A light-controlled nucleic acid detection device and detection method

Abstract

The invention discloses a light-controlled nucleic acid detection device and a detection method. The detection device comprises a light-controlled light source, an excitation light component, an emission light detection component, a temperature control component and a control component; the light-controlled light source is used to directly irradiate a sample tube to start a detection reaction after amplification; the excitation light component comprises an excitation light source, a first narrow-band filter and a dichroic mirror, the excitation light source, the first narrow-band filter and the dichroic mirror are located on the same straight line, the dichroic mirror is tilted, the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in sequence, and the dichroic mirror is used to reflect the excitation light into the sample tube; the emission light detection component comprises an emission light detector and a second narrow-band filter, the light emitted by the sample in the sample tube passes through the dichroic mirror and the second narrow-band filter in sequence into the emission light detector; the temperature control component is used to adjust the reaction temperature of the sample; the control component is used to control the operation of the light-controlled light source, the excitation light component, the emission light detection component and the temperature control component.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection instruments, and in particular to a light-controlled nucleic acid detection device and a detection method. Background Art

[0002] Nucleic acid detection technology is an effective means of preventing and controlling infectious diseases. In addition to real-time fluorescence quantitative PCR as the gold standard, there are also many isothermal amplification detection methods and nucleic acid detection technologies combined with CRISPR. Among them, CRISPR technology is considered to be the next generation of nucleic acid detection technology. Traditional CRISPR detection technology requires adding the amplification product to the CRISPR system after amplification. This process involves opening the lid, which may cause aerosol contamination and cause false positives.

[0003] One solution to prevent aerosol contamination is to mix the sample, amplification system, and CRISPR detection system in one tube to avoid opening the lid for sampling during the detection process. In this one-tube detection method, the CRISPR detection system continuously identifies and cuts the amplification template and amplification product from the beginning to the end of the amplification reaction. Therefore, there is a competitive relationship between the CRISPR detection system and the amplification system, which leads to a decrease in amplification efficiency and the inability to achieve high-sensitivity nucleic acid detection.

[0004] In order to achieve highly sensitive one-tube CRISPR nucleic acid detection, one method is to first silence the CRISPR detection system in the sample, amplification system, and CRISPR detection system to avoid cutting the amplification template during the amplification stage, which causes a decrease in amplification efficiency; after the amplification reaction is completed, use light of a specific wavelength to activate the CRISPR detection system, and then perform highly sensitive nucleic acid detection. However, there is currently no such integrated light-controlled CRISPR detection instrument on the market that can achieve automated program operation. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a light-controlled nucleic acid detection device, which solves the problem that the existing CRISPR nucleic acid detection device cannot achieve high-efficiency integrated and closed-tube nucleic acid detection.

[0006] Another object of the present invention is to provide a detection method for the above-mentioned light-controlled nucleic acid detection device.

[0007] The technical solution of the present invention is: a light-controlled nucleic acid detection device, comprising a light-controlled light source, an excitation light component, an emission light detection component, a temperature control component and a control component;

[0008] The light-controlled light source is used to directly illuminate the sample tube;

[0009] The excitation light assembly comprises an excitation light source, a first narrow-band filter and a dichroic mirror, wherein the excitation light source, the first narrow-band filter and the dichroic mirror are located on the same straight line, the dichroic mirror is tilted, and the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in sequence, and the dichroic mirror is used to reflect the excitation light into the sample tube;

[0010] The emission light detection assembly includes an emission light detector and a second narrow band filter, and the light emitted by the sample in the sample tube sequentially passes through the dichroic mirror and the second narrow band filter into the emission light detector;

[0011] The temperature control component is used to adjust the reaction temperature of the sample;

[0012] The light-controlled light source, the excitation light component, the emission light detection component and the temperature control component are all electrically connected to the control component, and the control component is used to control the operation of the light-controlled light source, the excitation light component, the emission light detection component and the temperature control component.

[0013] Furthermore, it also includes a sample table, the top surface of which is provided with a sample addition hole, the sample tube is placed in the sample addition hole, the side of the sample table is provided with a light intake hole, the light intake hole is connected to the sample addition hole, and the light control light source is installed in the light intake hole.

[0014] Furthermore, the temperature control component includes a heating tube and a temperature sensor. A mounting blind hole is provided on the side of the sample table. The heating tube and the temperature sensor are respectively installed in the mounting blind holes. The heating tube and the temperature sensor are both electrically connected to the control component. The temperature sensor is used to detect the temperature information of the sample table and transmit it to the control component. The control component controls the operation of the heating tube according to the temperature information.

[0015] Furthermore, it also includes a lens bracket, an excitation light source is installed to the side of the lens bracket, a first narrow-band filter is vertically installed to the lens bracket, a dichroic mirror is obliquely installed to the lens bracket, and the dichroic mirror is located above the sample tube, a second narrow-band filter is horizontally installed to the lens bracket, and the second narrow-band filter is located above the dichroic mirror, an emission light detector is installed to the top surface of the lens bracket and is located above the second narrow-band filter, and the emission light detector is parallel to the second narrow-band filter.

[0016] Furthermore, the lens bracket includes a first bracket and a second bracket which are symmetrical to each other. The first bracket and the second bracket are respectively provided with a plurality of slots. The slots of the first bracket and the slots of the second bracket are combined to form a slot for placing the first narrow-band filter, the dichroic mirror and the second narrow-band filter.

[0017] Furthermore, the control component includes a first control board and a second control board. The second control board is used to control the operation of the light control light source, the excitation light component, the emission light detection component and the temperature control component. The emission light detection component transmits the detection signal to the second control board, and the second control board transmits the detection signal to the first control board. The first control board analyzes the detection signal and displays the detection signal on the display terminal.

[0018] Furthermore, the dichroic mirror is tilted downward in a direction away from the first narrow-band filter, and the angle between the dichroic mirror and the horizontal plane is 45°.

[0019] Another technical solution of the present invention is: the detection method of the above-mentioned light-controlled nucleic acid detection device comprises the following steps:

[0020] Step S1: Add the sample into the sample tube, and adjust the temperature to the temperature required for the amplification reaction through the temperature control component;

[0021] Step S2: The excitation light component and the emission light detection component are started simultaneously by the control component, and the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in sequence. The dichroic mirror is used to reflect the excitation light into the sample tube, and irradiate for 10-30 minutes, and then turn off the excitation light component and the emission light detection component;

[0022] Step S3: the control component controls the light-controlled light source to start, the light-controlled light source directly illuminates the sample tube for 25-35 seconds, and then turns off the light-controlled light source;

[0023] Step S4: The control component controls the excitation light component and the emission light detection component to start simultaneously, and adjusts the temperature to the temperature required for the detection reaction through the temperature control component; after the excitation light is irradiated to the sample, the fluorescent molecules in the sample are excited to emit fluorescence, and the fluorescence is detected by the emission light detection component. The emission light detection component transmits the detection signal to the control component, and the control component analyzes the detection signal and displays it on the display terminal.

[0024] Furthermore, the amplification reaction is recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, helicase-dependent isothermal amplification, strand displacement amplification, rolling circle amplification, exponential isothermal amplification, nucleic acid sequence-dependent amplification, single primer isothermal amplification technology, chimeric primer-triggered nucleic acid isothermal amplification or strand exchange amplification technology.

[0025] Furthermore, the sample is DNA or RNA.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The light-controlled nucleic acid detection device of the present invention, by providing a light-controlled light source, an excitation light component and an emission light detection component, enables sample amplification and detection to be achieved through the light-controlled nucleic acid detection device of the present application, thereby improving detection efficiency.

[0028] The light-controlled nucleic acid detection device of the present invention has a mixed reagent in a sample tube, which includes a CRISPR Cas12 detection system, an isothermal amplification system and a sample. The sample tube is placed in the detection device, and nucleic acid detection can be completed without opening the cover, thereby avoiding aerosol contamination caused by opening the cover. Compared with the existing detection method, the detection device of the present invention does not need to take out the sample tube midway, and is easy to operate and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a structural schematic diagram of a light-controlled nucleic acid detection device.

[0030] Figure 2 This is a schematic diagram of the decomposition of a light-controlled nucleic acid detection device.

[0031] Figure 3 The present invention is a schematic diagram of the structure of a sample stage of a light-controlled nucleic acid detection device.

[0032] Figure 4 This is a schematic diagram of the optical system of a light-controlled nucleic acid detection device.

[0033] Figure 5 The present invention is a schematic diagram of the control system design of a light-controlled nucleic acid detection device.

[0034] Figure 6 The present invention is a schematic diagram of a detection method of a light-controlled nucleic acid detection device.

[0035] Light-controlled light source 1, excitation light component 2, excitation light source 21, first narrow-band filter 22, dichroic mirror 23, emission light detection component 3, emission light detector 31, second narrow-band filter 32, temperature control component 4, heating tube 41, temperature sensor 42, sample stage 5, control component 6, first control board 61, second control board 62, substrate 7, lens bracket 81, nylon column 82, sample tube 83, sample addition hole 84, light inlet hole 85. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0037] Example

[0038] This embodiment provides a light-controlled nucleic acid detection device, including a light-controlled light source 1, an excitation light component 2, an emission light detection component 3, a temperature control component 4, a control component 6, a sample stage 5, a lens bracket 81 and a substrate 7.

[0039] The sample stage, control component and lens bracket are all installed on the base plate, the light control light source and temperature control component are all installed on the sample stage, and the excitation light component and emission light detection component are all installed on the lens bracket.

[0040] A sample loading hole 84 is provided on the top surface of the sample stage, and the sample tube is placed in the sample loading hole. A light inlet hole 85 is provided on the side of the sample stage, and the light inlet hole is connected to the sample loading hole. A light-controlled light source is installed in the light inlet hole, and the light-controlled light source directly illuminates the sample tube to start the detection reaction after amplification.

[0041] In one embodiment, the light control light source is preferably a 365nm monochromatic light emitting diode.

[0042] In one embodiment, the sample addition hole is a tapered hole with a larger diameter at the top and a smaller diameter at the bottom, so as to ensure that the sample tube can be placed stably.

[0043] In one embodiment, the distance between the light control light source and the sample tube is as short as possible to reduce light path loss.

[0044] The temperature control component includes a heating tube 41 and a temperature sensor 42. A blind hole is provided on the side of the sample stage. The heating tube and the temperature sensor are respectively installed in the blind hole. The heating tube and the temperature sensor are both electrically connected to the control component. The temperature sensor is used to detect the temperature information of the sample stage and transmit it to the control component. The control component controls the operation of the heating tube according to the temperature information. The heating tube is used to heat the sample stage, and the temperature sensor is used to measure the temperature of the sample stage.

[0045] In one embodiment, the sample stage is provided with a threaded hole, which is connected to a mounting blind hole for placing the heating tube. A screw passes through the threaded hole and abuts against the heating tube to limit the position of the heating tube.

[0046] In one embodiment, the temperature sensor is NTC100, which is placed in the installation blind hole and fixed by a silicone plug, so that the environment where the temperature sensor is located is sealed, ensuring that the temperature sensor is not affected by the outside world, and the temperature measurement is more accurate.

[0047] In one embodiment, when the temperature of the sample stage is lower than a preset temperature, the heating tube heats up; when the sample temperature is higher than the preset temperature, the heating is stopped to allow the sample stage to cool down naturally, and the temperature control algorithm is preferably a PID control algorithm.

[0048] The excitation light assembly includes an excitation light source 21, a first narrowband filter 22 and a dichroic mirror 23. The excitation light source, the first narrowband filter and the dichroic mirror are located on the same straight line. The dichroic mirror is tilted. The excitation light emitted by the excitation light source passes through the first narrowband filter and the dichroic mirror in sequence. The dichroic mirror is used to reflect the excitation light into the sample tube. The first narrowband filter is used to filter out other stray light emitted by the light source.

[0049] In one embodiment, the dichroic mirror is located above the sample tube, and the dichroic mirror is tilted downward in a direction away from the first narrow-band filter, and the angle between the dichroic mirror and the horizontal plane is 45°.

[0050] In one embodiment, the excitation light source is a monochromatic LED lamp.

[0051] The emission light detection assembly includes an emission light detector 31 and a second narrow-band filter 32. The light emitted by the sample in the sample tube sequentially passes through the dichroic mirror and the second narrow-band filter into the emission light detector. The second narrow-band filter is located above the dichroic mirror, and the emission light detector is located above the second narrow-band filter.

[0052] In one embodiment, the emission light detector is a photodiode, and may also be a photomultiplier tube or a CCD / CMOS chip.

[0053] In one embodiment, the excitation light source is mounted to the side of the lens holder, the first narrow-band filter is vertically mounted to the lens holder, the dichroic mirror is tiltedly mounted to the lens holder, and the dichroic mirror is located above the sample tube, the second narrow-band filter is horizontally mounted to the lens holder, and the second narrow-band filter is located above the dichroic mirror, the emission light detector is mounted to the top surface of the lens holder and is located above the second narrow-band filter, and the emission light detector is parallel to the second narrow-band filter. The optical paths of the excitation light component and the emission light detection component are limited by the lens holder.

[0054] In one embodiment, the lens bracket includes a first bracket and a second bracket that are symmetrical to each other, the first bracket and the second bracket are both fixed to the substrate, and the mating surfaces of the first bracket and the second bracket are respectively provided with a plurality of slots, and the slots of the first bracket and the slots of the second bracket are combined to form a slot, and the slot is used to place the first narrow-band filter, the dichroic mirror, and the second narrow-band filter. By providing a split first bracket and a second bracket, it is convenient to fix and remove the first narrow-band filter, the dichroic mirror, and the second narrow-band filter, and at the same time, the light path is not blocked.

[0055] The light-controlled light source, the excitation light component, the emission light detection component and the temperature control component are all electrically connected to the control component, and the control component is used to control the operation of the light-controlled light source, the excitation light component, the emission light detection component and the temperature control component.

[0056] In one embodiment, the control component includes a first control board 61 and a second control board 62. The second control board is used to control the operation of the light control light source, the excitation light component, the emission light detection component and the temperature control component. The emission light detection component transmits the detection signal to the second control board, and the second control board transmits the detection signal to the first control board. The first control board analyzes the detection signal and displays the detection signal on the display terminal.

[0057] In one embodiment, the input end of the light-controlled light source is connected to the output end of the second control board, and the second control board controls the light-controlled light source to start or shut down. The excitation light emitted by the excitation light component is used to excite the fluorescent group in the sample, and the input end of the excitation light source is connected to the output end of the second control board, and the second control board controls the excitation light source to start or shut down; the emission light detection component is used to detect the fluorescence emitted from the sample, and the emission light detection group outputs a detection signal, and the output end of the emission light detection group is connected to the input end of the second control board, and the first control board and the second control board use serial communication.

[0058] In one embodiment, a power module is further included, and the power module is used to supply power to the first control board and the second control board.

[0059] In one embodiment, a nylon column 82 is further included, one end of the nylon column is connected to the base plate, and the other end of the nylon column is connected to the first control board. The first control board and the second control board are coupled via a pin header.

[0060] The working method of the above-mentioned light-controlled nucleic acid detection device comprises the following steps:

[0061] Step S1: adding the sample into the sample tube, placing the sample tube into the sample loading hole of the loading platform, and the second control board controls the temperature control component to adjust the temperature of the loading platform to the temperature required for the amplification reaction;

[0062] Step S2: Control the excitation light component and the emission light detection component to start simultaneously through the second control board, the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in turn, the dichroic mirror is used to reflect the excitation light into the sample tube, the light emitted by the sample in the sample tube passes through the dichroic mirror and the second narrow-band filter in turn into the emission light detector, the emission light detection component transmits the detection signal to the second control board, the second control board then transmits the detection signal to the first control board, the first control board analyzes the detection signal and displays the detection signal in real time on the display terminal; irradiate for 15 minutes, turn off the excitation light component and the emission light detection component;

[0063] Step S3: The second control board controls the light-controlled light source to start, and the light-controlled light source directly irradiates the sample tube. Since the emission light detection component is turned off, the display terminal does not update the screen. The light-controlled light source is turned off for 30 seconds.

[0064] Step S4: the second control board controls the excitation light component and the emission light detection component to start at the same time, and adjusts the temperature of the sample stage to the temperature required for the detection reaction through the temperature control component; after the excitation light is irradiated to the sample, the fluorescent molecules in the sample are excited to emit fluorescence, and the fluorescence is detected by the emission light detection component, and the emission light detection component transmits the detection signal to the second control board, and the second control board transmits the detection signal to the first control board, and the first control board analyzes the detection signal and displays the detection signal in real time to the display terminal; after 15 minutes of irradiation, the reaction ends, the second control board controls the excitation light component and the emission light detection component to turn off, and the first control board analyzes all detection signals and outputs the final result to the display terminal.

[0065] The detection method of the present application does not require opening the cover or taking out the sample tube during the detection process, and the operation is convenient.

[0066] In one embodiment, the amplification reaction is recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, helicase-dependent isothermal amplification, strand displacement amplification, rolling circle amplification, exponential isothermal amplification, nucleic acid sequence-dependent amplification, single primer isothermal amplification technology, chimeric primer-triggered nucleic acid isothermal amplification or strand exchange amplification technology.

[0067] In one embodiment, the sample is DNA or RNA.

[0068] In one embodiment, the sample tube is an EP tube.

[0069] Detection principle Figure 6 As shown, the sample tube contains a mixed reagent, which includes a CRISPR Cas12 detection system, an isothermal amplification system and a sample, wherein the crRNA in the CRISPR detection system is silenced by a sequence modified with a PC-Linker, so the trans-cleavage activity of the CRISPR Cas12 enzyme will not be activated; after a period of amplification, the sample sequence is replicated on a large scale; at this time, 365nm light is applied to cut the PC-Linker, activating the trans-cleavage activity of the CRISPR Cas12 enzyme, and the CRISPR Cas12 enzyme with the activated trans-cleavage activity cuts the fluorescent probe in the system, and the cut fluorescent probe emits fluorescence.

[0070] Obviously, those skilled in the art should understand that the light-controlled light source, excitation light component, and emission light detection component in the above-mentioned embodiment of the present invention can be installed as an integrated module in a mobile platform to realize multi-channel detection, or the excitation light component and the emission light detection component can be installed as a whole on the mobile platform separately with the light-controlled light source, so as to realize the light-controlled activation of the CRISPR system and the fluorescence detection system in time-sharing, thereby further shortening the detection time while realizing multi-channel detection; the above-mentioned first control board and the second control board can be integrated into a hardware board. The above-mentioned light-controlled light source can select light-emitting devices such as LEDs, lasers, or halogen lamps with corresponding wavelengths according to different reaction systems.

[0071] As described above, the present invention can be better implemented. The above embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention; that is, all equivalent changes and modifications made according to the content of the present invention are covered by the scope of protection required by the claims of the present invention.

Claims

1. A light-controlled nucleic acid detection device, characterized in that: It includes a light-controlled light source, an excitation light component, an emission light detection component, a temperature control component and a control component; The light-controlled light source is used to directly illuminate the sample tube to cut the PC-Linker; The excitation light assembly comprises an excitation light source, a first narrow-band filter and a dichroic mirror, wherein the excitation light source, the first narrow-band filter and the dichroic mirror are located on the same straight line, the dichroic mirror is tilted, and the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in sequence, and the dichroic mirror is used to reflect the excitation light into the sample tube; The emission light detection assembly includes an emission light detector and a second narrow band filter, and the light emitted by the sample in the sample tube sequentially passes through the dichroic mirror and the second narrow band filter into the emission light detector; The temperature control component is used to adjust the reaction temperature of the sample; The light-controlled light source, the excitation light component, the emission light detection component and the temperature control component are all electrically connected to the control component, and the control component is used to control the operation of the light-controlled light source, the excitation light component, the emission light detection component and the temperature control component.

2. The light-controlled nucleic acid detection device according to claim 1, characterized in that: It also includes a sample stage, the top surface of which is provided with a sample addition hole, the sample tube is placed in the sample addition hole, the side of the sample stage is provided with a light intake hole, the light intake hole is connected with the sample addition hole, and the light control light source is installed in the light intake hole.

3. The light-controlled nucleic acid detection device according to claim 2, characterized in that: The temperature control component includes a heating tube and a temperature sensor. A mounting blind hole is provided on the side of the sample table. The heating tube and the temperature sensor are respectively installed in the mounting blind holes. The heating tube and the temperature sensor are both electrically connected to the control component. The temperature sensor is used to detect the temperature information of the sample table and transmit it to the control component. The control component controls the operation of the heating tube according to the temperature information.

4. The light-controlled nucleic acid detection device according to claim 1, characterized in that: It also includes a lens bracket, an excitation light source is installed on the side of the lens bracket, a first narrow-band filter is vertically installed on the lens bracket, a dichroic mirror is obliquely installed on the lens bracket, and the dichroic mirror is located above the sample tube, a second narrow-band filter is horizontally installed on the lens bracket, and the second narrow-band filter is located above the dichroic mirror, an emission light detector is installed on the top surface of the lens bracket and is located above the second narrow-band filter, and the emission light detector is parallel to the second narrow-band filter.

5. The light-controlled nucleic acid detection device according to claim 4, characterized in that: The lens bracket includes a first bracket and a second bracket which are symmetrical to each other. The first bracket and the second bracket are respectively provided with a plurality of slots. The slots of the first bracket and the slots of the second bracket are combined to form a slot for placing a first narrow-band filter, a dichroic mirror and a second narrow-band filter.

6. The light-controlled nucleic acid detection device according to claim 1, characterized in that: The control component includes a first control board and a second control board. The second control board is used to control the operation of the light control light source, the excitation light component, the emission light detection component and the temperature control component. The emission light detection component transmits the detection signal to the second control board, and the second control board transmits the detection signal to the first control board. The first control board analyzes the detection signal and displays the detection signal on the display terminal.

7. The light-controlled nucleic acid detection device according to claim 1, characterized in that: The dichroic mirror is tilted downward in a direction away from the first narrow-band filter, and the angle between the dichroic mirror and the horizontal plane is 45°.

8. A detection method of the light-controlled nucleic acid detection device according to any one of claims 1 to 7, characterized in that: The following steps are involved: Step S1: Add the sample into the sample tube, and adjust the temperature to the temperature required for the amplification reaction through the temperature control component; Step S2: The excitation light component and the emission light detection component are started simultaneously by the control component, and the excitation light emitted by the excitation light source passes through the first narrow-band filter and the dichroic mirror in sequence. The dichroic mirror is used to reflect the excitation light into the sample tube, and irradiate for 10-30 minutes, and then turn off the excitation light component and the emission light detection component; Step S3: The control component controls the light-controlled light source to start, and the light-controlled light source directly illuminates the sample tube for 25-35 seconds, and then turns off the light-controlled light source; Step S4: The control component controls the excitation light component and the emission light detection component to start simultaneously, and adjusts the temperature to the temperature required for the detection reaction through the temperature control component; after the excitation light is irradiated to the sample, the fluorescent molecules in the sample are excited to emit fluorescence, and the fluorescence is detected by the emission light detection component. The emission light detection component transmits the detection signal to the control component, and the control component analyzes the detection signal and displays it on the display terminal.

9. The detection method of the light-controlled nucleic acid detection device according to claim 8, characterized in that: The amplification reaction is recombinase polymerase isothermal amplification, loop-mediated isothermal amplification, helicase-dependent isothermal amplification, strand displacement amplification, rolling circle amplification, exponential isothermal amplification, nucleic acid sequence-dependent amplification, single primer isothermal amplification, nucleic acid isothermal amplification initiated by chimeric primers, or strand exchange amplification.

10. The detection method of the light-controlled nucleic acid detection device according to claim 8, characterized in that: The sample is DNA or RNA.

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