 16, sample, continuous phase will reach "+word in the presence of negative pressure by runner 14 and 17
Type " micro-structural 13.Here, sample will form nanoliter level droplet 20 one by one because of the shearing of continuous phase, then pass through runner
18 enter individual layer droplet tiling cavity 15, are laid into droplet individual layer 19.
The technical method has the advantage that:Droplet individual layer 19, which is suitable for use with CCD, to carry out image conversion fluorescence and takes pictures detection,
With higher flux and speed;It is bent that the change of droplet fluorescence with circulation now can be obtained by continuous fluorescence image detection
Line, single drop qPCR detections are carried out, there is higher sensitivity and specificity;, can be easily by increasing excitation source and optical filter
Realize multiple digital pcr;Whole process integration is completed, in the absence of droplet loss and extraneous pollution.Finally, individual layer droplet will
With larger temperature control area and less thermal inertia, PCR cycle proliferation time can be reduced within half an hour.
In order to carry out detection of nucleic acids using the digital pcr chip, on the one hand need to carry out the digital pcr micro-fluidic chip
Temperature cycles expand, and on the other hand need to excite digital pcr drop individual layer progress real time laser and fluorescence imaging detects.Per temperature
Circulation primary is spent, a drop fluoroscopic image detection is just carried out, to obtain the fluorescence consecutive variations curve of each drop.To improve
Flux, on one chip, multiple integral type digital pcr micro-fluidic structures can be integrated, form 32,48 even 96 samples
While detect (reference picture 3), to improve flux, so just need chip to carry out one-dimensional or two-dimentional motion, so as to
Picture of large image scale collection can be carried out by scan mode.In addition, to improve detection efficiency, using a kind of new algorithm, first pass through
Small multiplying power carries out rough big visual field viewing, has found that it is likely that after drop phosphor dot being present, switches to the fine small field of view observation of high magnification
Mode, to improve efficiency.But existing detection means such as fluorescence microscope etc., can not meet above-mentioned testing requirements.
The content of the invention
It is an object of the invention to solve at least the above and/or defect, and provide at least will be described later it is excellent
Point.
It is a still further object of the present invention to provide a kind of large area digital pcr droplet fluorescence high pass amount detecting device, and it is logical
Cross system light path design and split detection zone continuous scanning, can realize digital pcr high flux, in real time and precisely
Quantitative detection, in addition, fast positioning and the detection of drop phosphor dot can be achieved by the switching of size surface sweeping visual field.
A further object of the invention is by a kind of large area digital pcr droplet fluorescence high-flux detection method, can be obtained
The accuracy of get Geng Gao detection efficiency and large area high flux detection.
In order to realize according to object of the present invention and further advantage, there is provided a kind of large area digital pcr droplet is glimmering
Light high pass amount detecting device, including fluorescence excitation light path and fluorescence detection optical path;
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to the exciting light of light supply apparatus transmitting successively
It is placed on the digital pcr chip of objective table;
Along the fluorescence detection optical path, the digital pcr chip is by fluorescence caused by the exciting light successively through the thing
Transmitted after lens device, the focusing arrangement and imaging device imaging to image acquiring sensor;
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip in real time
The circulating temperature of reaction.
Preferably, wherein, the objective table is fixed on optical circuit path, or at least has a translation freedoms,
To meet the needs of picture of large image scale collection.
Preferably, wherein, the objective apparatus includes:
One object lens, it is fixed in light path;
Or the object lens of several different multiplyings, it is configured into light path respectively in the form of changeable, so as to realize not
With the digital pcr droplet fluorescence imaging of multiplying power.
Preferably, wherein, the multiplying power of the object lens is selected from 2X, 4X, 5X or 10X.
Preferably, wherein, the light supply apparatus includes:
Light source assembly, it provides parallel mixing light source;
Light source optical filter box, it has:
One light source optical filter, it is fixed in the fluorescence excitation light path, or
Several light source optical filters, it is configured into the fluorescence excitation light path respectively in the form of changeable.
Light source optical filter can be used for carrying out optical filtering processing to mixing light source, can be used for exciting PCR droplets dyestuff institute to provide
The monochromatic source needed.Monochromatic source is produced using mixing light source and optical filter in this programme, can be provided with relatively low cost
The monochromatic excitation light source of multiple wavelength, realize multiple digital pcr function.
Preferably, wherein, the light supply apparatus includes several sets of monochromatic light sources;
Along light path direction of advance, the sets of monochromatic light sources includes successively:Monochromatic source, shutter and light lens;Wherein, institute
State shutter break-make and control the fluorescence excitation light path.
Preferably, wherein, the light supply apparatus includes:
At least two sets of monochromatic light sources, to provide stronger excitation source;;
Several first dichronic mirrors, the transmitting light path of the sets of monochromatic light sources is closed beam and imports the fluorescent exciting by it
Road.
Dichronic mirror is a branch of available for two-way excitation source is merged into, can be by all exciting lights using multiple dichroic mirrors
Source is merged into a branch of, so as to provide the excitation source of multiple different wave lengths, realizes multiple digital pcr function.
Preferably, wherein, focusing arrangement includes:
Second dichronic mirror, the exciting light of its reflection source device transmitting are excited to produce through the digital pcr chip simultaneously
Fluorescence.
Preferably, wherein, focusing arrangement also includes:
First focus lens assembly, it is located between the light supply apparatus and second dichronic mirror, for shaping and expansion
Shu Suoshu laser, so as to obtain preferable exciting light.
Preferably, wherein, focusing arrangement also includes:
Second focus lens assembly, it is located between second dichronic mirror and the imaging device, for shaping and expansion
Shu Suoshu fluorescence, so as to obtain preferable image quality.
Preferably, wherein, along the fluorescence detection optical path, the imaging device filters out component and glimmering including fluorescence successively
Photoimaging lens;
Preferably, along the fluorescence detection optical path, the imaging device includes single fluorescence filtering assembly and glimmering successively
Photoimaging lens;
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has:
Light source light blocking piece, it filters out the exciting light of the light supply apparatus transmitting;And
Fluorescent optical filter, it filters out the veiling glare of the non-wavelength of fluorescence.
Preferably, wherein, along the fluorescence detection optical path, the imaging device includes several fluorescence optical filtering groups successively
Part and fluorescence imaging lens;
Wherein, several described fluorescence filtering assemblies include:
The light source light blocking piece of several different wave lengths, it is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the exciting light of the light supply apparatus transmitting respectively;And
The fluorescent optical filter of several different wave lengths, it is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the veiling glare of the non-wavelength of fluorescence respectively.
The technical program is filtered out by light supply apparatus exciting light and the non-wavelength of fluorescence veiling glare, can improve system
Signal to noise ratio, switched by the combination between the light source light blocking piece and fluorescent optical filter of several different wave lengths, PCR droplets can be gathered
The fluoroscopic image of the different wave length distributed, realize multiple digital pcr function.
The purpose of the present invention can also be further by the method for large area digital pcr droplet fluorescence high flux detection Lai real
Existing, this method comprises the following steps:
Step 1:The digital pcr chip for having completed droplet processing is placed on objective table, the digital pcr chip and temperature control
Device connects;
Step 2:A PCR temperature cycles are carried out, process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s;
Step 3:Objective apparatus is switched into minimum magnification objective;
Step 4:Light supply apparatus, imaging device are switched into light path corresponding to the first fluorescent dye or probe;
Step 5:Decile is carried out to the dimension of level two of the digital pcr chip, obtains some rectangles or rectangular region,
Each region area is S, and carries out droplet fluoroscopic image acquisition to each region, wherein the S is 1mm2~16mm2;
Step 6:By light supply apparatus, imaging device switch to the second fluorescent dye or probe corresponding to light path, perform again
The step 5, complete the detection of the image of the second fluorescent dye;
Step 7:Repeating said steps 6, until all fluorescent dyes or probe have been detected and finished;
Step 8:Objective apparatus is switched into time low range object lens, repeating said steps 4, step 5, step 6 and step 7;
Step 9:Repeating said steps 8, until the switching of all multiplying powers of object lens finishes, all fluorescent dyes have detected
Finish;
Step 10:2~step 9 of repeat step, and count, often it is repeated once, counts once, is more than N until counting, wherein
N represents amplification cycles number, and the N is 30~40 times;
Step 11:Test is completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, and statistics is negative
Positive droplet number, and calculate concentration.
Preferably, wherein, in the step 5,
Any dimension of the digital pcr chip in the horizontal direction is subjected to decile, obtains the strip that some width are W
Shape detection zone, the strip detection zone is continuously scanned, the consecutive image of each detection zone is obtained, to digital pcr
Micro-fluidic chip image is continuously acquired, wherein, the width W is 1mm~3mm.
The present invention comprises at least following beneficial effect:
The present invention can realize the real-time digital qPCR detections of high flux, high sensitivity, high specific, for improving numeral
PCR detecting instruments performance and precision, flux are significant., can be to the nucleic acid of low concentration using technical solution of the present invention
Sample carries out quick high accuracy detection, has great importance for cancer, infection disease, pre-natal diagnosis etc., for changing
Philanthropists' accuracy of detection and time limit, the medical detection technique of raising, improvement clinical testing procedure tool have very important significance.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is the structural representation of integrated digital pcr chip in an example;
Fig. 2 is the structural representation of integrated digital pcr chip in another example;
Fig. 3 is high flux integrated digital pcr chip structural representation (48 array) in another example;
Fig. 4 is large area digital pcr droplet fluorescence high flux structure of the detecting device block diagram in another example;
Fig. 5 is large area digital pcr droplet fluorescence high flux structure of the detecting device schematic diagram in another example;
Fig. 6 is the large area digital pcr droplet fluorescence high pass for having in another example motion in one dimension mechanism objective table device
Amount detecting device structural representation;
Fig. 7 is the large area digital pcr droplet fluorescence high pass for having in another example two-dimensional motion mechanism objective table device
Amount detecting device structural representation;
Fig. 8 is to have the large area digital pcr droplet fluorescence of multiple different multiplying objective switch-over devices high in another example
Flux structure of the detecting device schematic diagram;
Fig. 9 is the large area digital pcr droplet fluorescence high flux structure of the detecting device with light source optical filter switching mechanism
Schematic diagram;
The large area digital pcr droplet fluorescence high flux structure of the detecting device that Figure 10 has multiple sets of monochromatic light sources is illustrated
Figure;
Figure 11 is that the large area digital pcr droplet fluorescence high flux in another example with exciting light focus lens assembly is examined
Survey apparatus structure schematic diagram;
Figure 12 is that the large area digital pcr droplet fluorescence high flux in another example with fluorescent foci lens subassembly detects
Apparatus structure schematic diagram;
Figure 13 is to have the large area digital pcr droplet of exciting light and fluorescent foci lens subassembly glimmering simultaneously in another example
Light high flux structure of the detecting device schematic diagram;
Figure 14 is the large area digital pcr droplet fluorescence high flux for having in another example fluorescence filtering assembly switching mechanism
Structure of the detecting device schematic diagram;
Figure 15 is the schematic diagram that digital pcr chip is divided into hough transform region by bidimensional in another example;
Figure 16 is digital pcr chip in another example by the one-dimensional schematic diagram for being divided into strip detection zone;
Figure 17 is the large area digital pcr droplet fluorescence high pass amount detecting device for being used for double digital pcr in another example
Structural representation;
Figure 18 is the large area digital pcr droplet fluorescence high pass amount detecting device for being used for sixfold digital pcr in another example
Structural representation;
Figure 19 is the structural representation of light source optical filter fixing device in another example;
Figure 20 is the structural representation of fluorescent optical filter fixing device in imaging device in another example.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of individual other elements or its combination.
Reference picture 4 and Fig. 5, as a kind of way of realization of the present invention, the detection of large area digital pcr droplet fluorescence high flux
Device includes:
Objective apparatus 200, it is located at directly over digital pcr chip, for the collection of whole digital pcr fluoroscopic image most before
End;
Light supply apparatus 300, the fluorescence excitation for digital pcr droplet;
Focusing arrangement 400, its one side converge to the light that light supply apparatus 300 is sent on digital pcr chip 1, the opposing party
Phosphor collection caused by digital pcr chip 1 is transmitted to imaging optical path device 500 and image acquiring sensor 600 by face, so as to
For light source illumination and the coupling of fluorescence imaging;
Imaging optical path device 500, it is used to fluoroscopic image carrying out shaping and optical filtering, and by imaging and IMAQ biography
600 on sensor, so as to the fluorescence imaging for digital pcr droplet;
Image acquiring sensor 600, it is used for the acquisition to fluoroscopic image and is converted into digitized image, to digital pcr
The fluoroscopic image of droplet gathers in real time.
PCR temperature control devices 700, it is used to carry out accurate temperature cycles control to digital pcr chip 1, realizes droplet number
Word PCR amplification procedure;And
Objective table device 800, it is used to carry digital pcr droplet micro-fluidic chip and its temperature control device.
In this technical scheme, including fluorescence excitation light path and fluorescence detection optical path;
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to the exciting light of light supply apparatus transmitting successively
It is placed on the digital pcr chip of objective table;
Along the fluorescence detection optical path, the digital pcr chip is by fluorescence caused by the exciting light successively through the thing
Transmitted after lens device, the focusing arrangement and imaging device imaging to image acquiring sensor;
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip in real time
The circulating temperature of reaction.
By adding a variety of fluorescent dyes or probe to digital pcr reaction system, coordinate different mix, primer, it is possible to achieve
Multiple digital pcr.Wherein, PCR tuples are identical with the fluorescent dye or the species of probe that add, for example, double PCR, then need
Two fluorescent dyes or probe.
In another example, the objective table is fixed on optical circuit path, or at least has a translation freedoms, with
Realize that one-dimensional or two-dimensional scan mode carries out IMAQ, so as to expand detection zone.
Reference picture 6, in a kind of embodiment for realizing the one-dimensional free degree of objective table, objective table device 800 includes servo
Motor 803, transmission device 802, position detecting device 806, one-dimensional precise on the horizontal X direction of objective table 801 is transported with realizing
Dynamic control.By the motion in one dimension to objective table, to realize that imaging system detects to the one-dimensional scanning of digital pcr chip, to carry
High detection scope, realize that large area high flux detects.Wherein, servomotor 803 can be step-servo motor, DC servo electricity
Machine, AC servo motor, transmission device 802 can be ball leading screw driving device, and position sensor can be that linear grating is compiled
Code device, resistor type displacement sensor.
Reference picture 7, in a kind of embodiment of objective table two-dimensional freedom is realized, objective table device 800 includes a pair
Servomotor 803 and 805, a pair of transmission devices 802 and 804, a pair of position detecting devices 806 and 807, to realize to objective table
X, Y bidimensional precise flange in 801 horizontal direction.By the two-dimensional motion to objective table, to realize imaging system logarithm
The bidimensional Scanning Detction of word pcr chip, to improve detection range, realize that large area high flux detects.
In another example, the objective apparatus includes an object lens, and it is fixed in light path.
In another example, the objective apparatus includes the object lens of several different multiplyings, and it is divided in the form of changeable
It is not configured into light path.
Reference picture 8, a kind of embodiment that different multiplying object lens automatically switch, which can be achieved, is:Objective apparatus 200 is included and watched
Take the control being depicted without in motor 205, transmission device 204, turning joint 203,202, one groups of object lens 201 of object lens fixed plate and figure
Unit processed.Wherein, the isometrical distribution centered on turning joint 203 in object lens fixed plate 202 of object lens 201.Servomotor 205 is logical
Transmission device 204, turning joint 203 are crossed, object lens fixed plate 202 can be driven to be rotated, and then can be by required for
Enlargement ratio object lens switch to the front end of focusing arrangement 400 in light path.Wherein, servomotor 205 can be step-servo motor,
DC servo motor, AC servo motor, transmission device 204 can be belt wheel transmission device, gear drive etc., rotating hinge
Chain 203 can be swivel bearing.The droplet fluorogram of different enlargement ratios can be realized by automating switching using this scheme
As obtaining.Also, this mode is a kind of explanation of preferred embodiments, but is not limited thereto., can be with when implementing of the invention
The different switching aspect of object lens is implemented according to user's demand.
In another example, the multiplying power of the object lens is selected from 2X, 4X, 5X or 10X.
In another example, reference picture 9, the light supply apparatus 300 includes:
Light source assembly, it is by mixing light source emission element 301 and convex lens 302, there is provided parallel mixing light source;
Light source optical filter box, it has:
One light source optical filter 303, it is fixed in the fluorescence excitation light path, or
Several light source optical filters 303, it is respectively configured in the form of changeable into the fluorescence excitation light path.
In the technical scheme, realize that the embodiment that light sources with different wavelengths optical filter automatically switches is:Light supply apparatus 300
By Halogen lamp LED 301, lens 302, a series of optical filters 303, optical filter fixed plate 305, turning joint 304, transmission device 306,
Servomotor 307 and control unit (being not drawn into figure) composition.Lens 302 are used to be collimated the light that light source 301 is sent, shape
Into collimated light beam.Servomotor 307 drives optical filter fixed plate 305 to do rotation fortune by transmission device 306 and turning joint 304
It is dynamic, the optical filter 303 of different wave length is switched into the front end of lens 302, realizes that the monochromatic light of the different wave length of light supply apparatus 300 is defeated
Go out.Wherein, transmission device 306 can be belt wheel transmission, gear drive, and turning joint 304 can be that the dynamic hinge of rotation is held.Servo
Motor can be step-servo motor, DC servo motor or AC servo motor.Also, this mode is a kind of preferably real
The explanation of example, but be not limited thereto.When implementing of the invention, the difference of light source optical filter can be implemented according to user's demand
Switch aspect.
In another example, reference picture 10, the light supply apparatus 300 includes several sets of monochromatic light sources;
Along light path direction of advance, the sets of monochromatic light sources includes successively:Monochromatic source 301, shutter 308 and light lens
302;Wherein, the break-make of shutter 308 controls the fluorescence excitation light path.
In another example, the light supply apparatus 300 includes:
Two and above sets of monochromatic light sources;
Several first dichronic mirrors 309, the transmitting light path of the sets of monochromatic light sources is closed beam and import the fluorescence by it to swash
Luminous road.
In this embodiment, light supply apparatus 300 is by multiple monochromatic sources 301, lens 302, shutter 308, the first color separation
Mirror 309 forms.Wherein, each light source 301 corresponds to a shutter 308, for controlling the break-make of the light source.First dichronic mirror
The 309 conjunction beam for realizing two-beam, the quantity of the first dichronic mirror 309 subtract 1 for the quantity of light source 301, realize that all light sources are sent
Light conjunction beam.Which road monochrome light output is controlled by shutter 308 and introduces system.Light source can be lasing light emitter, LED etc., but
Not limited to this.
In another example, reference picture 5, focusing arrangement includes:
Second dichronic mirror, the exciting light of its reflection source device transmitting are excited to produce through the digital pcr chip simultaneously
Fluorescence.
In this embodiment, focusing arrangement 400 only includes second dichronic mirror 401, and the second dichronic mirror 401 is on the one hand
Light for light-source system 300 to be sent reflexes to digital pcr chip 1, and digital pcr drop is excited;On the other hand
For passing through fluorescence caused by drop on digital pcr chip, and feed them into imaging device 500.
In another example, referring to Figure 11, focusing arrangement 400 also includes:
First focus lens assembly, it includes lens 402 and lens 403, and the first focus lens assembly is located at the light source
Between device 300 and second dichronic mirror 401, the light for light supply apparatus 300 to be sent is collimated and expanded, to change
Excite spot size and quality.
In another example, referring to Figure 12, focusing arrangement also includes:
Second focus lens assembly, it includes lens 404 and lens 405, and the second focus lens assembly by object lens to filling
Put 200 fluorescence sent to be collimated and expanded, coordinate with imaging len 501, improve image quality.
In another example, referring to Figure 13, focusing arrangement also simultaneously comprising lens 402, lens 403, lens 404 and 405, changes
Change excites spot size, improves image quality.
In another example, along the fluorescence detection optical path, the imaging device 500 includes single fluorescence filtering assembly successively
With fluorescence imaging lens 501;
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has:
Light source light blocking piece, it filters out the exciting light of the light supply apparatus transmitting;And
Fluorescent optical filter, it filters out the veiling glare of the non-wavelength of fluorescence.
In another example, reference picture 14, along the fluorescence detection optical path, the imaging device includes fluorescence optical filtering group successively
Part and fluorescence imaging lens 501;
Wherein, the fluorescence filtering assembly includes:
The light source light blocking piece of several different wave lengths, it is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the exciting light of the light supply apparatus transmitting respectively;And
The fluorescent optical filter of several different wave lengths, it is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the veiling glare of the non-wavelength of fluorescence respectively.
In this embodiment, comprising multigroup optical filter, (every group of optical filter includes a light source light blocking piece to imaging device 500
With a fluorescent optical filter), and the optical filter fixed plate 505 for switching between each group optical filter, turning joint 504, transmission
Device 506, servomotor 507, and control unit (being not drawn into figure).Servomotor 507 passes through transmission device 506 and rotating hinge
Chain 505 drives the optical filter 502 being located in optical filter fixed plate 505 and optical filter 503 to rotate, by required any group
Optical filter 502 and optical filter 503 switch to the front end of image acquiring sensor 700, realize filtering out and required fluorescence ripple for exciting light
The collection of section.Each group optical filter 502 and optical filter 503 mutually collect with each monochromatic light that excitation source is sent in described imaging device
Close, it is possible to achieve the digital pcr fluorescence excitation of different dyes and detection, and then realize multiple digital pcr.
In another example, a kind of method for realizing the detection of large area digital pcr droplet fluorescence high flux, including following step
Suddenly:
Step 1), the micro-fluidic chip 1 for having completed the generation of digital pcr droplet is placed to objective table 801, with PCR temperature controls
Device 700 connects.
Step 2), carries out a PCR temperature cycles, and process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s;
Step 3), objective apparatus 200 switch to minimum magnification objective;
Step 4), light supply apparatus, imaging device switch to light path corresponding to the first dyestuff or probe.
Step 5), referring to Figure 15, by digital pcr micro-fluidic chip 1, X, Y carry out decile in the vertical direction of level two, are
Some rectangles or rectangular region 101, each region has long L and wide W, and carries out droplet fluoroscopic image to each region and obtain
Take.The long L is 1mm~4mm, and the wide W is 1mm~4mm;
Step 6), light supply apparatus 300, imaging device 500 switch to light path corresponding to next dyestuff.Step is performed again
5) detection of the image of next fluorescent dye or probe, is completed;
Step 7), repeat step 6), until all fluorescent dyes or probe detection finish;
Step 8), objective apparatus 200 switch to time low range object lens;Repeat step 4), step 5), step 6), step 7);
Step 9), repeat step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dyes or probe have detected
Finish;
Step 10), repeat step 2)~step 9), and count, often it is repeated once, counts once, is more than N until counting;
The N represents amplification cycles number, is 30~40;
Step 11), all tests are completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
In another example, a kind of method for realizing the detection of large area digital pcr droplet fluorescence high flux, including following step
Suddenly:
Step 1), the micro-fluidic chip 1 for having completed the generation of digital pcr droplet is placed to objective table 801, with PCR temperature controls
Device 700 connects.
Step 2), carries out a PCR temperature cycles, and process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s;
Step 3), objective apparatus 200 switch to minimum magnification objective;
Step 4), light supply apparatus, imaging device switch to light path corresponding to the first dyestuff or probe.
Step 5), referring to Figure 16, the dimension of digital pcr chip in the horizontal direction is subjected to decile, is divided into one
Serial width is W strip detection zone, by continuous scan mode, obtains the consecutive image of each detection zone, right
Digital pcr micro-fluidic chip image is continuously acquired, and the width W is 1mm~3mm;
Step 6), light supply apparatus 300, imaging device 500 switch to light path corresponding to next dyestuff.Step is performed again
5) detection of the image of next fluorescent dye or probe, is completed;
Step 7), repeat step 6), until all fluorescent dyes or probe detection finish;
Step 8) objective apparatus 200 switches to time low range object lens;Repeat step 4), step 5), step 6), step 7);
Step 9), repeat step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dyes or probe have detected
Finish;
Step 10), repeat step 2)~step 9), and count, often it is repeated once, counts once, is more than N until counting;
The N represents amplification cycles number, is 30~40;
Step 11), all tests are completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
With reference to specific embodiment, the present invention is described in further detail:
<Example 1>
The digital pcr micro-fluidic chip parameter of required detection is:Double digital pcr is realized, uses the first dyestuff and second
Dyestuff, excitation wavelength are respectively 488nm, 532nm, and wavelength of fluorescence is respectively 515nm and 560nm, the micro-fluidic core of droplet digital pcr
Piece is integrated with 48 monocyte sample detection structures, integral core length of a film 150mm, wide 100mm.
Referring to Figure 17, for this chip, propose that a kind of real-time fluorescence image for carvel-built digital pcr drop obtains
Device is taken, including:
Objective apparatus 200, two object lens 2011,2012 comprising 4X and 10X enlargement ratios;
Light supply apparatus 300, for the fluorescence excitation of digital pcr droplet, including two lasers 3011,3012, two groups fast
Door 3081,3082, two groups of lens 3021,3022, and a dichroic mirror 3091;Wherein, the wavelength of laser 3011 is 488nm, laser
The wavelength of device 3012 is 532nm, and lens 3021,3022 have front end focal length 50mm, rear end focal length 47mm.
Focusing arrangement 400, for light source illumination and the coupling of fluorescence imaging, including 401, two groups of lens 402 of dichroic mirror,
403、404、405;The light that lens 402,403 send light source is expanded, and is allowed to be formed diameter 3mm circle hot spots and is exposed to numeral
On PCR droplet micro-fluidic chips;The fluorescence shaping that lens 404,405 will distribute from digital pcr droplet micro-fluidic chip, with into
As light path is combined, diameter 2mm image illuminations are made to image acquiring sensor 700.
Imaging optical path device 500, for the fluorescence imaging of digital pcr droplet, including two groups of optical filters 5021,5031,
5022、5032;With plus lens 501, optical filter fixed plate 505, turning joint 504, transmission device 506,507 groups of servomotor
Into.Wherein, filter out optical source wavelength optical filter 5031,5032 be respectively long wave pass filter, cut-off frequency be respectively 500nm,
540nm, fluorescent optical filter 5021,5022 respectively centre wavelength 515nm, 560nm, bandwidth 30nm optical filters, lens 501 have
Front end focal length 50mm, rear end focal length 47mm;
Image acquiring sensor 600, the fluoroscopic image for digital pcr droplet gather in real time, are sensed for Array CCD
Device;
PCR temperature control devices 700, for the temperature cycles control of digital pcr droplet, including temperature sensor 702, heating
Using Peltier as heating actuator 704 and cooling actuator 703, heat transfer fast 701;Wherein, 701 be to be formed by machining
Al-alloy parts, heating actuator 704 be resistive heater, cool actuator 703 be fan, temperature sensor 702 is thermoelectricity
Even sensor.
Objective table device, for carrying digital pcr droplet micro-fluidic chip and its temperature control device, including objective table 801, watch
Take motor 803,805, transmission device 802,804, position sensor 806,807.Wherein, servomotor 803,805 is two-phase four
Line step-servo motor, transmission device 802,804 are ball screw structure, and stroke is respectively 200mm and 150mm, position sensing
Device 806,807 is linear grating encoder, and range is 200mm and 150mm, precision 0.01mm.
Specific detection process is as follows:
Step 1), the micro-fluidic chip for having completed the generation of digital pcr droplet is placed to objective table, with PCR temperature control devices
Connect.
Step 2), carries out a PCR temperature cycles, and process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s;
Step 3), objective apparatus switch to minimum magnification objective;
Step 4), light supply apparatus, imaging device switch to light path corresponding to the first dyestuff.
Step 5), digital pcr micro-fluidic chip is subjected to decile in the vertical direction of level two, is some rectangles or rectangular
Shape region, each region area are S, and carry out droplet fluoroscopic image acquisition to each region.The rectangular area is wide 2mm's
Square;
Step 6), light supply apparatus, imaging device switch to light path corresponding to next dyestuff.Step 5) is performed again, is completed
The detection of the image of next fluorescent dye;
Step 7), repeat step 6), until all fluorescent dyes have been detected and finished;
Step 8), objective apparatus switch to time low range object lens;Repeat step 4), step 5), step 6), step 7);
Step 9), repeat step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dye detections finish;
Step 10), repeat step 2)~step 9), and count, often it is repeated once, counts once, is more than N until counting;
The N represents amplification cycles number, is 30~40;
Step 11), all tests are completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
<Example 2>
The digital pcr micro-fluidic chip parameter of required detection is:Sixfold digital pcr, dyestuff used are respectively:
Fluorescent dye 1:Excitation wavelength 390nm, wavelength of fluorescence 420nm;
Fluorescent dye 2:Excitation wavelength 475nm, wavelength of fluorescence 522nm;
Fluorescent dye 3:Excitation wavelength 525nm, wavelength of fluorescence 577nm;
Fluorescent dye 4:Excitation wavelength 572nm, wavelength of fluorescence 628nm;
Fluorescent dye 5:Excitation wavelength 630nm, wavelength of fluorescence 675nm;
Fluorescent dye 6:Excitation wavelength 650nm, wavelength of fluorescence 710nm;
Droplet digital pcr micro-fluidic chip is integrated with 48 monocyte samples detection structures, integral core length of a film 150mm, wide
100mm.For this chip, referring to Figure 18, propose that a kind of real-time fluorescence image for carvel-built digital pcr drop obtains
Device, including:
Objective apparatus 200, two object lens 2011,2012 comprising 4X and 10X enlargement ratios;
Light supply apparatus 300, for the fluorescence excitation of digital pcr droplet, including halogen tungsten lamp light source 301, lens 302, filter
Piece fixed plate 305, turning joint 3041, transmission device 306, servomotor 307, and the filter in optical filter fixed plate 305
Mating plate 3031,3032,3033,3034,3035,3036, is shown in Figure 19.Filter center wavelength be respectively 390nm, 475nm,
525nm, 572nm, 630nm, 650nm, bandwidth 20nm;Wherein, lens 302 have front end focal length 50mm, rear end focal length 47mm.Watch
It is the line step-servo motor of two-phase four to take motor 307, and transmission device 306 is belt wheel transmission mechanism, and turning joint is rolling bearing;
Focusing arrangement 400, for light source illumination and the coupling of fluorescence imaging, including 401, two groups of lens 402 of dichroic mirror,
403、404、405;The light that lens 402,403 send light source is expanded, and is allowed to be formed diameter 3mm circle hot spots and is exposed to numeral
On PCR droplet micro-fluidic chips;The fluorescence shaping that lens 404,405 will distribute from digital pcr droplet micro-fluidic chip, with into
As light path is combined, diameter 2mm image illuminations are made to image acquiring sensor 700.
Imaging optical path device 500, for the fluorescence imaging of digital pcr droplet, include filtering out optical source wavelength referring to Figure 20
Band resistance optical filter 5021,5022,5053,5024,5025,5026, and through droplet fluorescence fluorescent optical filter 5031,5032,
5033rd, 5034,5035,5036, plus lens 501, optical filter fixed plate 505, turning joint 504, transmission device 506, servo
Motor 507 forms.Wherein, cardiac wave in the band resistance optical filter 5021,5022,5053,5024,5025,5026 of optical source wavelength is filtered out
Long is respectively 390nm, 475nm, 525nm, 572nm, 630nm, 650nm, bandwidth 20nm, fluorescent optical filter 5031,5032,
5033rd, 5034,5035,5036 centre wavelengths are respectively 420nm, 522nm, 577nm, 628nm, 675nm, 710nm, bandwidth 20nm
Optical filter, lens 501 have front end focal length 50mm, rear end focal length 47mm;
Image acquiring sensor 700, the fluoroscopic image for digital pcr droplet gather in real time, are sensed for Array CCD
Device;
PCR temperature control devices 700, for the temperature cycles control of digital pcr droplet, including temperature sensor 702, heating
Using Peltier as heating actuator 704 and cooling actuator 703, heat transfer fast 701;Wherein, 701 be to be formed by machining
Al-alloy parts, heating actuator 704 be resistive heater, cool actuator 703 be fan, temperature sensor 702 is thermoelectricity
Even sensor.
Objective table device, for carrying digital pcr droplet micro-fluidic chip and its temperature control device, including objective table 801, watch
Take motor 803,805, transmission device 802,804, position sensor 806,807.Wherein, servomotor 803,805 is two-phase four
Line step-servo motor, transmission device 802,804 are ball screw structure, and stroke is respectively 200mm and 150mm, position sensing
Device 806,807 is linear grating encoder, and range is 200mm and 150mm, precision 0.01mm.
Specific detection process is as follows:
Step 1), the micro-fluidic chip for having completed the generation of digital pcr droplet is placed to objective table, with PCR temperature control devices
Connect.
Step 2), carries out a PCR temperature cycles, and process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s;
Step 3), objective apparatus switch to minimum magnification objective;
Step 4), light supply apparatus, imaging device switch to light path corresponding to the first dyestuff.
Step 5), the dimension of digital pcr chip in the horizontal direction is subjected to decile, being divided into a series of width is
W strip detection zone, pass through continuous scan mode, the consecutive image of each detection zone of acquisition, to digital pcr miniflow
Control chip image is continuously acquired.The width W is 2mm;
Step 6), light supply apparatus, imaging device switch to light path corresponding to next dyestuff.Step 5) is performed again, is completed
The detection of the image of next fluorescent dye;
Step 7), repeat step 6), until all fluorescent dyes have been detected and finished;
Step 8), objective apparatus switch to time low range object lens;Repeat step 4), step 5), step 6), step 7);
Step 9), repeat step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dye detections finish;
Step 10), repeat step 2)~step 9), and count, often it is repeated once, counts once, is more than N until counting;
The N represents amplification cycles number, is 30~40;
Step 11), all tests are completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
Number of devices and treatment scale described herein are the explanations for simplifying the present invention.To the large area of the present invention
The application of digital pcr droplet fluorescence high pass amount detecting device and method, modifications and variations are to one skilled in the art
Obviously.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With.It can be applied to various suitable the field of the invention completely., can be easily for those skilled in the art
Realize other modification.Therefore it is of the invention and unlimited under the universal limited without departing substantially from claim and equivalency range
In specific details and shown here as the legend with description.
Claims (14)
1. a kind of large area digital pcr droplet fluorescence high pass amount detecting device, it is characterised in that including fluorescence excitation light path and glimmering
Light light path;
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to and are placed in the exciting light of light supply apparatus transmitting successively
On the digital pcr chip of objective table;
Along the fluorescence detection optical path, the digital pcr chip is filled through the object lens successively by fluorescence caused by the exciting light
Put, transmitted after the imaging of the focusing arrangement and imaging device to image acquiring sensor;
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip to react in real time
Circulating temperature.
2. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that the load
Thing platform is fixed on optical circuit path, or at least has a translation freedoms.
3. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that the thing
Lens device includes:
One object lens, it is fixed in light path;
Or the object lens of several different multiplyings, it is configured into light path respectively in the form of changeable.
4. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 3, it is characterised in that the thing
The multiplying power of mirror is selected from 2X, 4X, 5X or 10X.
5. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that the light
Source device includes:
Light source assembly, it provides parallel mixing light source;
Light source optical filter box, it has:
One light source optical filter, it is fixed in the fluorescence excitation light path, or
Several light source optical filters, it is configured into the fluorescence excitation light path respectively in the form of changeable.
6. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that the light
Source device includes several sets of monochromatic light sources;
Along light path direction of advance, the sets of monochromatic light sources includes successively:Monochromatic source, shutter and light lens;Wherein, it is described fast
Door break-make controls the fluorescence excitation light path.
7. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 6, it is characterised in that the light
Source device includes:
At least two sets of monochromatic light sources;
Several first dichronic mirrors, the transmitting light path of the sets of monochromatic light sources is closed beam and imports the fluorescence excitation light path by it
In.
8. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that focus on dress
Put including:
Second dichronic mirror, it reflects the exciting light of the light supply apparatus transmitting is excited to produce through the digital pcr chip simultaneously
Fluorescence.
9. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 8, it is characterised in that focus on dress
Putting also includes:
First focus lens assembly, it is located between the light supply apparatus and second dichronic mirror, for shaping and expands institute
State laser.
10. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 9, it is characterised in that focus on dress
Putting also includes:
Second focus lens assembly, it is located between second dichronic mirror and the imaging device, for shaping and expands institute
State fluorescence.
11. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that along described
Fluorescence detection optical path, the imaging device include single fluorescence filtering assembly and fluorescence imaging lens successively;
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has:
Light source light blocking piece, it filters out the exciting light of the light supply apparatus transmitting;And
Fluorescent optical filter, it filters out the veiling glare of the non-wavelength of fluorescence.
12. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 1, it is characterised in that along described
Fluorescence detection optical path, the imaging device include several fluorescence filtering assemblies and fluorescence imaging lens successively;
Wherein, several described fluorescence filtering assemblies include:
The light source light blocking piece of several different wave lengths, it is respectively configured in the form of changeable to the fluorescence detection optical path, with
The exciting light of the light supply apparatus transmitting is filtered out respectively;And
The fluorescent optical filter of several different wave lengths, it is respectively configured in the form of changeable to the fluorescence detection optical path, with
The veiling glare of the non-wavelength of fluorescence is filtered out respectively.
13. one kind realizes large area digital pcr droplet fluorescence height using the detection means as any one of claim 1-12
The method of flux detection, it is characterised in that comprise the following steps:
Step 1:The digital pcr chip for having completed droplet processing is placed on objective table, the digital pcr chip and temperature control device
Connect;
Step 2:A PCR temperature cycles are carried out, process is:92~96 degree are warming up to, 25~40s is stopped, is cooled to 53~57
Degree, 25~35s is stopped, be warming up to 70~74 degree, stop 35~45s;
Step 3:Objective apparatus is switched to the object lens of minimum multiplying power;
Step 4:Light supply apparatus, imaging device are switched into light path corresponding to the first fluorescent dye or probe;
Step 5:Decile is carried out to the dimension of level two of the digital pcr chip, obtains some rectangles or rectangular region, each
Region area is S, and carries out droplet fluoroscopic image acquisition to each region, wherein the S is 1mm2~16mm2;
Step 6:Light supply apparatus, imaging device are switched into light path corresponding to the second fluorescent dye or probe, again described in execution
Step 5, the detection of the image of the second fluorescent dye is completed;
Step 7:Repeating said steps 6, until all fluorescent dyes or probe have been detected and finished;
Step 8:Objective apparatus is switched into time low range object lens, repeating said steps 4, step 5, step 6 and step 7;
Step 9:Repeating said steps 8, until the switching of all multiplying powers of object lens finishes, all fluorescent dye detections finish;
Step 10:2~step 9 of repeat step, and count, often it is repeated once, counts once, is more than N, wherein N generations until counting
Table amplification cycles number, the N are 30~40 times;
Step 11:Test is completed, the qPCR for carrying out successive image processing and each digital pcr drop is calculated, and counts negative positive
Droplet number, and calculate concentration.
14. the method for large area digital pcr droplet fluorescence high flux detection as claimed in claim 13, it is characterised in that
In the step 5,
Any dimension of the digital pcr chip in the horizontal direction is subjected to decile, the strip that some width are W is obtained and examines
Region is surveyed, continuously scans the strip detection zone, the consecutive image of each detection zone is obtained, to digital pcr miniflow
Control chip image is continuously acquired, wherein, the width W is 1mm~3mm.
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