CN212894754U - PCR fluorescent nucleic acid detection device - Google Patents

Abstract

The utility model provides a PCR fluorescence nucleic acid detection device, the device includes microfluid chip, blue light LED subassembly, yellow light LED subassembly, PCR fluorescence detector, temperature control device and control circuit board, wherein, microfluid chip is used for carrying on the PCR nucleic acid amplification reaction of nucleic acid, and blue, yellow light LED subassembly are located microfluid chip top, are used for emiting blue light, yellow light to microfluid chip surface respectively; the PCR fluorescence detector is positioned above the microfluid chip and between the blue LED component and the yellow LED component and is used for collecting fluorescence signals from the microfluid chip; the temperature control device is positioned below the microfluidic chip and used for adjusting the temperature of the microfluidic chip; the control circuit board is connected with the blue and yellow LED components, the PCR fluorescence detector and the temperature control device. The utility model discloses a PCR fluorescence nucleic acid detection device is based on microfluid chip, has miniaturization, chipization, detects the highly integrated characteristics of flow, can be applied to the PCR ultrafast fluorescence detection of the nucleic acid material in virus, bacterium, cell, the body fluid.

Description

PCR fluorescent nucleic acid detection device

Technical Field

The utility model belongs to the technical field of gene detection, a PCR fluorescence nucleic acid detection device is related to.

Background

There are three general approaches to virus detection: whole gene sequencing, nucleic acid detection and immune protein detection. In addition, the CT technique is also a very important detection technique, but it is expensive and inconvenient for outdoor use, such as customs and general clinics. In comparison, nucleic acid detection has significant advantages, including automated detection procedures, high-throughput, large platform-based detection of sample in and out, and real-time in-situ detection. However, most of the existing PCR (polymerase chain reaction) instruments for detecting nucleic acids on the market are not portable, have a slow speed (30 minutes or more for PCR amplification), and are expensive.

Therefore, how to provide a nucleic acid PCR ultrafast fluorescence detection technology and a portable device based on the miniaturization, chipization and high integration of detection process of silicon-based microfluidic chips becomes an important technical problem to be solved urgently by those skilled in the art.

SUMMERY OF THE UTILITY MODEL

In view of the above-mentioned shortcomings of the prior art, the present invention is directed to a PCR fluorescence nucleic acid detection device, which is used to solve the problems of the prior art, such as the lack of portability, the slow speed and the high price of the PCR instrument.

To achieve the above and other related objects, the present invention provides a PCR fluorescent nucleic acid detecting device, comprising:

a microfluidic chip for performing a PCR nucleic acid amplification reaction of nucleic acid;

the blue light LED component is positioned above the microfluid chip and used for emitting blue light to the surface of the microfluid chip;

the yellow light LED component is positioned above the microfluid chip and used for emitting yellow light to the surface of the microfluid chip;

the PCR fluorescence detector is positioned above the microfluidic chip, positioned between the blue LED component and the yellow LED component and used for collecting fluorescence signals from the microfluidic chip;

the temperature control device is positioned below the microfluidic chip and used for adjusting the temperature of the microfluidic chip;

and the control circuit board is connected with the blue light LED assembly, the yellow light LED assembly, the PCR fluorescence detector and the temperature control device.

Optionally, the blue light LED assembly includes a blue light LED lamp and a blue light collimation part disposed in front of a light exit surface of the blue light LED lamp, and the yellow light LED assembly includes a yellow light LED lamp and a yellow light collimation part disposed in front of a light exit surface of the yellow light LED lamp.

Optionally, the blue light collimating component and the yellow light collimating component include any one of a collimating lens and a reflective cup.

Optionally, the PCR fluorescence nucleic acid detecting apparatus further includes a dual-band-pass filter, and the dual-band-pass filter is located in front of the light incident surface of the PCR fluorescence detecting apparatus.

Optionally, at least one silicon column is arranged on the top of the temperature control device.

Optionally, a coupling heat conducting material layer is further disposed between the microfluidic chip and the temperature control device.

Optionally, the PCR fluorescence detector includes any one of a CCD camera, a CMOS camera, a photomultiplier tube, and an avalanche photodiode.

Optionally, the CMOS camera comprises a USB CMOS camera, and the photomultiplier comprises a silicon photomultiplier.

Optionally, the PCR fluorescence nucleic acid detecting apparatus further comprises a display, and the display is connected to the control circuit board and is configured to display fluorescence image data.

Optionally, the PCR fluorescence nucleic acid detection device further comprises a wireless transmission module, and the wireless transmission module is connected to the control circuit board and used for interacting with a wireless intelligent terminal.

Optionally, the PCR fluorescence nucleic acid detection device further comprises a wireless transmission module, wherein the wireless transmission module is connected to the control circuit board and is used for uploading a nucleic acid detection result to a cloud server.

Optionally, the microfluidic chip includes a substrate, at least one set of microfluidic structures and at least one set of heat insulation groove structures, the microfluidic structures are located in the substrate and include a liquid inlet, a liquid input channel, a PCR reaction chamber, a liquid output channel and a liquid outlet, which are sequentially communicated, the heat insulation groove structures penetrate through the substrate from top to bottom, one set of heat insulation groove structures surround at least one receiving area in the substrate, and at least one set of microfluidic structures is disposed in one receiving area.

As above, the utility model discloses a PCR fluorescence nucleic acid detection device includes microfluid chip and is located blue light LED subassembly, PCR fluorescence detector and the yellow light LED subassembly of microfluid chip top, and microfluid chip below is equipped with temperature control device, blue light LED subassembly the yellow light LED subassembly PCR fluorescence detector reaches temperature control device all is connected with control circuit board. The utility model discloses a PCR fluorescence nucleic acid detection device is based on the microfluid chip, has miniaturization, chipization, detects the highly integrated characteristics of flow, can realize the ultrafast fluorescence detection of nucleic acid PCR, can be applied to the detection of the nucleic acid material in virus, bacterium, cell, the body fluid.

Drawings

FIG. 1 is a schematic diagram showing the structure of a PCR fluorescent nucleic acid detecting apparatus.

Fig. 2 a-2 b are schematic diagrams showing the coupling between the temperature control device and the microfluidic chip via the coupling thermally conductive material layer.

Fig. 3 a-3 b are schematic diagrams showing top views of two microfluidic chips.

Fig. 4a and 4b are plan views showing the arrangement of the thermal isolation channel structure, the liquid input flow channel and the liquid output flow channel in the microfluidic chip shown in fig. 3a and 3 b.

Fig. 5 is a schematic top view of a second microfluidic chip.

Fig. 6 is a plan view of the thermal sink structure, liquid inlet flow channels, and liquid outlet flow channels of the microfluidic chip shown in fig. 5.

FIG. 7 is a plan view of a third microfluidic chip showing the structure of thermal isolation trenches, liquid inlet channels and liquid outlet channels.

Fig. 8 is a schematic top view of a fourth microfluidic chip.

Fig. 9 is a schematic diagram showing the collimation of blue (yellow) light emitted from a blue (yellow) LED lamp by a collimating lens.

Fig. 10 shows a schematic view of the collimation of blue (yellow) light emitted by a blue (yellow) LED lamp by a reflector cup.

Fig. 11 is a schematic view showing the collimation of blue light (yellow light) emitted from a blue light (yellow light) LED lamp by a reflective lens.

FIG. 12 is a graph showing the G-channel data extracted after one cycle of PCR nucleic acid amplification.

FIGS. 13 a-13 b show PCR reaction curves.

Fig. 14 is a schematic diagram illustrating experimental results of different PCR devices can be uploaded to the cloud for different local devices to obtain.

Description of the element reference numerals

1 microfluidic chip

101 substrate

102 microfluidic structure

1021 liquid inlet

1022 liquid input flow passage

1023 PCR reaction chamber

1024 liquid output flow channel

1025 liquid outlet

103 heat insulation groove structure

1031 accommodating area

1032 Heat insulation groove

104 cover plate

2 blue light LED assembly

201 blue light LED lamp

202 blue light collimation component

202a collimating lens

202b reflecting cup

202c reflective lens

203 blue light filter

3 yellow LED assembly

301 yellow light LED lamp

302 yellow light collimation component

303 yellow light filter

4 PCR fluorescence detector

401 lens module

402 USB imaging module

403 focal plane

5 temperature control device

6 control circuit board

7 double band-pass filter

8 silicon column

9 display

10 coupled layers of thermally conductive material

11 Heat sink

Detailed Description

The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The present invention can also be implemented or applied through other different specific embodiments, and various details in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Please refer to fig. 1 to 14. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the invention in a schematic manner, and only the components related to the invention are shown in the drawings rather than being drawn according to the number, shape and size of the components in actual implementation, and the form, quantity and proportion of the components in actual implementation may be changed at will, and the layout of the components may be more complicated.

Example one

The present embodiment provides a PCR fluorescence nucleic acid detection apparatus, please refer to fig. 1, which shows a schematic structural diagram of the PCR fluorescence nucleic acid detection apparatus, including a microfluidic chip 1, a blue LED component 2, a yellow LED component 3, a PCR fluorescence detector 4, a temperature control device 5 and a control circuit board 6, wherein the microfluidic chip 1 is used for performing PCR nucleic acid amplification reaction, and the blue LED component 1 is located above the microfluidic chip 1 and is used for emitting blue light to the surface of the microfluidic chip 1; the yellow LED component 3 is positioned above the microfluidic chip 1 and used for emitting yellow light to the surface of the microfluidic chip 1; the PCR fluorescence detector 4 is positioned above the microfluidic chip 1, is positioned between the blue LED component 2 and the yellow LED component 3, and is used for collecting fluorescence signals from the microfluidic chip 1; the temperature control device 5 is positioned below the microfluidic chip 1 and used for adjusting the temperature of the microfluidic chip 1; the control circuit board 6 is connected with the blue light LED component 2, the yellow light LED component 3, the PCR fluorescence detector 4 and the temperature control device 5.

As an example, the micro fluid chip 1 is provided with a heat insulation groove structure, so that the rapid temperature rise and drop can be realized, the PCR reaction time can be greatly shortened, the PCR amplification process which usually requires more than 30 minutes can be shortened to less than 5 minutes, and for example, the time required for 50 cycles can be about 3 minutes.

Specifically, the ultrafast PCR is realized by a tec (thermoelectric cooler) temperature control device. As an example, as shown in fig. 1, at least one silicon column 8 is disposed on the top of the temperature control device 5 for connecting with the microfluidic chip 1.

As an example, referring to fig. 2a, a coupling heat conducting material layer 10 is further disposed between the microfluidic chip 1 and the temperature control device 5, and a heat sink 11 is further disposed below the temperature control device 5, wherein the microfluidic chip 1 is coupled with the temperature control device 5 through the coupling heat conducting material layer 10, and the coupling heat conducting material layer 10 can prevent an air gap from being formed between the microfluidic chip 1 and the temperature control device 5, which is helpful for increasing the heat transfer performance between the microfluidic chip 1 and the temperature control device 5. It should be noted that when the top of the temperature control device 5 is provided with the silicon pillar 8 (not shown in fig. 2 a), the coupling thermal conductive material layer 10 is located between the silicon pillar 8 and the interface of the microfluidic chip 1.

By way of example, the coupling thermal conductive material layer 10 includes, but is not limited to, a thermal conductive silicone grease, a graphene gasket, and the like. The coupling heat conducting material layer 10 can be coupled with the microfluidic chip 1 as a whole (as shown in fig. 2 a), or can be separately coupled with the microfluidic channels in the microfluidic chip 1, that is, the separate heat conducting materials are respectively disposed right below the microfluidic channels in the microfluidic chip 1 (as shown in fig. 2 b).

Referring to fig. 3a, fig. 3b and fig. 4, wherein fig. 3a and fig. 3b are schematic top views of two microfluidic chips, FIGS. 4a and 4b are plan views of the structure of the thermal sink, the liquid inlet channels and the liquid outlet channels in the microfluidic chip 1 shown in FIGS. 3a and 3b, the microfluidic chip 1 comprises a substrate 101, at least one set of microfluidic structures 102 and at least one set of thermal isolation trench structures 103, wherein, the micro-fluid structure 102 is located in the substrate 101 and comprises a liquid inlet 1021, a liquid input channel 1022, a PCR reaction chamber 1023, a liquid output channel 1024 and a liquid outlet 1025 which are sequentially communicated, the heat insulation groove structure 103 vertically penetrates through the substrate 101, wherein a set of said thermal shield slot structures 103 encloses at least one receiving area 1031 in said substrate 101, at least one set of said microfluidic structures 102 being provided in one of said receiving areas 1031. Fig. 3a shows a set of the heat shield structure 103 enclosing a receiving area 1031 in the substrate 101, and two sets of the microfluidic structure 102 are arranged in one receiving area 1031. Fig. 3b shows a set of the heat sink structures 103 enclosing a receiving area 1031 in the substrate 101, wherein four sets of the microfluidic structures 102 are arranged in one receiving area 1031. The PCR chamber 1023, the liquid inlet flow path 1022, and the liquid outlet flow path 1024 may be opened from the upper surface of the substrate 101 and extend toward the lower surface of the substrate 101, but do not penetrate the lower surface of the substrate 101, and the liquid inlet 1021 and the liquid outlet 1025 both penetrate the substrate 101. The microfluidic chip may further include a cover plate 104, wherein the cover plate 104 is disposed on the upper surface of the substrate 101 and covers the liquid inlet 1021, the PCR reaction chamber 1023, the liquid input channel 1022, the liquid output channel 1024, and the liquid outlet 1025. The material of the cover plate 104 includes, but is not limited to, a transparent material such as glass. The heat insulation slot structure 103 may comprise at least two heat insulation slots 1032 arranged separately, wherein at least two of the heat insulation slots are partially overlapped in the horizontal direction. In this embodiment, the heat insulation groove structure 103 includes four heat insulation grooves 1032, two of which are located in the inner ring, and the other two of which are located in the outer ring, two of which are not connected to allow the flow passage to pass through, and two of which are also not connected to allow the flow passage to pass through, wherein the passage of the inner ring is blocked by the heat insulation groove of the outer ring at the outer side, and the passage of the outer ring is blocked by the heat insulation groove of the inner ring at the inner side, so that the heat insulation groove structure 103 can enclose a substantially closed accommodating area 1031, thereby enhancing the heat insulation effect. It should be noted that the heat insulation groove structure 103 may also include only one heat insulation groove, but the heat insulation effect is slightly inferior to that of the multi-layer heat insulation groove. The heat insulation groove can comprise at least one bending angle which is arc-shaped, for example, the bending angle adopts a round design scheme, which is beneficial to reducing the stress of the chip, increasing the pressure resistance of the chip in the use process and preventing the chip from being cracked. The liquid inlet 1021 and the liquid outlet 1025 can be located at the periphery of the heat insulation groove structure 103, and the liquid input flow channel 1022 and the liquid output flow channel 1024 can both pass through the area between the two heat insulation grooves to enter the housing area 1031 to connect with the PCR reaction chamber 1023. The liquid inlet 1021 and the liquid outlet 1025 can be configured in different shapes for easy differentiation, for example, the liquid inlet 1021 is configured in a circle, and the liquid outlet 1025 is configured in a square, with the diameter of the circle or the side length of the square being between 0.5mm and 2 mm. PCR reaction chamber 1023 can be the snakelike that makes a snaking round and round, and the design of coiled pipe PCR reaction chamber 1023 can effectively avoid the production of bubble to improve space utilization. In this embodiment, the width of the PCR reaction chamber 1023 is in the range of 0.1mm to 2mm, the length is in the range of 1mm to 10mm, and the area of the entire microfluidic chip is in the range of 10mm × 10mm to 50mm × 50 mm. It should be noted that, in this embodiment, one housing area 1031 enclosed by the heat insulation slot structure 103 includes two sets of the microfluidic structures 102, and the lengths of the PCR reaction chambers 1023 of the two sets of the microfluidic structures 102 are the same. However, in other embodiments, the heat insulation slot structure 103 may also include other numbers of the microfluidic structures, such as 1-10 sets, in a housing area 1031 enclosed by the heat insulation slot structure, and when at least two sets of the microfluidic structures are included, the lengths of the PCR reaction chambers 1023 of different microfluidic structures may also be different. In addition, in other embodiments, the shape and size of the PCR reaction chamber 1023 and the area of the microfluidic chip can be adjusted according to needs, for example, the PCR reaction chamber 1023 is a square with a side length of 2mm-20mm, which should not unduly limit the scope of the present invention. In addition, the substrate 101 may include a silicon substrate, wherein the cover plate 104 may be coupled to the silicon substrate by a direct bonding method, the microfluidic structure 102 may be formed by etching, and the heat insulation groove structure 103 may be formed by etching through the silicon substrate, such that the PCR reaction chamber 1023 and most of the flow channels are thermally insulated from the peripheral silicon substrate. It is noted that the substrate material may be aluminum nitride, ceramic, metal or plastic in addition to silicon.

Referring to fig. 5 and 6, fig. 5 is a schematic top view of a second microfluidic chip, and fig. 6 is a plan view of a thermal isolation structure, a liquid inlet channel and a liquid outlet channel in the microfluidic chip. Unlike the microfluidic chip shown in fig. 3a and 3b, which uses an independent heat sink structure and only encloses one housing region in the substrate, the heat sink structure of the microfluidic chip shown in fig. 5 uses a shared heat sink, i.e., one heat sink structure encloses a plurality of housing regions. Fig. 5 and fig. 6 show that the heat insulation groove structure 103 encloses four receiving areas 1031 in the substrate 101, the four receiving areas 1031 are respectively provided with a PCR reaction chamber 1023, each PCR reaction chamber 1023 is respectively provided with an independent liquid inlet 1021, a liquid input flow channel 1022, a liquid output flow channel 1024 and a liquid outlet 1025, the areas of the four receiving areas 1031 are the same, and the four PCR reaction chambers 1023 have the same size and are symmetrically arranged. That is, the microfluidic chip has 4-channel reaction chambers, which can be used to test positive samples, negative samples as control groups, and two samples to be tested, respectively. It should be noted that, in other embodiments, one of the heat insulation slot structures 103 may also enclose other numbers of receiving areas 1031 in the substrate 101, for example, 2 to 10, the sizes of the receiving areas 1031 may be the same or different, the number of the PCR reaction chambers 1023 in each receiving area 1031 is also not limited to 1, for example, 1 to 10, and the sizes and shapes of the PCR reaction chambers 1023 may also be adjusted as required, which should not unduly limit the protection scope of the present invention.

FIG. 7 is a plan view of a third microfluidic chip showing the structure of the thermal isolation channel, the liquid inlet channel and the liquid outlet channel. Unlike the microfluidic chip of fig. 4 which employs a shared insulating slot structure, the microfluidic chip of fig. 7 employs a discrete insulating slot structure, i.e., the microfluidic chip includes a plurality of separately disposed insulating slot structures. Fig. 6 shows a microfluidic chip comprising four independent thermal well structures 103, each thermal well structure 103 enclosing a receiving area 1031 in the substrate 101. It should be noted that, in other embodiments, the microfluidic chip may also include other numbers of independent thermal insulation slot structures 103, for example, 2 to 10, each independent thermal insulation slot structure 103 may not only enclose one receiving area 1031, for example, 2 to 10, the sizes of the receiving areas 1031 may be the same, or may not be the same, the number of the PCR reaction chambers 1023 in each receiving area 1031 is also not limited to 1, for example, 1 to 10, and the sizes and shapes of the PCR reaction chambers 1023 may also be adjusted as required, which should not unduly limit the scope of the present invention.

Referring to fig. 8, which is a schematic top view of a fourth microfluidic chip, different from the situation that the microfluidic chip shown in fig. 3a includes two or four PCR reaction chambers 1023, the microfluidic chip shown in fig. 8 includes four PCR reaction chambers 1023, and the four PCR reaction chambers 1023 are asymmetrically arranged, wherein four PCR reaction chambers 1023 are respectively arranged in the housing area 1031 surrounded by the heat insulation groove structure 103, two of the PCR reaction chambers 1023 have a longer length (about 2/3 of the whole reaction area), and the other two PCR reaction chambers 1023 have a shorter length (about 1/3 of the whole reaction area). In practical applications, the two PCR reaction chambers 1023 with shorter length can be used as a control test for the negative sample and the positive sample, respectively, and the two PCR reaction chambers 1023 with longer length can be used as a measured sample channel. It should be noted that, in this embodiment, the channels of the inner ring are not completely blocked by the heat insulation grooves of the outer ring on the outer side, while a part of the channels of the outer ring are completely blocked by the heat insulation grooves of the inner ring on the inner side, and another part of the channels are not blocked by the heat insulation grooves of the inner ring on the inner side, but because the width of the unblocked channels is very small, a strong heat insulation effect can still be achieved. Of course, in other embodiments, the design of the heat insulation slot may be more complicated, so that there is no unblocked passage to obtain better heat insulation effect, and the protection scope of the present invention should not be limited too much here.

As an example, the blue LED assembly 2 includes a blue LED lamp 201 and a blue collimating component 202 disposed in front of a light emitting surface of the blue LED lamp 201, and the yellow LED assembly 3 includes a yellow LED lamp 301 and a yellow collimating component 302 disposed in front of a light emitting surface of the yellow LED lamp 301.

By way of example, the blue light collimating component 202 and the yellow light collimating component 302 include, but are not limited to, any one of a collimating lens and a reflective cup. Referring to fig. 9 to 11, fig. 9 is a schematic diagram illustrating that blue light emitted by the blue LED lamp 201 is collimated by a collimating lens 202a, fig. 10 is a schematic diagram illustrating that blue light emitted by the blue LED lamp 201 is collimated by a reflecting cup 202b, and fig. 11 is a schematic diagram illustrating that blue light emitted by the blue LED lamp 201 is collimated by a reflecting lens 202 c. The same principle can be used for the yellow light path design.

As an example, the center wavelength of the blue LED lamp is 470nm, the center wavelength of the yellow LED lamp is 585nm, and the power of the LED can be 200mW-600 mW.

As an example, the blue LED assembly 2 further includes a blue filter 203, the blue filter 203 is located in front of the blue collimating component 202, and a bandpass center wavelength of the blue filter 203 is 470 nm; the yellow LED component 3 further comprises a yellow light filter 303, the yellow light filter 303 is located in front of the yellow light collimation component 302, and the band-pass center wavelength of the yellow light filter 303 is 585 nm.

By way of example, the PCR fluorescence detector 4 includes, but is not limited to, any one of a CCD (Charge Coupled Device) camera, a CMOS camera, a photomultiplier tube, and an Avalanche Photodiode (APD), wherein the photomultiplier tube may be a Silicon photomultiplier tube (SiPM).

In this embodiment, the PCR fluorescence detector 4 preferably uses a USB CMOS camera in consideration of portability and cost. As shown in fig. 1, the PCR fluorescence detector 4 includes a lens module 401 facing the microfluidic chip 1 and a USB imaging module 402 located behind the lens module 401, wherein a focal plane 403 of the imaging chip is also shown in fig. 2.

By way of example, in consideration of image definition and operation amount, the pixel resolution of the USB CMOS camera is preferably 2 Mp-10 Mp, and preferably a low-illumination camera, the illumination range of the camera is 0.0001 lux-0.2 lux, the smaller the aperture coefficient f/# of the camera, the higher the light collection efficiency, the smaller the f/# should be less than f/3.0, the larger the numerical aperture NA of the camera is 0.17, the higher the light collection efficiency. The field of view of the camera is selected in preference to the size of the sample (microfluidic chip) to be imaged and the distance of the camera from the sample. For example, if the required imaging area of the microfluidic chip is 12mm × 12mm, the field angle of the diagonal line of the microfluidic chip can be selected from 90 ° -150 °; the focal distance range can be selected from 2.6mm to 3.6mm by adopting a micro-focus camera, and the corresponding object distance range is 1cm to 5 cm. The CMOS chip of the camera plays a key role in the signal-to-noise ratio of the detected signal, and the size of the selected pixel is between 1.2 and 3 mu m. In order to obtain a larger test signal-to-noise ratio, the longer the exposure time of the camera, the better the exposure time is, the longer the exposure time is, the more than 320ms is, and the exposure time depends on the synchronization of the camera, the LED excitation system and the temperature control system and the overall time requirement of PCR detection.

It should be noted that the above camera parameters are only examples, and can be adjusted as needed in practical applications, and the protection scope of the present invention should not be limited too much here.

As an example, in order to remove the influence of the excitation light, the PCR fluorescence nucleic acid detecting apparatus further includes a dual band-pass filter 7, the dual band-pass filter 7 is located in front of the light incident surface of the PCR fluorescence detecting apparatus 4 for filtering, and the band-pass is: 510 nm-550nm and 610nm-650 nm.

By way of example, the PCR fluorescent nucleic acid detection device further comprises a display 9, and the display 9 is connected with the control circuit board 6 and is used for displaying fluorescent image data and displaying an operation interface.

For example, the PCR fluorescence detection detects nucleic acid by testing an internal standard and a target of a sample, in this embodiment, the yellow LED module 3 is used to detect the internal standard of the sample, and the blue LED module 2 is used to detect the target of the sample. The fluorescently labeled molecules used for the target and internal standards may be FAM and ROX, respectively. The excitation wavelength of the FAM channel is 450nm-490nm after passing through the blue light filter 203, the excited fluorescence wavelength is 515nm-530nm, and similarly, the excitation wavelength of the ROX channel is 555nm-585nm after passing through the yellow light filter 303, and the excited fluorescence wavelength is 610nm-650 nm.

Please refer to fig. 12, which shows a color image obtained by the imaging camera after one cycle of PCR nucleic acid amplification experiment, which is decomposed into three color channels of R (red), G (green), and B (blue), and the G channel image data is extracted. Please refer to fig. 13a and 13b, which show the average value of the pixel values in the PCR reaction chamber region (coiled pipe) in fig. 12, and the same processing of the pictures in each cycle, and the curve of the average value and the cycle number is drawn to obtain the classic PCR reaction curve, and it is noted that the PCR curve in fig. 13a is the curve processed by the algorithm of smoothing, denoising, baseline removal, etc.; FIG. 13b is a graph showing the logarithm of the PCR curve, and linear fitting is performed on the linear portion of the obtained typical PCR curve to obtain the PCR amplification rate and relative initial concentration, and further, a threshold parameter is given through clinical trials, as shown in FIG. 13b, the intersection point of the threshold line and the PCR logarithm curve is defined as the experimental Ct value, wherein the selection of the clinical threshold should ensure that the intersection point of the threshold curve is on the linear portion of the PCR curve. And comparing the obtained experimental Ct value with a clinical reference Ct _ ref value, and if the Ct is less than Ct _ ref, determining that the sample is a positive sample, otherwise, determining that the sample is negative. It should be noted that the specific judgment standard can be adjusted as required, and the protection scope of the present invention should not be limited excessively here.

By way of example, the PCR fluorescence nucleic acid detection apparatus further includes a wireless transmission module, where the wireless transmission module is connected to the control circuit board, and is used for interacting with a wireless intelligent terminal, for example, fluorescent image data can be read through a mobile phone, a computer or other terminals, and control of each module of the system and image analysis, processing and data sharing can be realized through an application APP installed on the intelligent terminal. In addition, PCR fluorescence nucleic acid detection device can also pass through wireless transmission module and upload the nucleic acid testing result to the cloud end server, and other terminals can obtain test data from the cloud end server. Referring to fig. 14, it is shown that the experimental results of different PCR devices (denoted as PCR 1, PCR 2, PCR 3, …, and PCR n) can be uploaded to the cloud for different local devices (denoted as PCR 1, PCR 2, …, and PCR n), where n is an integer. In practical application, other users, such as research institutions, quarantine departments and government institutions, can use the local equipment to acquire data from the cloud server, check and analyze the data, and timely and efficiently provide nucleic acid detection support for epidemic situation prevention and control.

Example two

This example applies the PCR fluorescence nucleic acid detecting apparatus described in the first example to gene detection, such as detection of nucleic acids in viruses, bacteria, cells, and body fluids, including but not limited to the novel coronavirus COVID-19. Wherein, the PCR amplification process of the nucleic acid detection process is performed in the PCR reaction chamber 1023 of the microfluidic chip.

To sum up, the utility model discloses a PCR fluorescence nucleic acid detection device includes the microfluid chip and is located blue light LED subassembly, PCR fluorescence detector and the yellow light LED subassembly of microfluid chip top, and microfluid chip below is equipped with temperature control device, the blue light LED subassembly the yellow light LED subassembly PCR fluorescence detector reaches temperature control device all is connected with control circuit board. The utility model discloses a PCR fluorescence nucleic acid detection device is based on the microfluid chip, has miniaturization, chipization, detects the highly integrated characteristics of flow, can realize PCR nucleic acid ultrafast fluorescence detection, can be applied to the detection of the nucleic acid material in virus, bacterium, cell, the body fluid, helps carrying out quick examination to the crowd under the epidemic situation state. Therefore, the utility model effectively overcomes various defects in the prior art and has high industrial utilization value.

The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (12)

1. A PCR fluorescent nucleic acid detecting apparatus, comprising:

a microfluidic chip for performing a PCR nucleic acid amplification reaction of nucleic acid;

the blue light LED component is positioned above the microfluid chip and used for emitting blue light to the surface of the microfluid chip;

the yellow light LED component is positioned above the microfluid chip and used for emitting yellow light to the surface of the microfluid chip;

the PCR fluorescence detector is positioned above the microfluidic chip, positioned between the blue LED component and the yellow LED component and used for collecting fluorescence signals from the microfluidic chip;

the temperature control device is positioned below the microfluidic chip and used for adjusting the temperature of the microfluidic chip;

and the control circuit board is connected with the blue light LED assembly, the yellow light LED assembly, the PCR fluorescence detector and the temperature control device.

2. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the blue light LED assembly comprises a blue light LED lamp and a blue light collimation part arranged in front of a light outlet surface of the blue light LED lamp, and the yellow light LED assembly comprises a yellow light LED lamp and a yellow light collimation part arranged in front of a light outlet surface of the yellow light LED lamp.

3. The PCR fluorescent nucleic acid detecting apparatus according to claim 2, characterized in that: the blue light collimation component and the yellow light collimation component comprise any one of a collimation lens and a reflecting cup.

4. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the PCR fluorescent virus detection device further comprises a double-band-pass filter, and the double-band-pass filter is located in front of the light incident surface of the PCR fluorescent detector.

5. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the top of the temperature control device is provided with at least one silicon column.

6. The PCR fluorescent nucleic acid detecting apparatus according to claim 1 or 5, wherein: and a coupling heat conduction material layer is also arranged between the microfluidic chip and the temperature control device.

7. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the PCR fluorescence detector comprises any one of a CCD camera, a CMOS camera, a photomultiplier and an avalanche photodiode.

8. The PCR fluorescent nucleic acid detecting apparatus according to claim 7, characterized in that: the CMOS camera comprises a USB CMOS camera, and the photomultiplier comprises a silicon photomultiplier.

9. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the PCR fluorescent nucleic acid detection device also comprises a display, and the display is connected with the control circuit board and is used for displaying fluorescent image data.

10. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the PCR fluorescent nucleic acid detection device also comprises a wireless transmission module, wherein the wireless transmission module is connected with the control circuit board and is used for interacting with a wireless intelligent terminal.

11. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the PCR fluorescent nucleic acid detection device further comprises a wireless transmission module, wherein the wireless transmission module is connected with the control circuit board and used for uploading a virus detection result to a cloud server.

12. The PCR fluorescent nucleic acid detecting apparatus according to claim 1, characterized in that: the microfluidic chip comprises a substrate, at least one group of microfluidic structures and at least one group of heat insulation groove structures, wherein the microfluidic structures are positioned in the substrate and comprise a liquid inlet, a liquid input runner, a PCR reaction cavity, a liquid output runner and a liquid outlet which are sequentially communicated, the heat insulation groove structures penetrate through the substrate from top to bottom, at least one accommodating area is defined by the group of heat insulation groove structures in the substrate, and at least one group of microfluidic structures is arranged in one accommodating area.

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