Two-color fluorescence detection device suitable for CRISPR (clustered regularly interspaced short palindromic repeats) molecular diagnosis technology

Abstract

The utility model discloses a two-color fluorescence detection device suitable for a CRISPR (clustered regularly interspaced short palindromic repeats) molecular diagnosis technology, which is characterized in that a detection chamber shell and a detection chamber upper cover are matched to form a closed detection space to create a darkroom environment for the whole reaction, so that the interference of natural light on the whole detection reaction is avoided, and the detection efficiency is improved. The internal sample test tube rack is driven by the driving unit to centrifugally rotate, a centrifugal environment is provided for to-be-detected samples, the to-be-detected samples are distributed along the circumferential direction of the sample test tube rack, 48 samples can be detected at the same time, and multi-flux detection is achieved. On the premise that the cavity is not opened and the sample is not transferred, collection of double-gene fluorescence images of the sample can be achieved, the device achieves multi-flux during detection, meanwhile, a tube-in-tube structure is used, the pollution problem caused by nucleic acid transfer of the amplified sample, uncovering and other operations is avoided, the sample can be obtained, and the detection efficiency is improved. The efficiency and the accuracy of nucleic acid detection are improved.

Description

Technical Field

[0001] This invention belongs to the field of CRISPR molecular detection technology and relates to a dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology. Background Technology

[0002] Rapid, accurate, sensitive, and quantitative detection of specific nucleic acid sequences plays an increasingly important role in the diagnosis of human infectious diseases, food security, pathogen identification, global biosafety, and in environmental analysis, including tracking biocontamination and monitoring environmental quality. The COVID-19 pandemic has posed a significant threat to human life. The rapid outbreak of COVID-19 was due to the lack of effective detection methods; nucleic acid detection is currently the "gold standard" for COVID-19 detection, and real-time quantitative polymerase chain reaction (PCR) is the gold standard. PCR and its variant reverse transcription polymerase chain reaction (RT-PCR) are currently the most commonly used pathogen nucleic acid detection techniques due to their high specificity, low purity requirements, and high sensitivity. However, these methods typically require expensive equipment and highly trained personnel, and have long reaction times, making them unsuitable for simple, rapid, and targeted molecular diagnosis of SARS-CoV-2. Therefore, in recent decades, methods such as recombinase polymerase amplification (RPA), recombinase-mediated chain substitution amplification (RAA), and loop-mediated isothermal amplification (LAMP) have been widely used in point-of-care molecular diagnostics due to their simplicity, speed, and low cost. While these isothermal amplification detection techniques eliminate reliance on large instruments and hold great potential for rapid on-site diagnosis and screening, they still have certain limitations. For example, RPA and RAA techniques have high detection efficiency but low sensitivity, failing to meet testing requirements; LAMP technology offers high sensitivity and specificity, but its primer design is complex and requires precise temperature control. Therefore, a highly efficient nucleic acid detection method is lacking to achieve rapid, simple, and highly sensitive nucleic acid detection.

[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) originated from the adaptive immune system of prokaryotes. A CRISPR system consists of a CRISPR array containing regular repeat sequences and spacer sequences, along with its associated protein (Cas) genome. CRISPR nucleic acid detection technology is based on CRISPR-related proteins that non-specifically cleave the target sequence after recognizing it, thus recognizing foreign genes to exert an immune function. This technology has been used to detect nucleic acids of various pathogens. With the advent of the CRISPR / Cas system, the nucleic acid detection industry has ushered in a new era of development. Compared with traditional nucleic acid detection methods, CRISPR detection technology not only shows significant advantages in terms of detection cost, efficiency, portability, specificity, and simplicity, but also possesses good biocompatibility and can be combined with other technologies to achieve simpler and more sensitive nucleic acid detection. Therefore, this technology is hailed as a next-generation novel nucleic acid detection technology. Fluorescence analysis, with its advantages of high specificity, less interference from non-fluorescent substances, high selectivity for trace analytes, fast response, and environmental friendliness, is the most commonly used signal output in CRISPR / Cas-based nucleic acid detection. CRISPR technology typically requires nucleic acid amplification before nucleic acid detection, followed by the addition of the amplified nucleic acid to the CRISPR reaction system to detect the target nucleic acid, thereby improving detection sensitivity. These two processes need to be performed separately, involving reagent transfer in a two-step process. This not only easily leads to reagent contamination and waste but also requires significant human resources. Currently, most technologies do not employ this approach. Therefore, developing a simple and sensitive integrated device based on CRISPR technology will be of great significance for nucleic acid detection.

[0004] The "Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 9)" stipulates that the criteria for lifting isolation management and discharge from the hospital are that the Ct values ​​of the N gene and ORF gene in two consecutive novel coronavirus nucleic acid tests are both ≥35 (for the quantitative real-time PCR method, the cutoff value is 40, and the sampling time interval is at least 24 hours). Both the N gene and ORF gene need to be tested. Currently, most detection technologies for the novel coronavirus are limited to single-gene detection. (Tian Tian, ​​Zhiqiang Qiu, Yongzhong Jiang, et al. Exploiting the orthogonal CRISPR-Cas12a / Cas13a trans-cleavage for dual-gene virus detection using a handheld device[J]. Biosensors and...) Bioelectronics, 2022, 196:113701-113701. [A group proposed a highly efficient dual-gene diagnostic technique based on the orthogonal DNA / RNA side branch cleavage mechanism of the CRISPR-Cas12a / Cas13a system. This technique simultaneously detects dual-gene amplification products from multiplex recombinase polymerase amplification (RPA) in a single tube using Cas12a and Cas13a assays. The resulting orthogonal DNA / RNA side branch cleavage can specifically cleave two different DNA and RNA probes respectively. Reliable dual-gene detection of the N gene and ORF1ab gene of SARS-CoV-2 was verified by integration with smartphone-based fluorescence readings. However, this technique cannot detect multiple samples simultaneously and suffers from transfer reagent contamination issues.] Utility Model Content

[0005] The purpose of this invention is to solve the problems of existing technologies, such as the inability to simultaneously detect two genes, low throughput, and transfer reagent contamination, and to provide a dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology includes a detection chamber shell, a sample tube rack and a heating unit are arranged inside the detection chamber shell, the sample tube rack is used to place the sample to be tested, the sample to be tested is distributed along the circumference of the sample tube rack, and a corresponding detection chamber cover is provided on the detection chamber shell;

[0008] A drive unit is provided at the lower end of the detection chamber shell. The output end of the drive unit extends into the interior of the detection chamber shell. The drive unit can drive the sample tube rack to rotate circumferentially.

[0009] A fluorescence excitation detection unit is provided on one side of the detection chamber shell. The fluorescence excitation detection unit is connected to the inside of the detection chamber shell and is used to collect fluorescence images inside the detection chamber shell.

[0010] The further improvement of this utility model is as follows:

[0011] The sample tube rack includes a bottom connecting plate, with side plates distributed circumferentially on the bottom connecting plate. Several test tube holes are opened on the side plates, and test tubes to be tested are placed in the test tube holes.

[0012] The side plate is inclined outward relative to the bottom connecting plate at an angle of 25°.

[0013] The heating unit includes a heating element and a heat sink. The heating element is fixed to the bottom of the detection chamber housing, and the heat sink is fixed to the heating element.

[0014] A temperature sensor is also installed inside the housing of the detection chamber.

[0015] The output end of the drive unit is connected to the rotating platform of the motor output end, and the sample tube rack is fixed on the rotating platform of the motor output end.

[0016] It also includes a motor shaft platform, which is located at the upper end of the drive unit, and the motor output end rotating platform is located above the motor shaft platform;

[0017] The output end of the drive unit passes through the motor shaft platform and is connected to the motor output end rotating platform.

[0018] The drive unit includes a stepper motor, which is fixed to the lower end of the detection chamber housing by a stepper motor mounting bracket.

[0019] A fluorescence detection port is provided on the side wall of the detection chamber shell, and the image acquisition end of the fluorescence excitation detection unit is connected to the fluorescence detection port.

[0020] The fluorescence excitation detection unit includes a first light source channel mounting part, a second light source channel mounting part, a detection channel mounting part, a CMOS mounting part, a first CMOS camera, and a second CMOS camera connected in sequence.

[0021] The first light source channel mounting section is used to transmit a blue excitation light source;

[0022] The second light source channel mounting section is used to transmit the green excitation light source;

[0023] The first CMOS camera is used to detect green fluorescence generated by a sample excited by a blue excitation light source; the second CMOS camera is used to detect red fluorescence generated by a sample excited by a green excitation light source.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discloses a dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology. The detection chamber shell and the detection chamber cover cooperate to form a closed detection space, creating a dark environment for the entire reaction and avoiding interference from natural light. The sample tube rack inside is centrifugally rotated under the drive of the drive unit, providing a centrifugal environment for the samples to be tested. The samples to be tested are distributed circumferentially along the sample tube rack, and up to 48 samples can be detected simultaneously, realizing multi-throughput detection. The detection chamber shell is connected to the fluorescence excitation detection unit, and the image of the sample can be acquired without opening the chamber or transferring the sample. This device achieves simultaneous multi-throughput detection, avoiding contamination problems caused by opening the lid, transferring sample nucleic acid, closing the lid, etc. It can realize sample input and result output, improving the efficiency and accuracy of nucleic acid detection.

[0026] Furthermore, in this invention, the side plate is tilted outward relative to the bottom connecting plate to prevent the sample from detaching from the test tube rack during rotation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this utility model, the drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this utility model and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is an overall structural diagram of the present invention;

[0029] Figure 2 A schematic diagram of the device structure for removing the top cover and the shell of this utility model;

[0030] Figure 3 This is an assembly drawing of the centrifugal drive unit of this utility model;

[0031] Figure 4 This is a schematic diagram of the sample tube rack structure of this utility model;

[0032] Figure 5 This is a schematic diagram of the test chamber cover and shell structure of this utility model;

[0033] Figure 6This is a schematic diagram of the tube sleeve of this utility model;

[0034] Figure 7 This is a schematic diagram of the fluorescence excitation detection unit of this utility model;

[0035] Figure 8 This is a schematic cross-sectional view of the fluorescence excitation detection unit of this utility model.

[0036] Wherein: 1-Sample tube rack; 2-Detection chamber top cover; 3-Motor shaft platform; 4-Stepper motor mounting bracket; 5-Motor; 6-Device support plate; 7-Fluorescence excitation detection unit; 8-Detection chamber shell; 9-Heating plate; 10-Heat sink; 11-Supporting foot pad; 12-Motor output rotating platform; 13-Detection channel mounting part; 14-Optical path channel locking piece; 15-First light source channel mounting part; 16-Light source mounting base plate; 17-Blue LED light source; 18-Second light source channel mounting part; 19-Green LED light source; 20-Optical path module pad; 21-Optical path support base; 22-First CMOS camera; 23-Second CMOS camera; 24-CMOS mounting part; 25- 470nm filter; 26-Blue LED bead lens; 27-First monochromatic DM; 28-570nm filter; 29-Green LED bead lens; 30-525nm filter; 31-610nm filter; 32-Second monochromatic DM; 33-First dual-color DM; 34-Test tube hole; 35-Sample rack mounting hole; 36-Pin; 37-Stepper motor mounting bracket mounting hole; 38-Stepper motor mounting hole; 39-Motor shaft platform mounting hole; 40-Temperature sensor socket; 41-Fluorescent through hole; 42-Spring snap; 43-Stepper motor output shaft hole; 44-Inner gasket of cover; 45-Tube cap; 46-Outer reaction tube; 47-Inner reaction tube; 48-Sleeve; 49-Drainage hole. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of this utility model clearer, the technical solutions of the embodiments of this utility model will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this utility model, and not all embodiments. The components of the embodiments of this utility model described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0039] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0040] In the description of the embodiments of this utility model, it should be noted that if terms such as "upper," "lower," "horizontal," or "inner" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the utility model product is in use, they are only for the convenience of describing the utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the utility model. Furthermore, terms such as "first" and "second" are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0041] Furthermore, the use of the term "horizontal" does not imply that the component must be absolutely horizontal, but rather that it can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal than "vertical," and does not mean that the structure must be completely horizontal, but can be slightly tilted.

[0042] In the description of the embodiments of this utility model, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this utility model according to the specific circumstances.

[0043] The present invention will now be described in further detail with reference to the accompanying drawings:

[0044] See Figures 1 to 8This utility model discloses a dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology, which solves the problems of existing technologies that can only detect one target at a time, have low throughput, and are contaminated with transfer reagents. Combining RPA technology and CRISPR detection technology with a tube-in-tube structure, it provides a high-throughput centrifugal rotary dual-color fluorescence detection device and method suitable for CRISPR molecular diagnostic technology. It can simultaneously detect the N gene and ORF gene of the novel coronavirus, realizing automated high-throughput dual-target nucleic acid detection function, and can detect 48 samples simultaneously. After sample addition, once the test tube is placed in the instrument, no manual operation is required, and the test results can be obtained after 55 minutes. It has high sensitivity and strong specificity, realizes automated detection, and improves the efficiency of nucleic acid detection.

[0045] See Figures 1 to 2 A dual-color fluorescence detection device suitable for CRISPR molecular diagnostic technology includes a detection chamber housing 8. A sample tube rack 1 and a heating unit are disposed within the detection chamber housing 8. The sample tube rack 1 is used to hold the sample to be tested, and the sample to be tested is distributed circumferentially along the sample tube rack 1. A corresponding detection chamber cover 2 is disposed on the detection chamber housing 8. A driving unit is disposed at the lower end of the detection chamber housing 8, and the output end of the driving unit extends into the interior of the detection chamber housing 8. The driving unit can drive the sample tube rack 1 to rotate circumferentially. A fluorescence excitation detection unit 7 is disposed on one side of the detection chamber housing 8, and the fluorescence excitation detection unit 7 is connected to the interior of the detection chamber housing 8 for acquiring fluorescence images inside the detection chamber housing 8.

[0046] Furthermore, in this embodiment, a device support plate 6 is provided at the lower end of the entire device. The device support plate 6 provides support for the entire device. Supporting feet 11 are installed at the lower end of the device support plate for support. The supporting feet 11 are made of rubber material.

[0047] See Figure 2 Furthermore, in this embodiment, the heating unit includes a heating element 9 and a heat sink 10. The heating element 9 and the heat sink 10 are stacked and fixed on the detection chamber housing 8. The heat sink 10 is fixed on the heating element 9. The heating element 9 can heat the environment and provide a suitable temperature for the entire reaction. The heat sink 10 can increase the contact area between the heating element and the air, improve the heat conduction rate, and increase the speed at which the temperature in the detection chamber rises. A temperature sensor is also provided inside the detection chamber housing 8. The temperature sensor, the heating element 9, and the heat sink 10 can provide a suitable reaction temperature for the reagents during the detection process.

[0048] Furthermore, in this embodiment, the temperature sensor is fixed to the bottom of the detection chamber housing 8, and two temperature sensor sockets 40 are opened at the bottom of the detection chamber housing 8. The temperature sensor is attached to the inner wall of the detection chamber housing 8 by adhesive.

[0049] Furthermore, in this embodiment, the temperature sensor is preferably a PT100 platinum resistance temperature sensor, the heating element 9 is preferably a silicone rubber heating element, and the heat sink 10 is made of aluminum.

[0050] See Figure 3 Furthermore, in this embodiment, the driving unit includes a stepper motor 5, with stepper motor mounting brackets 4 on both sides of the stepper motor 5. A motor shaft platform 3 is mounted on the upper end of the stepper motor mounting brackets 4. The stepper motor mounting brackets 4, stepper motor 5, and motor shaft platform 3 provide support for the entire nucleic acid detection chamber. The motor shaft platform 3 and the sample tube rack 1 are separated by the detection chamber shell 8. The two stepper motor mounting brackets 4 are fixed to the motor shaft platform 3 with screws. The output end of the stepper motor 5 passes through the motor shaft platform 3, and the output end of the stepper motor 5 is fixed to the motor output end rotation platform 12. The sample tube rack 1 is fixed to the motor output end rotation platform 12. The stepper motor 5 rotates the sample tube rack 1 through the motor output end rotation platform 12, thereby centrifuging and mixing the RPA and CRISPR reagents placed in the test tubes. The stepper motor 5 is installed and fixed to the motor shaft platform 3, the detection chamber shell 8, and the motor output end rotation platform 12 through clearance fit and screw connection.

[0051] Furthermore, in this embodiment, the stepper motor 5 is a closed-loop stepper motor with a brake, the motor model is 57CME12X-BZ, the body length is 109mm, the rated current is 5A, and the compatible driver is CL1-507.

[0052] See Figure 4 Furthermore, in this embodiment, the sample tube rack 1 includes a bottom connecting plate with side plates distributed circumferentially on the bottom connecting plate. This sample tube rack 1 is mainly used as a carrier for mounting test tubes and is used in conjunction with a stepper motor 5 to achieve mixing and homogenization of reagents inside the test tubes. To achieve high-throughput simultaneous detection, the side plates of the sample tube rack 1 have an octagonal structure, forming eight side plates. Each side plate has six test tube holes 34 for placing test tubes. The test tubes are fitted with the test tube holes 34 with a clearance. The entire sample tube rack can achieve a total detection throughput of 48. Each side plate is set as a trapezoid with a certain slope of 25° to prevent the test tubes from being thrown out during centrifugation.

[0053] Furthermore, in this embodiment, two sample rack mounting holes 35 are opened at the center of the bottom of the sample tube rack 1, and a groove is opened at the bottom of the sample tube rack 1 for connecting with the rotating platform 12 at the motor output end. The sample tube rack 1 is fixed on the stepper motor 5 by screws.

[0054] Furthermore, in this embodiment, the sample tube rack 1 is 3D printed from PLA black material.

[0055] See Figure 5 Furthermore, in this embodiment, the detection chamber cover 2 is connected to the detection chamber housing 8 by a pin 36. The inner side wall of the detection chamber housing 8 has a groove at the position where it mates with the cover. The inner gasket 44 is installed in the groove. The detection chamber cover 2 and the detection chamber housing 8 can be fastened together by a spring buckle 42, creating a dark room environment for the entire reaction, avoiding interference from natural light on the entire detection reaction, and also having a certain heat preservation effect.

[0056] Furthermore, in this embodiment, both the top cover 2 of the detection chamber and the shell 8 of the detection chamber are 3D printed from PLA black material.

[0057] See Figure 5 Furthermore, in this embodiment, the bottom of the detection chamber housing 8 is provided with one stepper motor output shaft hole 43, two stepper motor mounting bracket holes 37, four stepper motor mounting holes 38 and two motor shaft platform mounting holes 39, which are used to fix the sample tube rack 1, the stepper motor 5 and the stepper motor mounting bracket 4 with screws.

[0058] See Figure 6 Furthermore, in this embodiment, the test tube includes a cap 45, an outer reaction tube 46, and an inner reaction tube 47. The openings of the outer reaction tube 46 and the inner reaction tube 47, which is sleeved in the outer reaction tube 46, are covered by the cap 45. In this invention, the CRISPR reagent is in the inner reaction tube 47, and the RPA reagent is in the outer reaction tube 46. The cap 45 is placed on top, and the outer reaction tube and the test tube rack are fitted with a gap.

[0059] A sleeve 48 is provided inside the outer reaction tube 46. The inner reaction tube 47 is fixed inside the outer reaction tube 46 by the sleeve 48. The outer diameter of the sleeve 48 is the same as the inner diameter of the connection part of the outer reaction tube 46. The sleeve 48 can hold the inner reaction tube 47, and play a role in fixing and supporting the inner reaction tube 47, ensuring that the inner reaction tube 47 is semi-suspended in the outer reaction tube 46 and does not fall off. The outer reaction tube 46 plays a role in fixing and supporting the sleeve 48.

[0060] In this invention, the sleeve 48 is preferably made of polymethyl methacrylate (PMMA) material; the sleeve 48 is preferably manufactured using injection molding.

[0061] Furthermore, several drainage holes 49 are provided at the bottom of the lower part of the inner reaction tube 47. The drainage holes 49 are hydrophobic and have a small aperture. Under the condition of no strong external interference, due to the surface tension of the droplets and atmospheric pressure, the drainage holes 49 can ensure that the nucleic acid amplification reagent will not flow from the inner reaction tube 47 to the outer reaction tube 46.

[0062] Furthermore, the function of the cap 45 is to seal the outer reaction tube 46, ensuring that the sample nucleic acid does not evaporate outside the tube and avoid aerosol contamination.

[0063] When the reaction tube is in operation, CRISPR reagents are added to the outer reaction tube 46, and the sleeve 48 is fixed inside the outer reaction tube 46. Nucleic acid amplification reagents are added to the inner reaction tube 47, and the inner reaction tube 47 is fixed inside the sleeve 48. The nucleic acid of the sample to be tested is added to the inner reaction tube 47, and the cap 45 of the outer reaction tube is closed. During detection, under the action of centrifugal force, the nucleic acid of the sample amplified by nucleic acid will flow into the interior of the outer reaction tube 46 through the drain hole 49 of the inner reaction tube 47 and mix with the CRISPR reagents.

[0064] The test tubes are evenly placed on the test tube holes 34 on the outside of the sample test tube rack 1, and are arranged outward from the center of the sample test tube rack 1.

[0065] Furthermore, in this embodiment, a fluorescence through-hole 41 is opened on the side wall of the detection chamber housing 8, corresponding to the direction of the fluorescence light on one side of the sample tube rack 1, in conjunction with the fluorescence excitation detection unit 7 to excite and detect fluorescence.

[0066] See Figures 7 to 8 Furthermore, in this embodiment, the fluorescence excitation detection unit 7 comprises two LED light sources, two monochrome DMs, one dual-color DM, two 4K high-definition 120° wide-angle CMOS cameras, and an optical path channel. The main function of the fluorescence detection unit is to excite the FAM group of the ORF1 ab gene and the ROX group of the N gene in the test tube to generate fluorescence, and at the same time transmit the fluorescence to the camera end for imaging.

[0067] The optical path channels within the fluorescence excitation detection unit 7 are all mounted with lenses and electrical components via grooves, and are all 3D printed from PLA black material. These include a first light source channel mounting section 15, a second light source channel mounting section 18, a detection channel mounting section 13, and a CMOS mounting section 24. Specifically, the first light source channel mounting section 15 is used to mount the blue LED light source 17, blue LED bead lens 26, 470nm filter 25, monochrome DM1 27, and dual-color DM1 33; the second light source channel mounting section 18 is used to mount the green LED light source 19, green LED bead lens 29, 570nm filter 28, and the first monochrome DM27; the detection channel mounting section 13 is used to mount the first dual-color DM 33, the second monochrome DM32, and the first CMOS camera 22; the CMOS mounting section 24 is used to mount the second monochrome DM32 and the second CMOS camera 23; and the light source mounting base plate 16 is used to engage with screws to secure the LED beads, LED bead lenses, and filters. The optical path module pad 20, together with the optical path support base 21, is used to support the fluorescence excitation detection unit.

[0068] Furthermore, the first channel mounting part 15 and the detection channel mounting part 13 are combined and locked with two optical path channel locking pieces 14 and screws, and the other optical path channel components are connected and locked with screws.

[0069] Furthermore, in this invention, the blue LED light source 17 on one side is preferably a blue light source with a wavelength of 470nm that excites FAM groups, and the green LED light source 19 below is preferably a green light source with a wavelength of 570nm that excites ROX groups. The light generated by the blue LED light source 17 and the green LED light source 19 is focused by the blue LED bead lens 26 and the green LED bead lens 29, respectively, and then filtered by the 470nm filter 25 and the 570nm filter 28 before being directly emitted. The power of the LED light source is 5W.

[0070] In this invention, the FAM group generates 525nm green fluorescence after excitation, and the ROX group generates 610nm red fluorescence after excitation.

[0071] The light generated by the blue LED light source 17 and the green LED light source 19 shines directly onto the first monochromatic DM27. The first monochromatic DM27 transmits a 570nm green light source and reflects and excites a 470nm blue light source. The first dual-color DM33 reflects the 570nm green light source and the 470nm blue light source to the sample tube rack 1 and transmits a 610nm red fluorescence and a 525nm green fluorescence. The second monochromatic DM32 transmits a 610nm red fluorescence and reflects a 525nm green fluorescence.

[0072] A 525nm filter 30 is installed at the front end of the lens of the first CMOS camera 22 below the second monochrome DM 32, and a 610nm filter 31 is installed at the front end of the lens of the second CMOS camera 23 on one side of the second monochrome DM 32. The filters filter the light entering the lens and retain the light of the desired wavelength. The 610nm red fluorescence is received by the second CMOS camera 23, and the 525nm green fluorescence is received by the first CMOS camera 22 below.

[0073] Furthermore, the fluorescence images captured by the CMOS camera are connected to a computer via a USB interface for display, and the fluorescence images are processed and identified using image processing software.

[0074] The working principle of this embodiment:

[0075] Using a tube-in-tube reaction mode, the RPA mixture is added to the outer reaction tube 46, the prepared CRISPR is added to the inner reaction tube 47, and then the inner reaction tube 47 is added to the outer reaction tube 46. The tube cap 45 is then closed. This step does not have any special limitations on the specific composition and concentration of the CRISPR and RPA reagents. Conventional CRISPR and RPA reagents in the art can be used.

[0076] Start the device, open the top cover 2 of the detection chamber, place the entire sleeve structure on the sample tube rack 1 in the instrument (up to 48 samples can be detected at the same time), control the heating element 9 and the temperature sensor to heat the entire detection chamber, and then maintain a constant temperature until the entire reaction is complete.

[0077] First, perform an isothermal RPA reaction. After the RPA reaction is complete, control the stepper motor 5 to centrifuge the reagent in the inner reaction tube 47 to the outer reaction tube 46. Then, adjust the forward and reverse rotation of the stepper motor 5 to mix the reagent in the test tube. Finally, perform an isothermal CRISPR reaction.

[0078] After the CRISPR reaction is complete, turn on the LED light source to excite the fluorescent group. Connect the USB interface of the CMOS camera to the monitor and use the CMOS camera to take fluorescence images. First, take a picture of one test tube containing reagents. Then, adjust the stepper motor 5 to rotate and fix the angle to take pictures of other test tubes. The results are displayed on the screen. The results can be output by manual operation. The acquired images can be processed using ImageJ to read the fluorescence value and display the positive or negative result of the virus detection.

[0079] In this embodiment of the invention, the constant temperature is 39-40°C, the duration of the constant temperature is 25-30 min, the RPA reaction is 25 min, and the CRISPR reaction is 30 min.

[0080] The centrifugation speed was 1200 rpm, and the centrifugation time was 1 min.

[0081] One rotation of the sample tube rack 1 clockwise and one rotation counterclockwise constitutes one cycle. The sample tube rack 1 is rotated 3 times in both directions, with a rotation speed of 350 rpm and a duration of 5 seconds for each rotation.

[0082] The device disclosed in this embodiment automates the process from RPA reaction to CRISPR reaction detection, completely avoiding the contamination problems caused by opening the lid, transferring the sample nucleic acid, and closing the lid, which were previously encountered when using CRISPR molecular diagnostic technology for nucleic acid detection. Furthermore, it allows for sample input and result output through a simple procedure, improving the efficiency of nucleic acid detection. During detection, it can simultaneously detect the N gene and ORF gene of the SARS-CoV-2 virus, achieving simultaneous detection at a throughput of 48. The device is simple to operate and solves the problems of existing technologies where only one target can be detected per test and the throughput is low. It also avoids the contamination problems caused by opening the lid, transferring the sample nucleic acid, and closing the lid, which were previously encountered when using CRISPR molecular diagnostic technology for nucleic acid detection. The simple procedure allows for sample input and result output, improving the efficiency of nucleic acid detection.

[0083] The above are merely preferred embodiments of this utility model and are not intended to limit the scope of this utility model. Various modifications and variations can be made to this utility model by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this utility model should be included within the protection scope of this utility model.

Claims

1. A dual-color fluorescence detection device suitable for CRISPR molecular diagnostic techniques, characterized in that, The application relates to a detection chamber shell (8) provided with a sample test tube rack (1) and a heating unit, the sample test tube rack (1) is used for placing samples to be detected, the samples to be detected are distributed in a circumferential direction along the sample test tube rack (1), and a corresponding detection chamber upper cover (2) is arranged on the detection chamber shell (8); A driving unit is arranged at the lower end of the detection chamber shell (8), the output end of the driving unit extends into the inside of the detection chamber shell (8), and the driving unit can drive the sample test tube rack (1) to rotate in the circumferential direction; A fluorescence excitation detection unit (7) is arranged on one side of the detection chamber shell (8), the fluorescence excitation detection unit (7) is communicated with the inside of the detection chamber shell (8), and is used for collecting a fluorescence image in the inside of the detection chamber shell (8).

2. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 1, wherein, The sample test tube rack (1) comprises a bottom connecting plate, side plates are distributed in the circumferential direction on the bottom connecting plate, a plurality of test tube holes (34) are formed in the side plates, and test tubes are placed in the test tube holes (34).

3. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 2, wherein, The side plates are inclined outward relative to the bottom connecting plate, and the inclination angle is 25 degrees.

4. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 1, wherein, The heating unit comprises a heating sheet (9) and a heat dissipation sheet (10), the heating sheet (9) is fixed at the bottom of the detection chamber shell (8), and the heat dissipation sheet (10) is fixed on the heating sheet (9).

5. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 4, wherein, A temperature sensor is further arranged in the inside of the detection chamber shell (8).

6. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 1, wherein, The output end of the driving unit is connected with a motor output end rotating platform (12), and the sample test tube rack (1) is fixed on the motor output end rotating platform (12).

7. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 6, wherein, A motor rotating shaft platform (3) is further arranged, the motor rotating shaft platform (3) is arranged at the upper end of the driving unit, and the motor output end rotating platform (12) is arranged above the motor rotating shaft platform (3). The output end of the driving unit is connected with the motor output end rotating platform (12) after penetrating through the motor rotating shaft platform (3).

8. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 1, wherein, The driving unit comprises a stepping motor (5), and the stepping motor (5) is fixed at the lower end of the detection chamber shell (8) through a stepping motor fixing support (4). 9.The dual-color fluorescence detection device for CRISPR molecular diagnostic technology according to claim 1, wherein, A fluorescence detection port is formed in the side wall of the detection chamber shell (8), and the image collecting end of the fluorescence excitation detection unit (7) is communicated with the fluorescence detection port.

10. The dual-color fluorescence detection device for CRISPR molecular diagnostic technique according to claim 9, wherein, The fluorescence excitation detection unit (7) comprises a first light source channel mounting portion (15), a second light source channel mounting portion (18), a detection channel mounting portion (13), a CMOS mounting portion (24), a first CMOS camera (22) and a second CMOS camera (23) which are communicated in sequence; The first light source channel mounting portion (15) is used for transmitting a blue excitation light source; The second light source channel mounting portion (18) is used for transmitting a green excitation light source; The first CMOS camera (22) is used for detecting green fluorescence generated by exciting samples by the blue excitation light source, and the second CMOS camera (23) is used for detecting red fluorescence generated by exciting samples by the green excitation light source.

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