CN107478629A - A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method - Google Patents

Abstract

The invention discloses a large-area digital PCR droplet fluorescence high-throughput detection device, which includes a fluorescence excitation light path and a fluorescence detection light path; On the digital PCR chip on the stage; along the fluorescence detection optical path, the fluorescence generated by the excited light of the digital PCR chip is sequentially imaged by the objective lens device, focusing device and imaging device and then transmitted to the image acquisition sensor; wherein, the stage is also equipped with The temperature control device controls the circulation temperature of the digital PCR chip reaction in real time. The invention discloses a large-area digital PCR droplet fluorescence high-throughput detection method, which can obtain higher detection efficiency and accuracy of large-area high-throughput detection. The invention can realize real-time digital qPCR detection with high throughput, high sensitivity and high specificity, and has great significance for improving the performance, precision and throughput of digital PCR detection instruments.

Description

A large-area digital PCR microdroplet fluorescence high-throughput detection device and method

technical field

The invention relates to the field of nucleic acid detection in the field of biomedical testing, in particular to a device and method capable of realizing large-area digital PCR droplet fluorescence high-throughput detection.

Background technique

In fields such as early diagnosis and screening of tumors, blood virus typing and load analysis, and noninvasive prenatal diagnosis, high-speed, high-sensitivity, and high-specificity detection of ultra-low-concentration nucleic acids is required, and conventional PCR is increasingly unable to meet the requirements. need. In order to achieve this function, people have developed a digital PCR technology on the basis of PCR, which improves the detection sensitivity by 1 to 2 orders of magnitude.

The "divide and conquer" detection strategy adopted by droplet digital PCR uses the sample and PCR reagent mixture as the dispersed phase, oil as the continuous phase, and uses microfluidic technology to suspend the sample mixture into droplets in the continuous phase. Furthermore, a standard PCR amplification is distributed into a large number of micro-droplets (nanoliters to picoliters), each containing only 0 or 1 copy of the target molecule (DNA template). Each droplet is independently amplified by PCR. After the amplification, the fluorescence of each droplet is detected, and the droplets with positive signals are counted to obtain the absolute copy number and nucleic acid concentration of the sample template. The size and number of droplets determine the detection sensitivity and lower limit of digital PCR. The number of existing digital PCR droplets is between 20,000 and 10 million.

The existing commercial droplet digital PCR technology is operated as follows: droplets are prepared on a dedicated droplet preparation chip; droplets are transferred to a centrifuge tube by a pipette, and a conventional PCR instrument is used for temperature cycle amplification; After the addition is completed, the fluorescence excitation and detection of each droplet are performed sequentially by flow cytometry, and the results are calculated. This detection method has the following problems:

(1) The flow detection speed is slow, the detection speed is 200-500 pieces/s, the detection process takes a long time, and the throughput is low;

(2) End-point detection can only be performed on a single droplet, and real-time qPCR is not possible, which reduces specificity and sensitivity;

(3) The optical path hardware is complicated, and it is difficult to realize multiple digital PCR detection (more than 4);

(4) The transfer process of the two droplets will result in the loss of droplets and possible external pollution;

(5) The thermal inertia of the heating block of the conventional PCR amplification instrument is large, and the thermal cycle time is long (about 2 hours).

In order to solve the above five problems, the applicant has proposed an integrated digital PCR chip, and has applied for an invention patent (application number 201510961967.0). Referring to accompanying drawing 1, integrated digital PCR chip comprises body 1, is positioned at continuous phase injection hole 11, sample injection hole 12, single-layer droplet tiling chamber 15, vacuum access hole 16, is used to generate droplet The "+-shaped" microstructure 13, and the liquid path 14 for connecting the continuous phase injection hole 11 and the "+-shaped" microstructure 13, and the liquid path 14 for connecting the sample injection hole 12 and the "+-shaped" microstructure 13 The first liquid path 17 and the second liquid path 18 for connecting the “+”-shaped microstructure 13 and the single-layer droplet tiling cavity 15 . All the microstructures are fabricated on a polymer substrate by MEMS technology, and then bonded with another polymer plate to form a closed microfluidic channel system. In the continuous phase injection hole 11, sample injection hole 12, vacuum interface The inlet hole 16 has three openings for injecting samples and applying negative pressure.

Referring to Fig. 2, the basic principle is: after the oil is injected into the continuous phase injection hole 11 and the sample is injected into the sample injection hole 12, a vacuum negative pressure is applied to the vacuum access hole 16, and the sample and the continuous phase will pass through under the action of negative pressure. Runners 14 and 17 reach the “+” microstructure 13 . Here, the sample will form nanoliter droplets 20 one by one due to the shearing of the continuous phase, and then enter the single-layer droplet flattening chamber 15 through the flow channel 18 to be flattened into a single layer 19 of droplets.

This technical method has the following advantages: the droplet monolayer 19 is suitable for using CCD to carry out image fluorescence photography detection, and has high throughput and speed; through continuous fluorescence image detection, the change curve of droplet fluorescence with circulation at this time can be obtained, Single droplet qPCR detection has high sensitivity and specificity; multiple digital PCR can be easily realized by adding excitation light sources and filters; the whole process is integrated, and there is no droplet loss and external pollution. Finally, the single-layer microdroplet will have a larger temperature control area and a smaller thermal inertia, which can reduce the PCR cycle amplification time to less than half an hour.

In order to use the digital PCR chip for nucleic acid detection, on the one hand, it is necessary to perform temperature cycle amplification on the digital PCR microfluidic chip, and on the other hand, it is necessary to perform real-time laser excitation and fluorescence imaging detection on the digital PCR droplet monolayer. Every time the temperature is cycled, the droplet fluorescence image detection is carried out to obtain the continuous change curve of the fluorescence of each droplet. In order to improve throughput, multiple integrated digital PCR microfluidic structures can be integrated on one chip to form simultaneous detection of 32, 48 or even 96 samples (see Figure 3) in order to improve throughput, which requires The chip can move in one or two dimensions, so that large-area image acquisition can be performed by scanning. In addition, in order to improve the detection efficiency, a new algorithm is adopted. Firstly, a small magnification is used to observe a rough large field of view. After finding that there may be liquid droplet fluorescent spots, it is switched to a high magnification fine small field of view observation method to improve efficiency. However, existing detection devices such as fluorescence microscopes cannot meet the above detection requirements.

Contents of the invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages as will be described hereinafter.

Another purpose of the present invention is to provide a large-area digital PCR droplet fluorescence high-throughput detection device, which can realize high-throughput, real-time and accurate quantification of digital PCR through the design of the system optical path and continuous scanning of the divided detection area Detection, in addition, the rapid positioning and detection of droplet fluorescent spots can be realized by switching the large and small scanning fields of view.

Another object of the present invention is to obtain higher detection efficiency and accuracy of large-area high-throughput detection through a large-area digital PCR droplet fluorescent high-throughput detection method.

In order to achieve these objects and other advantages according to the present invention, a large-area digital PCR droplet fluorescence high-throughput detection device is provided, including a fluorescence excitation light path and a fluorescence detection light path;

Along the fluorescent excitation optical path, the excitation light emitted by the light source device is incident on the digital PCR chip placed on the stage through the focusing device and the objective lens device in sequence;

Along the fluorescence detection optical path, the fluorescence generated by the digital PCR chip by the excitation light is sequentially imaged by the objective lens device, the focusing device and the imaging device, and then transmitted to the image acquisition sensor;

Wherein, the stage is further provided with a temperature control device, and the temperature control device controls the cycle temperature of the reaction of the digital PCR chip in real time.

Preferably, the object stage is fixedly arranged on the optical path, or has at least one translational degree of freedom, so as to meet the needs of large-area image acquisition.

Preferably, wherein, the objective lens device comprises:

An objective lens, which is fixedly arranged in the optical path;

Or several objective lenses with different magnifications, which are respectively arranged in the optical path in a switchable form, so as to realize digital PCR droplet fluorescence imaging with different magnifications.

Preferably, wherein, the magnification of the objective lens is selected from 2X, 4X, 5X or 10X.

Preferably, wherein, the light source device includes:

a light source assembly providing a parallel mixed light source;

A light source filter assembly having:

A light source filter, which is fixedly arranged in the fluorescence excitation light path, or

Several light source filters are respectively arranged in the fluorescence excitation light path in a switchable manner.

Light source filters can be used to filter mixed light sources to provide the monochromatic light needed to excite PCR droplet dyes. In this solution, a mixed light source and a filter are used to generate a monochromatic light source, which can provide multiple wavelengths of monochromatic excitation light sources at a lower cost, and realize multiple digital PCR functions.

Preferably, wherein, the light source device includes several monochromatic light source groups;

Along the forward direction of the optical path, the monochromatic light source group includes in sequence: a monochromatic light source, a shutter, and a light source lens; wherein, the shutter controls the fluorescent excitation optical path on and off.

Preferably, wherein, the light source device includes:

At least two groups of monochromatic light sources to provide a strong excitation light source;

Several first dichroic mirrors combine the emission light paths of the monochromatic light source groups and lead them into the fluorescence excitation light path.

Dichroic mirrors can be used to combine two excitation light sources into one beam, and multiple dichroic mirrors can be used to combine all excitation light sources into one beam, thereby providing multiple excitation light sources with different wavelengths and realizing multiple digital PCR functions.

Preferably, wherein the focusing device includes:

The second dichroic mirror reflects the excitation light emitted by the light source device and simultaneously transmits the fluorescent light generated by the digital PCR chip.

Preferably, wherein, the focusing device also includes:

The first focusing lens assembly, which is arranged between the light source device and the second dichroic mirror, is used for shaping and expanding the beam of the laser light, so as to obtain better excitation light.

Preferably, wherein, the focusing device also includes:

The second focusing lens assembly, which is arranged between the second dichroic mirror and the imaging device, is used for shaping and expanding the fluorescent light, so as to obtain better imaging quality.

Preferably, wherein, along the fluorescence detection optical path, the imaging device sequentially includes a fluorescence filter assembly and a fluorescence imaging lens;

Preferably, along the fluorescence detection optical path, the imaging device sequentially includes a single fluorescence filter assembly and a fluorescence imaging lens;

Wherein, the fluorescence filter assembly is fixed in the fluorescence detection optical path, and the fluorescence filter assembly has:

a light source light blocking sheet, which filters out the excitation light emitted by the light source device; and,

A fluorescence filter that filters out stray light at wavelengths other than the stated fluorescence.

Preferably, wherein, along the fluorescence detection optical path, the imaging device sequentially includes several fluorescence filter assemblies and fluorescence imaging lenses;

Wherein, several described fluorescence filter assemblies include:

Several light source light blocking plates with different wavelengths are respectively arranged in the fluorescence detection optical path in a switchable form, so as to respectively filter out the excitation light emitted by the light source device; and,

Fluorescence filters of different wavelengths are switchably arranged in the fluorescence detection light path respectively, so as to filter stray light of non-fluorescence wavelengths respectively.

This technical solution can improve the signal-to-noise ratio of the system by filtering out the excitation light of the light source device and the stray light of non-fluorescence wavelengths, and can improve the signal-to-noise ratio of the system through the combined switching between light blocking sheets and fluorescent filters of several different wavelengths. Collect fluorescence images of different wavelengths emitted by PCR droplets to realize multiple digital PCR functions.

The purpose of the present invention can also be further realized by the method of large-area digital PCR droplet fluorescence high-throughput detection, which method comprises the following steps:

Step 1: placing the digital PCR chip that has completed the droplet processing on the stage, and the digital PCR chip is connected to the temperature control device;

Step 2: Carry out a PCR temperature cycle, the process is: heat up to 92-96 degrees, stay for 25-40s, cool down to 53-57 degrees, stay for 25-35s, heat up to 70-74 degrees, stay for 35-45s;

Step 3: Switch the objective lens device to the lowest magnification objective lens;

Step 4: switch the light source device and imaging device to the optical path corresponding to the first fluorescent dye or probe;

Step 5: Equally divide the two horizontal dimensions of the digital PCR chip to obtain a number of rectangles or rectangular areas, each area is S, and perform microdroplet fluorescence image acquisition on each area, wherein S is 1mm ² ~16mm ² ;

Step 6: switch the light source device and the imaging device to the optical path corresponding to the second fluorescent dye or the probe, and perform the step 5 again to complete the detection of the image of the second fluorescent dye;

Step 7: Repeat the step 6 until all fluorescent dyes or probes have been detected;

Step 8: Switch the objective lens device to the second-lowest magnification objective lens, and repeat the steps 4, 5, 6 and 7;

Step 9: Repeat the step 8 until the switching of all magnifications of the objective lens is completed, and the detection of all fluorescent dyes is completed;

Step 10: Repeat steps 2 to 9, and count, and count once each time it is repeated, until the count is greater than N, where N represents the number of amplification cycles, and N is 30 to 40 times;

Step 11: Complete the test, perform subsequent image processing and qPCR calculation of each digital PCR droplet, count the number of negative and positive droplets, and calculate the concentration.

Preferably, wherein, in the step 5,

Divide the digital PCR chip equally in any dimension in the horizontal direction to obtain several strip-shaped detection areas with a width of W, continuously scan the strip-shaped detection areas, and obtain continuous images of each detection area. The images of the digital PCR microfluidic chip are continuously acquired, wherein the width W is 1 mm to 3 mm.

The present invention at least includes the following beneficial effects:

The invention can realize real-time digital qPCR detection with high throughput, high sensitivity and high specificity, and has great significance for improving the performance, precision and throughput of digital PCR detection instruments. Utilizing the technical scheme of the present invention, low-concentration nucleic acid samples can be detected quickly and with high precision, which is of great significance for cancer tumors, infectious diseases, prenatal diagnosis, etc. The detection method is of great significance.

Other advantages, objectives and features of the present invention will partly be embodied through the following descriptions, and partly will be understood by those skilled in the art through the study and practice of the present invention.

Description of drawings

Fig. 1 is a structural representation of an integrated digital PCR chip in an example;

Fig. 2 is a structural representation of an integrated digital PCR chip in another example;

Fig. 3 is a structural schematic diagram of a high-throughput integrated digital PCR chip (48 arrays) in another example;

Fig. 4 is a structural block diagram of a large-area digital PCR droplet fluorescent high-throughput detection device in another example;

Fig. 5 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device in another example;

Fig. 6 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with a one-dimensional motion mechanism stage device in another example;

Fig. 7 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with a two-dimensional motion mechanism stage device in another example;

Fig. 8 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with multiple different magnification objective lens switching devices in another example;

Fig. 9 is a schematic structural diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with a light source filter switching mechanism;

Fig. 10 is a schematic structural diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with multiple monochromatic light source groups;

Fig. 11 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with an excitation light focusing lens assembly in another example;

Fig. 12 is a structural schematic diagram of a large-area digital PCR droplet fluorescent high-throughput detection device with a fluorescent focusing lens assembly in another example;

Fig. 13 is a structural schematic diagram of a large-area digital PCR droplet fluorescence high-throughput detection device with both excitation light and fluorescence focusing lens assemblies in another example;

Fig. 14 is a structural schematic diagram of a large-area digital PCR droplet fluorescent high-throughput detection device with a fluorescent filter assembly switching mechanism in another example;

Fig. 15 is a schematic diagram of a digital PCR chip being divided into rectangular detection areas in two dimensions in another example;

Fig. 16 is a schematic diagram of a digital PCR chip being one-dimensionally divided into strip-shaped detection areas in another example;

Fig. 17 is a structural schematic diagram of a large-area digital PCR microdroplet fluorescence high-throughput detection device for double digital PCR in another example;

Fig. 18 is a structural schematic diagram of a large-area digital PCR droplet fluorescent high-throughput detection device for six-fold digital PCR in another example;

Fig. 19 is a schematic structural view of a light source filter fixing device in another example;

Fig. 20 is a schematic structural diagram of a fluorescence filter fixing device in an imaging device in another example.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

It should be understood that terms such as "having", "comprising" and "including" as used herein do not entail the presence or addition of one or more other elements or combinations thereof.

Referring to Figure 4 and Figure 5, as an implementation form of the present invention, the large-area digital PCR droplet fluorescence high-throughput detection device includes:

The objective lens device 200, which is located directly above the digital PCR chip, is used for the front end of the entire digital PCR fluorescence image acquisition;

A light source device 300, used for fluorescence excitation of digital PCR micro-droplets;

The focusing device 400, on the one hand, converges the light from the light source device 300 onto the digital PCR chip 1, and on the other hand collects the fluorescence generated by the digital PCR chip 1, and transmits it to the imaging optical path device 500 and the image acquisition sensor 600, thereby Used for the coupling of light source illumination and fluorescence imaging;

Imaging optical path device 500, which is used to shape and filter the fluorescence image, and image it on the image acquisition sensor 600, so as to be used for fluorescence imaging of digital PCR microdroplets;

The image acquisition sensor 600 is used for acquiring and converting the fluorescent image into a digital image, and collecting the fluorescent image of the digital PCR droplet in real time.

A PCR temperature control device 700, which is used to precisely control the temperature cycle of the digital PCR chip 1 to realize the amplification process of the micro-droplet digital PCR; and,

The stage device 800 is used to carry the digital PCR droplet microfluidic chip and its temperature control device.

In this technical solution, it includes a fluorescence excitation light path and a fluorescence detection light path;

Along the fluorescent excitation optical path, the excitation light emitted by the light source device is incident on the digital PCR chip placed on the stage through the focusing device and the objective lens device in sequence;

Along the fluorescence detection optical path, the fluorescence generated by the digital PCR chip by the excitation light is sequentially imaged by the objective lens device, the focusing device and the imaging device, and then transmitted to the image acquisition sensor;

Wherein, the stage is further provided with a temperature control device, and the temperature control device controls the cycle temperature of the reaction of the digital PCR chip in real time.

Multiplex digital PCR can be realized by adding a variety of fluorescent dyes or probes to the digital PCR reaction system with different mixes and primers. Wherein, the number of PCR multiplexes is the same as that of the added fluorescent dyes or probes, for example, two fluorescent dyes or probes are required for double PCR.

In another example, the object stage is fixed on the optical path, or has at least one translational degree of freedom, so as to realize image acquisition in a one-dimensional or two-dimensional scanning manner, thereby enlarging the detection area.

Referring to FIG. 6 , in an embodiment for realizing the one-dimensional degree of freedom of the stage, the stage device 800 includes a servo motor 803, a transmission device 802, and a position detection device 806 to realize the horizontal X direction of the stage 801. One-dimensional precision motion control. Through the one-dimensional movement of the stage, the one-dimensional scanning detection of the digital PCR chip by the imaging system is realized, so as to improve the detection range and realize large-area high-throughput detection. Wherein, the servo motor 803 may be a stepping servo motor, a DC servo motor, or an AC servo motor, the transmission device 802 may be a ball screw transmission device, and the position sensor may be a linear grating encoder or a resistive displacement sensor.

Referring to FIG. 7 , in an embodiment for realizing the two-dimensional freedom of the stage, the stage device 800 includes a pair of servo motors 803 and 805, a pair of transmission devices 802 and 804, and a pair of position detection devices 806 and 807. , so as to realize the X, Y two-dimensional precise motion control of the stage 801 in the horizontal direction. Through the two-dimensional movement of the stage, the two-dimensional scanning detection of the digital PCR chip by the imaging system is realized, so as to improve the detection range and realize large-area high-throughput detection.

In another example, the objective lens device includes an objective lens fixedly arranged in the optical path.

In another example, the objective lens device includes several objective lenses with different magnifications, which are respectively arranged in the optical path in a switchable manner.

With reference to Fig. 8, a kind of embodiment that can realize the automatic switching of objective lens of different magnifications is: objective lens device 200 comprises servo motor 205, transmission device 204, rotating hinge 203, objective lens fixed plate 202, a group of objective lens 201 and not drawn in the figure control unit. Wherein, the objective lenses 201 are equidiametrically distributed on the objective lens fixing plate 202 with the rotating hinge 203 as the center. The servo motor 205 can drive the objective lens fixed plate 202 to rotate through the transmission device 204 and the rotating hinge 203, and then can switch the required magnification objective lens to the front end of the focusing device 400 in the optical path. Wherein, the servo motor 205 can be a stepping servo motor, a DC servo motor, an AC servo motor, the transmission device 204 can be a pulley transmission device, a gear transmission device, etc., and the rotating hinge 203 can be a rotary bearing. With this scheme, the acquisition of microdroplet fluorescence images with different magnifications can be realized through automatic switching. Moreover, this manner is only an illustration of a preferred example, but is not limited thereto. When implementing the present invention, different switching modes of the objective lens can be implemented according to user requirements.

In another example, the magnification of the objective lens is selected from 2X, 4X, 5X or 10X.

In another example, referring to FIG. 9, the light source device 300 includes:

A light source component, which provides a parallel mixed light source by mixing the light source emitting part 301 and the convex mirror 302;

A light source filter assembly having:

A light source filter 303, which is fixedly arranged in the fluorescence excitation optical path, or

Several light source filters 303 are respectively arranged in the fluorescence excitation optical path in a switchable manner.

In this technical solution, the implementation mode to realize the automatic switching of different wavelength light source filters is as follows: the light source device 300 is composed of a halogen lamp 301, a lens 302, a series of filters 303, a filter fixing plate 305, a rotating hinge 304, a transmission Device 306, servo motor 307 and control unit (not shown in the figure). The lens 302 is used to collimate the light emitted by the light source 301 to form a parallel light beam. The servo motor 307 drives the filter fixing plate 305 to rotate through the transmission device 306 and the rotating hinge 304, and switches the filters 303 of different wavelengths to the front end of the lens 302 to realize the monochromatic light output of the light source device 300 with different wavelengths. Wherein, the transmission device 306 may be a pulley transmission or a gear transmission, and the rotating hinge 304 may be a rotating dynamic hinge bearing. The servo motor can be a stepping servo motor, a DC servo motor or an AC servo motor. Moreover, this manner is only an illustration of a preferred example, but is not limited thereto. When implementing the present invention, different switching modes of the light source filter can be implemented according to user requirements.

In another example, referring to FIG. 10, the light source device 300 includes several monochromatic light source groups;

Along the forward direction of the optical path, the monochromatic light source group includes in sequence: a monochromatic light source 301 , a shutter 308 and a light source lens 302 ; wherein the shutter 308 controls the fluorescence excitation optical path on and off.

In another example, the light source device 300 includes:

Two or more monochromatic light source groups;

Several first dichroic mirrors 309 combine the emission light paths of the monochromatic light source groups and lead them into the fluorescence excitation light path.

In this embodiment, the light source device 300 is composed of a plurality of monochromatic light sources 301 , a lens 302 , a shutter 308 , and a first dichroic mirror 309 . Wherein, each light source 301 corresponds to a shutter 308 for controlling the light source to be turned on and off. The first dichroic mirror 309 is used to combine the two beams of light, and the number of the first dichroic mirrors 309 is the number of the light sources 301 minus 1, so as to realize the beam combining of the light emitted by all the light sources. The shutter 308 controls which monochromatic light is output and introduced into the system. The light source may be a laser source, LED, etc., but is not limited thereto.

In another example, referring to Fig. 5, the focusing device includes:

The second dichroic mirror reflects the excitation light emitted by the light source device and simultaneously transmits the fluorescent light generated by the digital PCR chip.

In this embodiment, the focusing device 400 only includes a second dichroic mirror 401. On the one hand, the second dichroic mirror 401 is used to reflect the light from the light source system 300 onto the digital PCR chip 1, and the digital PCR droplet For excitation; on the other hand, it is used to pass through the fluorescence generated by the liquid droplets on the digital PCR chip, and make it enter the imaging device 500 .

In another example, referring to FIG. 11 , the focusing device 400 further includes:

The first focusing lens assembly includes a lens 402 and a lens 403, the first focusing lens assembly is arranged between the light source device 300 and the second dichroic mirror 401, and is used to collimate the light from the light source device 300 Straighten and expand beams to vary excitation spot size and quality.

In another example, referring to Fig. 12, the focusing device further includes:

The second focusing lens assembly includes a lens 404 and a lens 405. The second focusing lens assembly collimates and expands the fluorescence emitted by the objective lens device 200, and cooperates with the imaging lens 501 to improve imaging quality.

In another example, referring to FIG. 13 , the focusing device also includes a lens 402 , a lens 403 , and lenses 404 and 405 to change the size of the excitation spot and improve the imaging quality.

In another example, along the fluorescence detection optical path, the imaging device 500 sequentially includes a single fluorescence filter assembly and a fluorescence imaging lens 501;

Wherein, the fluorescence filter assembly is fixed in the fluorescence detection optical path, and the fluorescence filter assembly has:

a light source light blocking sheet, which filters out the excitation light emitted by the light source device; and,

A fluorescence filter that filters out stray light at wavelengths other than the stated fluorescence.

In another example, referring to FIG. 14 , along the fluorescence detection optical path, the imaging device sequentially includes a fluorescence filter assembly and a fluorescence imaging lens 501;

Wherein, the fluorescent filter assembly includes:

Several light source light blocking plates with different wavelengths are respectively arranged in the fluorescence detection optical path in a switchable form, so as to respectively filter out the excitation light emitted by the light source device; and,

Fluorescence filters of different wavelengths are switchably arranged in the fluorescence detection light path respectively, so as to filter stray light of non-fluorescence wavelengths respectively.

In this embodiment, the imaging device 500 includes multiple sets of filters (each set of filters includes a light source block and a fluorescence filter), and filters for switching between the sets of filters Fixed plate 505, rotating hinge 504, transmission device 506, servo motor 507, and control unit (not shown in the figure). The servo motor 507 drives the optical filter 502 and the optical filter 503 on the optical filter fixing plate 505 to rotate through the transmission device 506 and the rotating hinge 505, and any desired set of optical filters 502 and optical filters 503 Switch to the front end of the image acquisition sensor 700 to achieve filtering of excitation light and acquisition of required fluorescence wavelength bands. Each set of filters 502 and 503 in the imaging device are combined with the monochromatic lights emitted by the excitation light source, which can realize digital PCR fluorescence excitation and detection of different dyes, and then realize multiple digital PCR.

In another example, a method for realizing large-area digital PCR droplet fluorescence high-throughput detection includes the following steps:

Step 1), place the microfluidic chip 1 that has completed the digital PCR droplet generation on the stage 801, and connect with the PCR temperature control device 700.

Step 2), perform a PCR temperature cycle, the process is as follows: heat up to 92-96 degrees, stay for 25-40s, cool down to 53-57 degrees, stay for 25-35s, heat up to 70-74 degrees, stay for 35-45s;

Step 3), the objective lens device 200 is switched to the lowest magnification objective lens;

Step 4), the light source device and the imaging device are switched to the optical path corresponding to the first dye or probe.

Step 5), referring to FIG. 15 , the digital PCR microfluidic chip 1 is equally divided into horizontal and vertical directions X and Y to form several rectangular or rectangular areas 101, each area has a length L and a width W, and each Fluorescent images of microdroplets were acquired in each area. The length L is 1 mm to 4 mm, and the width W is 1 mm to 4 mm;

Step 6), the light source device 300 and the imaging device 500 switch to the optical path corresponding to the next dye. Perform step 5) again to complete the detection of the image of the next fluorescent dye or probe;

Step 7), repeat step 6), until all fluorescent dyes or probes are detected;

Step 8), the objective lens device 200 switches to the second low magnification objective lens; repeat step 4), step 5), step 6), step 7);

Step 9), repeat step 8), until the objective lenses of all magnifications are switched, and all fluorescent dyes or probes are detected;

Step 10), repeat steps 2) to 9), and count, and count once each time it is repeated, until the count is greater than N; said N represents the number of amplification cycles, which is 30 to 40;

Step 11), complete all tests, perform subsequent image processing and qPCR calculation of each digital PCR droplet, count the number of negative and positive droplets, and calculate the concentration.

In another example, a method for realizing large-area digital PCR droplet fluorescence high-throughput detection includes the following steps:

Step 1), place the microfluidic chip 1 that has completed the digital PCR droplet generation on the stage 801, and connect with the PCR temperature control device 700.

Step 2), perform a PCR temperature cycle, the process is as follows: heat up to 92-96 degrees, stay for 25-40s, cool down to 53-57 degrees, stay for 25-35s, heat up to 70-74 degrees, stay for 35-45s;

Step 3), the objective lens device 200 is switched to the lowest magnification objective lens;

Step 4), the light source device and the imaging device are switched to the optical path corresponding to the first dye or probe.

Step 5), referring to Figure 16, divide the digital PCR chip into equal parts in one dimension in the horizontal direction, and divide it into a series of strip-shaped detection areas with a width of W, and obtain the continuous detection area of each detection area through continuous scanning. Images, continuously acquiring images of the digital PCR microfluidic chip, the width W is 1 mm to 3 mm;

Step 6), the light source device 300 and the imaging device 500 switch to the optical path corresponding to the next dye. Perform step 5) again to complete the detection of the image of the next fluorescent dye or probe;

Step 7), repeat step 6), until all fluorescent dyes or probes are detected;

Step 8) The objective lens device 200 is switched to the next low magnification objective lens; Step 4), step 5), step 6), step 7) are repeated;

Step 9), repeat step 8), until the objective lenses of all magnifications are switched, and all fluorescent dyes or probes are detected;

Step 10), repeat steps 2) to 9), and count, and count once each time it is repeated, until the count is greater than N; said N represents the number of amplification cycles, which is 30 to 40;

Step 11), complete all tests, perform subsequent image processing and qPCR calculation of each digital PCR droplet, count the number of negative and positive droplets, and calculate the concentration.

Below in conjunction with specific embodiment the present invention is described in further detail:

<Example 1>

The parameters of the digital PCR microfluidic chip to be detected are: to achieve double digital PCR, use the first dye and the second dye, the excitation wavelengths are 488nm and 532nm, the fluorescence wavelengths are 515nm and 560nm, respectively, and the droplet digital PCR microfluidic The control chip integrates 48 single-sample detection structures, and the overall chip is 150mm long and 100mm wide.

Referring to Figure 17, for this chip, a real-time fluorescent image acquisition device for digital PCR droplets tiled on a single layer is proposed, including:

Objective lens device 200, including two objective lenses 2011, 2012 with magnifications of 4X and 10X;

The light source device 300 is used for fluorescence excitation of digital PCR microdroplets, including two lasers 3011, 3012, two sets of shutters 3081, 3082, two sets of lenses 3021, 3022, and a dichromatic mirror 3091; wherein, the wavelength of the laser 3011 is 488nm , the laser 3012 has a wavelength of 532nm, the lenses 3021 and 3022 have a front focal length of 50mm, and a rear focal length of 47mm.

The focusing device 400 is used for the coupling of light source illumination and fluorescence imaging, including a dichromatic mirror 401, two sets of lenses 402, 403, 404, and 405; the lenses 402, 403 expand the light beam from the light source to form a beam with a diameter of 3mm The circular light spot is irradiated onto the digital PCR droplet microfluidic chip; the lenses 404 and 405 shape the fluorescence emitted from the digital PCR droplet microfluidic chip, and combine it with the imaging optical path to make an image with a diameter of 2mm irradiated to the image acquisition sensor 700 on.

Imaging optical path device 500, used for fluorescence imaging of digital PCR microdroplets, includes two sets of optical filters 5021, 5031, 5022, 5032; The motor 507 is composed. Among them, the filters 5031 and 5032 for filtering the wavelength of the light source are long-wave pass filters respectively, and the cut-off frequencies are 500nm and 540nm respectively, and the fluorescence filters 5021 and 5022 are filters with a central wavelength of 515nm and 560nm and a bandwidth of 30nm, respectively. The lens 501 has a front focal length of 50 mm and a rear focal length of 47 mm;

Image acquisition sensor 600, used for real-time acquisition of fluorescence images of digital PCR droplets, is an area array CCD image sensor;

The PCR temperature control device 700 is used for temperature cycle control of digital PCR microdroplets, including a temperature sensor 702, a Peltier for heating as a heating actuator 704, a cooling actuator 703, and a heat transfer fast 701; wherein, 701 is a passing machine The processed aluminum alloy parts, the heating actuator 704 is a heating resistance wire, the cooling actuator 703 is a fan, and the temperature sensor 702 is a thermocouple sensor.

The stage device is used to carry the digital PCR droplet microfluidic chip and its temperature control device, including a stage 801, servo motors 803, 805, transmission devices 802, 804, and position sensors 806, 807. Among them, the servo motors 803 and 805 are two-phase four-wire stepping servo motors, the transmission devices 802 and 804 are ball screw structures, and the strokes are 200 mm and 150 mm respectively, and the position sensors 806 and 807 are linear grating encoders with a measuring range of 200 mm and 150mm, precision 0.01mm.

The specific detection process is as follows:

Step 1), place the microfluidic chip that has completed the generation of digital PCR droplets on the stage, and connect with the PCR temperature control device.

Step 2), perform a PCR temperature cycle, the process is as follows: heat up to 92-96 degrees, stay for 25-40s, cool down to 53-57 degrees, stay for 25-35s, heat up to 70-74 degrees, stay for 35-45s;

Step 3), the objective lens device is switched to the lowest magnification objective lens;

Step 4), the light source device and the imaging device are switched to the optical path corresponding to the first dye.

In step 5), the digital PCR microfluidic chip is equally divided in the horizontal and vertical directions into several rectangular or rectangular areas, each area is S, and microdroplet fluorescence images are acquired for each area. The rectangular area is a square with a width of 2 mm;

Step 6), the light source device and the imaging device switch to the optical path corresponding to the next dye. Perform step 5) again to complete the detection of the image of the next fluorescent dye;

Step 7), repeat step 6), until all fluorescent dyes have been detected;

Step 8), the objective lens device is switched to the next low magnification objective lens; repeat step 4), step 5), step 6), step 7);

Step 9), repeat step 8), until the switching of objective lenses of all magnifications is completed, and the detection of all fluorescent dyes is completed;

Step 10), repeat steps 2) to 9), and count, and count once each time it is repeated, until the count is greater than N; said N represents the number of amplification cycles, which is 30 to 40;

Step 11), complete all tests, perform subsequent image processing and qPCR calculation of each digital PCR droplet, count the number of negative and positive droplets, and calculate the concentration.

<Example 2>

The parameters of the digital PCR microfluidic chip to be detected are: six-fold digital PCR, and the dyes used are:

Fluorescent dye 1: excitation wavelength 390nm, fluorescence wavelength 420nm;

Fluorescent dye 2: excitation wavelength 475nm, fluorescence wavelength 522nm;

Fluorescent dye 3: excitation wavelength 525nm, fluorescence wavelength 577nm;

Fluorescent dye 4: excitation wavelength 572nm, fluorescence wavelength 628nm;

Fluorescent dye 5: excitation wavelength 630nm, fluorescence wavelength 675nm;

Fluorescent dye 6: excitation wavelength 650nm, fluorescence wavelength 710nm;

The droplet digital PCR microfluidic chip integrates 48 single-sample detection structures, and the overall chip is 150mm long and 100mm wide. For this chip, see Figure 18, a real-time fluorescent image acquisition device for digital PCR droplets tiled on a single layer is proposed, including:

Objective lens device 200, including two objective lenses 2011, 2012 with magnifications of 4X and 10X;

The light source device 300 is used for the fluorescence excitation of the digital PCR droplet, including a tungsten-halogen light source 301, a lens 302, an optical filter fixing plate 305, a rotating hinge 3041, a transmission device 306, a servo motor 307, and an optical filter fixing plate The optical filters 3031, 3032, 3033, 3034, 3035, 3036 on the 305 are shown in FIG. 19 . The central wavelengths of the filters are 390nm, 475nm, 525nm, 572nm, 630nm, 650nm respectively, and the bandwidth is 20nm; wherein, the lens 302 has a front focal length of 50mm and a rear focal length of 47mm. The servo motor 307 is a two-phase four-wire stepping servo motor, the transmission device 306 is a pulley transmission mechanism, and the rotating hinge is a rotating bearing;

The focusing device 400 is used for the coupling of light source illumination and fluorescence imaging, including a dichromatic mirror 401, two sets of lenses 402, 403, 404, and 405; the lenses 402, 403 expand the light beam from the light source to form a beam with a diameter of 3mm The circular light spot is irradiated onto the digital PCR droplet microfluidic chip; the lenses 404 and 405 shape the fluorescence emitted from the digital PCR droplet microfluidic chip, and combine it with the imaging optical path to make an image with a diameter of 2mm irradiated to the image acquisition sensor 700 on.

The imaging optical path device 500 is used for fluorescence imaging of digital PCR microdroplets, referring to FIG. Optical sheets 5031, 5032, 5033, 5034, 5035, 5036, converging lens 501, filter fixing plate 505, rotating hinge 504, transmission device 506, and servo motor 507 are formed. Among them, the band-stop filters 5021, 5022, 5053, 5024, 5025, and 5026 that filter out the wavelength of the light source are respectively 390nm, 475nm, 525nm, 572nm, 630nm, and 650nm, with a bandwidth of 20nm, and the fluorescence filters 5031, 5032, 5033, 5034, 5035, and 5036 have central wavelengths of 420nm, 522nm, 577nm, 628nm, 675nm, and 710nm, respectively, and filters with a bandwidth of 20nm. The lens 501 has a front focal length of 50mm and a rear focal length of 47mm;

Image acquisition sensor 700, used for real-time acquisition of fluorescence images of digital PCR droplets, is an area array CCD image sensor;

The PCR temperature control device 700 is used for temperature cycle control of digital PCR microdroplets, including a temperature sensor 702, a Peltier for heating as a heating actuator 704, a cooling actuator 703, and a heat transfer fast 701; wherein, 701 is a passing machine The processed aluminum alloy parts, the heating actuator 704 is a heating resistance wire, the cooling actuator 703 is a fan, and the temperature sensor 702 is a thermocouple sensor.

The stage device is used to carry the digital PCR droplet microfluidic chip and its temperature control device, including a stage 801, servo motors 803, 805, transmission devices 802, 804, and position sensors 806, 807. Among them, the servo motors 803 and 805 are two-phase four-wire stepping servo motors, the transmission devices 802 and 804 are ball screw structures, and the strokes are 200 mm and 150 mm respectively, and the position sensors 806 and 807 are linear grating encoders with a measuring range of 200 mm and 150mm, precision 0.01mm.

The specific detection process is as follows:

Step 1), place the microfluidic chip that has completed the generation of digital PCR droplets on the stage, and connect with the PCR temperature control device.

Step 2), perform a PCR temperature cycle, the process is as follows: heat up to 92-96 degrees, stay for 25-40s, cool down to 53-57 degrees, stay for 25-35s, heat up to 70-74 degrees, stay for 35-45s;

Step 3), the objective lens device is switched to the lowest magnification objective lens;

Step 4), the light source device and the imaging device are switched to the optical path corresponding to the first dye.

Step 5), the digital PCR chip is equally divided into one dimension in the horizontal direction, and is equally divided into a series of strip-shaped detection areas with a width of W, and the continuous images of each detection area are obtained by continuous scanning. PCR microfluidic chip images were acquired continuously. The width W is 2mm;

Step 6), the light source device and the imaging device switch to the optical path corresponding to the next dye. Perform step 5) again to complete the detection of the image of the next fluorescent dye;

Step 7), repeat step 6), until all fluorescent dyes have been detected;

Step 8), the objective lens device is switched to the next low magnification objective lens; repeat step 4), step 5), step 6), step 7);

Step 9), repeat step 8), until the switching of objective lenses of all magnifications is completed, and the detection of all fluorescent dyes is completed;

Step 10), repeat steps 2) to 9), and count, and count once each time it is repeated, until the count is greater than N; said N represents the number of amplification cycles, which is 30 to 40;

Step 11), complete all tests, perform subsequent image processing and qPCR calculation of each digital PCR droplet, count the number of negative and positive droplets, and calculate the concentration.

The number of devices and processing scales described here are used to simplify the description of the present invention. The application, modification and change of the large-area digital PCR microdroplet fluorescence high-throughput detection device and method of the present invention will be obvious to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the applications set forth in the specification and examples. It can be fully applied to various fields suitable for the present invention. Additional modifications can readily be made by those skilled in the art. Therefore, the invention should not be limited to the specific details and examples shown and described herein, without departing from the general concept defined by the claims and their equivalents.

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.