CN114672411A - Light-operated nucleic acid detection device and detection method - Google Patents
Light-operated nucleic acid detection device and detection method Download PDFInfo
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Abstract
The invention discloses a light-operated nucleic acid detection device and a detection method, wherein the detection device comprises a light-operated light source, an exciting light component, an emitting light detection component, a temperature control component and a control component; the light-operated light source is used for directly irradiating the sample tube and starting the detection reaction after amplification; the excitation light assembly comprises an excitation light source, a first narrow-band light filter and a dichroic mirror, the excitation light source, the first narrow-band light filter and the dichroic mirror are positioned on the same straight line, the dichroic mirror is obliquely arranged, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band light filter and the dichroic mirror, and the dichroic mirror is used for reflecting the excitation light into the sample tube; the emission light detection assembly comprises an emission light detector and a second narrow-band filter, and light emitted by the sample in the sample tube enters the emission light detector through the dichroic mirror and the second narrow-band filter in sequence; the temperature control component is used for adjusting the reaction temperature of the sample; the control component is used for controlling the light-operated light source, the exciting light component, the emitted light detection component and the temperature control component to work.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection instruments, in particular to a light-operated nucleic acid detection device and a detection method.
Background
The nucleic acid detection technology is an effective means for preventing and controlling infectious diseases, and besides real-time fluorescence quantitative PCR (polymerase chain reaction) serving as a gold standard, a plurality of isothermal amplification detection methods and a nucleic acid detection technology combined with CRISPR (clustered regularly interspaced short palindromic repeats) appear at present, wherein the CRISPR technology is considered as a next-generation nucleic acid detection technology. Traditional CRISPR detection techniques require the addition of amplification products to the CRISPR system after amplification, a process that involves decapping and therefore potentially causes aerosol contamination, leading to false positives.
One solution to prevent aerosol contamination is to mix the sample, the amplification system, and the CRISPR detection system in one tube, avoiding sampling while uncapping during the detection process. In the one-tube detection method, the CRISPR detection system continuously identifies and cuts the amplification template and the amplification product from the beginning to the end of the amplification reaction, so that the CRISPR detection system and the amplification system have a mutual competition relationship, the amplification efficiency is reduced, and the high-sensitivity nucleic acid detection cannot be realized.
In order to realize high-sensitivity one-tube CRISPR nucleic acid detection, one method is to silence a CRISPR detection system in a sample, an amplification system and a CRISPR detection system, so that the reduction of the amplification efficiency caused by cutting an amplification template in an amplification stage is avoided; after the amplification reaction is finished, the CRISPR detection system is activated by light with a specific wavelength, and then high-sensitivity nucleic acid detection is carried out. However, there is no integrated optically controlled CRISPR detection instrument available on the market that can implement automated program operation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a light-operated nucleic acid detection device, and solves the problem that the existing CRISPR nucleic acid detection device cannot realize high-efficiency integration and closed-tube nucleic acid detection.
Another object of the present invention is to provide a detection method of the above light-operated nucleic acid detection device.
The technical scheme of the invention is as follows: a light-operated nucleic acid detection device comprises a light-operated light source, an exciting light component, an emitting light detection component, a temperature control component and a control component;
the light-operated light source is used for directly irradiating the sample tube;
the excitation light assembly comprises an excitation light source, a first narrow-band light filter and a dichroic mirror, the excitation light source, the first narrow-band light filter and the dichroic mirror are positioned on the same straight line, the dichroic mirror is obliquely arranged, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band light filter and the dichroic mirror, and the dichroic mirror is used for reflecting the excitation light into the sample tube;
the emission light detection assembly comprises an emission light detector and a second narrow-band filter, and light emitted by a sample in the sample tube enters the emission light detector through the dichroic mirror and the second narrow-band filter in sequence;
the temperature control component is used for adjusting the reaction temperature of the sample;
the light-operated light source, the exciting light component, the emission light detection component and the temperature control component are electrically connected with the control component, and the control component is used for controlling the light-operated light source, the exciting light component, the emission light detection component and the temperature control component to work.
Further, still include the sample platform, the top surface of sample platform is equipped with the application of sample hole, and the sample cell is placed in the application of sample hole, and the side of sample platform is equipped with into light through-hole, advances light through-hole and application of sample hole intercommunication, and light-operated light source installs to advancing light through-hole.
Further, temperature control unit includes heating tube and temperature sensor, the side of sample platform is equipped with the installation blind hole, and heating tube and temperature sensor install respectively to the installation blind hole in, heating tube and temperature sensor all with control assembly electric connection, and temperature sensor is used for detecting the temperature information of sample platform and transmits to control assembly, and control assembly controls the heating tube work according to temperature information.
The excitation light source is installed on the side face of the lens support, the first narrow-band optical filter is vertically installed on the lens support, the dichroic mirror is installed on the lens support in an inclined mode and located above the sample tube, the second narrow-band optical filter is horizontally installed on the lens support and located above the dichroic mirror, the emission light detector is installed on the top face of the lens support and located above the second narrow-band optical filter, and the emission light detector is parallel to the second narrow-band optical filter.
Further, the lens support comprises a first support and a second support which are symmetrical to each other, the first support and the second support are respectively provided with a plurality of grooves, the grooves of the first support and the grooves of the second support surround to form clamping grooves, and the clamping grooves are used for placing the first narrow-band optical filter, the dichroic mirror and the second narrow-band optical filter.
Further, the control assembly comprises a first control panel and a second control panel, the second control panel is used for controlling the light-operated light source, the exciting light assembly, the emitting light detection assembly and the temperature control assembly to work, the emitting light detection assembly transmits the detection signal to the second control panel, the second control panel transmits the detection signal to the first control panel, and the first control panel analyzes the detection signal and displays the detection signal to the display terminal.
Further, the dichroic mirror is arranged in a downward inclination manner towards a direction far away from the first narrow-band filter, and an included angle between the dichroic mirror and a horizontal plane is 45 degrees.
The other technical scheme of the invention is as follows: the detection method of the light-operated nucleic acid detection device comprises the following steps:
step S1: adding a sample into a sample tube, and regulating the temperature to the temperature required by the amplification reaction through a temperature control assembly;
step S2: the control assembly controls the excitation light assembly and the emission light detection assembly to be started simultaneously, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band filter and the dichroic mirror, the dichroic mirror is used for reflecting the excitation light into the sample tube, irradiating for 10-30 minutes, and closing the excitation light assembly and the emission light detection assembly;
step S3: the control component controls the light-operated light source to start, the light-operated light source directly irradiates the sample tube for 25-35 seconds, and the light-operated light source is closed;
step S4: the control component controls the excitation light component and the emission light detection component to start simultaneously, and the temperature is adjusted to the temperature required by the detection reaction through the temperature control component; after the exciting light irradiates the sample, fluorescent molecules in the sample are excited to emit fluorescence, the fluorescence is detected by the emitted light detection assembly, the emitted light detection assembly transmits a detection signal to the control assembly, and the control assembly analyzes the detection signal and displays the detection signal to the display terminal.
Further, 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-initiated nucleic acid isothermal amplification or strand exchange amplification technology.
Further, the sample is DNA or RNA.
Compared with the prior art, the invention has the following beneficial effects:
according to the light-operated nucleic acid detection device, the light-operated light source, the exciting light assembly and the emitted light detection assembly are arranged, so that sample amplification and detection can be realized through the light-operated nucleic acid detection device, and the detection efficiency is improved.
According to the light-operated nucleic acid detection device, the mixed reagent is filled in the sample tube, the CRISPR Cas12 detection system, the isothermal amplification system and the sample are contained in the mixed reagent, the sample tube is placed in the detection device, nucleic acid detection can be completed without opening a cover, aerosol pollution caused by opening the cover is avoided, and compared with the existing detection mode, the light-operated nucleic acid detection device disclosed by the invention is free from taking out the sample tube midway, is convenient to operate and has high efficiency.
Drawings
FIG. 1 is a schematic structural diagram of a light-operated nucleic acid detecting device.
FIG. 2 is an exploded view of a light-operated nucleic acid detecting device.
FIG. 3 is a schematic diagram of a sample stage of a light-operated nucleic acid detecting apparatus.
FIG. 4 is a schematic diagram of an optical system of a light-operated nucleic acid detecting device.
FIG. 5 is a schematic diagram of the control system of an optically controlled nucleic acid detection device.
FIG. 6 is a schematic diagram of a detection method of an optically controlled nucleic acid detection device.
The device comprises a light-operated light source 1, an excitation light assembly 2, an excitation light source 21, a first narrowband filter 22, a dichroic mirror 23, an emission light detection assembly 3, an emission light detector 31, a second narrowband filter 32, a temperature control assembly 4, a heating tube 41, a temperature sensor 42, a sample stage 5, a control assembly 6, a first control plate 61, a second control plate 62, a substrate 7, a lens support 81, a nylon column 82, a sample tube 83, a sample adding hole 84 and a light inlet through hole 85.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Examples
The embodiment provides a light-operated nucleic acid detection device, which comprises a light-operated light source 1, an excitation light assembly 2, an emission light detection assembly 3, a temperature control assembly 4, a control assembly 6, a sample stage 5, a lens support 81 and a substrate 7.
Sample platform, control assembly and lens support all install to the base plate, and light-operated light source and temperature control assembly all install to the sample platform, and exciting light subassembly, emission light detection subassembly all install to the lens support.
The top surface of the sample table is provided with a sample adding hole 84, the sample tube is placed in the sample adding hole, the side surface of the sample table is provided with a light inlet through hole 85, the light inlet through hole is communicated with the sample adding hole, the light-operated light source is installed in the light inlet through hole and directly irradiates the sample tube, and detection reaction after amplification is started.
In one embodiment, the light-controlled light source is preferably a 365nm monochromatic light emitting diode.
In one embodiment, the sample adding hole is a conical hole with a large diameter at the top and a small diameter at the bottom, so that the sample tube can be stably placed.
In one embodiment, the distance between the optically controlled light source and the sample tube is as short as possible to reduce optical path loss.
The temperature control assembly comprises a heating tube 41 and a temperature sensor 42, an installation blind hole is formed in the side face of the sample table, the heating tube and the temperature sensor are installed in the installation blind hole respectively, the heating tube and the temperature sensor are electrically connected with the control assembly, the temperature sensor is used for detecting temperature information of the sample table and transmitting the temperature information to the control assembly, and the control assembly controls the heating tube to work according to the temperature information. The heating tube is used for heating the sample table, and the temperature sensor is used for measuring the temperature of the sample table.
In one embodiment, the sample platform is provided with a threaded hole, the threaded hole is communicated with a mounting blind hole for placing the heating tube, and the screw penetrates through the threaded hole to abut against the heating tube to limit the heating tube.
In one embodiment, the temperature sensor is an NTC100, and the temperature sensor is placed in the mounting blind hole and fixed by a silica gel plug. The environment where the temperature sensor is located is closed, the temperature sensor is not affected by the outside, and the temperature measurement is more accurate.
In one embodiment, when the temperature of the sample table is lower than a preset temperature, the heating tube is heated; and when the temperature of the sample is higher than the preset temperature, stopping heating, naturally cooling the sample table, wherein the temperature control algorithm is preferably a PID control algorithm.
The exciting light component comprises an exciting light source 21, a first narrow-band filter 22 and a dichroic mirror 23, the exciting light source, the first narrow-band filter and the dichroic mirror are located on the same straight line, the dichroic mirror is obliquely arranged, exciting light emitted by the exciting light source sequentially passes through the first narrow-band filter and the dichroic mirror, and the dichroic mirror is used for reflecting the exciting light into the sample tube. The first narrow-band filter is used for filtering other stray light emitted by the light source.
In one embodiment, the dichroic mirror is located above the sample tube, the dichroic mirror is arranged to be inclined downwards in a direction away from the first narrow-band filter, and an included angle between the dichroic mirror and the horizontal plane is 45 °.
In one embodiment, the excitation light source is a monochromatic LED lamp.
The emission light detection assembly comprises an emission light detector 31 and a second narrow band filter 32, and light emitted by the sample in the sample tube enters the emission light detector through the dichroic mirror and the second narrow band filter in sequence. The second narrowband filter is positioned above the dichroic mirror, and the emission light detector is positioned above the second narrowband filter.
In one embodiment, the emission photodetector is a photodiode, which may also be a photo-amplifier or a CCD/CMOS chip.
In one embodiment, the excitation light source is mounted to a side of the lens support, the first narrowband filter is vertically mounted to the lens support, the dichroic mirror is obliquely mounted to the lens support, the dichroic mirror is located above the sample tube, the second narrowband filter is horizontally mounted to the lens support, the second narrowband filter is located above the dichroic mirror, the emission light detector is mounted to a top surface of the lens support and above the second narrowband filter, and the emission light detector is parallel to the second narrowband filter. The light paths of the excitation light assembly and the emitted light detection assembly are limited through the lens support.
In one embodiment, the lens support includes a first support and a second support that are symmetrical to each other, the first support and the second support are both fixed to the substrate, a plurality of slots are respectively provided on mating surfaces of the first support and the second support, the slots of the first support and the slots of the second support surround to form a slot, and the slot is used for placing the first narrowband optical filter, the dichroic mirror and the second narrowband optical filter. Through setting up split type first support and second support, make things convenient for first narrowband filter, dichroic mirror and second narrowband filter's fixed and take out, do not shelter from the light path simultaneously.
The light-operated light source, the exciting light component, the emission light detection component and the temperature control component are electrically connected with the control component, and the control component is used for controlling the light-operated light source, the exciting light component, the emission light detection component and the temperature control component to work.
In one embodiment, the control assembly includes a first control board 61 and a second control board 62, the second control board is used for controlling the operation of the light-controlled light source, the exciting light assembly, the emitted light detection assembly and the temperature control assembly, the emitted light detection assembly transmits the detection signal to the second control board, 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 to the display terminal.
In one embodiment, the input end of the light-operated light source is connected with the output end of the second control panel, and the second control panel controls the light-operated light source to be started or stopped. The exciting light emitted by the exciting light assembly is used for exciting the fluorescent group in the sample, the input end of the exciting light source is connected with the output end of the second control board, and the second control board controls the exciting light source to be started or closed; the emission light detection assembly is used for detecting fluorescence emitted in a sample, the emission light detection assembly outputs a detection signal, the output end of the emission light detection assembly is connected with the input end of the second control board, and the first control board and the second control board are communicated through a serial port.
In one embodiment, the power supply module is further included and is used for supplying power to the first control board and the second control board.
In one embodiment, a nylon column 82 is further included, one end of the nylon column is connected to the base plate, the other end of the nylon column is connected to the first control board, and the first control board and the second control board are coupled by a pin header.
The working method of the light-operated nucleic acid detection device comprises the following steps:
step S1: adding a sample into a sample tube, placing the sample tube in a sample adding hole of a sample adding table, and controlling a temperature control assembly to adjust the temperature of the sample adding table to a temperature required by amplification reaction by a second control board;
step S2: the excitation light component and the emission light detection component are controlled to be started simultaneously through the second control board, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band light filter and the dichroic mirror, the dichroic mirror is used for reflecting the excitation light into the sample tube, the light emitted by the sample in the sample tube sequentially passes through the dichroic mirror and the second narrow-band light filter and enters the emission light detector, the emission light detection component transmits a detection signal to the second control board, 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 to the display terminal in real time; irradiating for 15 minutes, and turning off the exciting light component and the emitted light detection component;
step S3: the second control panel controls the light-operated light source to be started, the light-operated light source directly irradiates the sample tube, and the light-operated light source is turned off after the display terminal does not update the picture and irradiates for 30 seconds as the emission light detection assembly is turned off;
step S4: the second control panel controls the excitation light assembly and the emission light detection assembly to be started simultaneously, and adjusts the temperature of the sample stage to the temperature required by the detection reaction through the temperature control assembly; after the exciting light irradiates the sample, fluorescent molecules in the sample are excited to emit fluorescence, the fluorescence is detected by the emitted light detection assembly, the emitted light detection assembly transmits a detection signal to the second control board, 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 to the display terminal in real time; and after the irradiation is carried out for 15 minutes, the reaction is finished, the second control board controls the excitation light assembly and the emission light detection assembly to be closed, the first control board analyzes all detection signals, and a final result is output to the display terminal.
According to the detection method, a cover does not need to be opened in the detection process, the sample tube does not need to be taken out, and the operation is convenient and fast.
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 technique, chimeric primer-primed nucleic acid isothermal amplification, or strand-exchange amplification technique.
In one embodiment, the sample is DNA or RNA.
In one embodiment, the sample tube is an EP tube.
The detection principle is shown in fig. 6, the sample tube contains a mixed reagent, and the mixed reagent contains a CRISPR Cas12 detection system, an isothermal amplification system and a sample, wherein crRNA in the CRISPR detection system is modified with sequence silencing of PC-Linker, so that the trans-cleavage activity of the CRISPR Cas12 enzyme is not activated; after a period of amplification, the sample sequence is replicated in large scale; at the moment, 365nm light is applied to cut the PC-Linker, the trans-cutting activity of the CRISPR Cas12 enzyme is activated, the fluorescent probe in the CRISPR Cas12 enzyme cutting system with the trans-cutting activity is activated, and the cut fluorescent probe emits fluorescence.
Obviously, it should be understood by those skilled in the art that, in the above-described embodiment of the present invention, the light-controlled light source, the excitation light module, and the emission light detection module may be installed as an integrated module in the mobile platform to implement multi-channel detection, or the excitation light module and the emission light detection module may be integrated and installed with the light-controlled light source to the mobile platform respectively to implement the light-controlled activation CRISPR system and the fluorescence detection system in a time-sharing manner, so as to further shorten the detection time while implementing multi-channel detection; the first control board and the second control board can be integrated into a hardware board card. The light-operated light source can select light-emitting devices such as LEDs, lasers or halogen lamps with corresponding wavelengths according to different reaction systems.
As mentioned above, the present invention can be better realized, and the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; all equivalent changes and modifications made according to the present disclosure are intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. A light-operated nucleic acid detection device is characterized by comprising a light-operated light source, an exciting light component, an emitting light detection component, a temperature control component and a control component;
the light-operated light source is used for directly irradiating the sample tube;
the excitation light assembly comprises an excitation light source, a first narrow-band light filter and a dichroic mirror, the excitation light source, the first narrow-band light filter and the dichroic mirror are positioned on the same straight line, the dichroic mirror is obliquely arranged, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band light filter and the dichroic mirror, and the dichroic mirror is used for reflecting the excitation light into the sample tube;
the emission light detection assembly comprises an emission light detector and a second narrow-band filter, and light emitted by a sample in the sample tube enters the emission light detector through the dichroic mirror and the second narrow-band filter in sequence;
the temperature control component is used for adjusting the reaction temperature of the sample;
the light-operated light source, the exciting light component, the emission light detection component and the temperature control component are electrically connected with the control component, and the control component is used for controlling the light-operated light source, the exciting light component, the emission light detection component and the temperature control component to work.
2. The light-operated nucleic acid detecting device according to claim 1, further comprising a sample stage, wherein a sample hole is formed in a top surface of the sample stage, the sample tube is placed in the sample hole, a light inlet through hole is formed in a side surface of the sample stage, the light inlet through hole is communicated with the sample hole, and the light-operated light source is installed in the light inlet through hole.
3. The light-operated nucleic acid detecting device according to claim 2, wherein the temperature control assembly comprises a heating tube and a temperature sensor, a blind mounting hole is formed in a side surface of the sample stage, the heating tube and the temperature sensor are respectively mounted in the blind mounting hole, the heating tube and the temperature sensor are both electrically connected with the control assembly, the temperature sensor is used for detecting temperature information of the sample stage and transmitting the temperature information to the control assembly, and the control assembly controls the heating tube to operate according to the temperature information.
4. The light-operated nucleic acid detecting device according to claim 1, further comprising a lens holder, wherein the excitation light source is mounted to a side surface of the lens holder, the first narrow-band filter is vertically mounted to the lens holder, the dichroic mirror is obliquely mounted to the lens holder, the dichroic mirror is located above the sample tube, the second narrow-band filter is horizontally mounted to the lens holder, the second narrow-band filter is located above the dichroic mirror, the emission photodetector is mounted to a top surface of the lens holder and located above the second narrow-band filter, and the emission photodetector is parallel to the second narrow-band filter.
5. The light-operated nucleic acid detecting device according to claim 4, wherein the lens holder includes a first holder and a second holder that are symmetrical to each other, the first holder and the second holder are respectively provided with a plurality of slots, the slots of the first holder and the slots of the second holder surround to form a slot, and the slot is used for placing the first narrow-band filter, the dichroic mirror and the second narrow-band filter.
6. The light-operated nucleic acid detecting device according to claim 1, wherein the control unit comprises a first control board and a second control board, the second control board is configured to control the operation of the light-operated light source, the excitation light unit, the emission light detecting unit and the temperature control unit, the emission light detecting unit transmits the detection signal to the second control board, 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 on the display terminal.
7. The light-operated nucleic acid detecting device according to claim 1, wherein the dichroic mirror is disposed to be inclined 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. The method for detecting the light-operated nucleic acid detection device as claimed in any one of claims 1 to 7, which comprises the following steps:
step S1: adding a sample into a sample tube, and regulating the temperature to the temperature required by the amplification reaction through a temperature control assembly;
step S2: the control assembly controls the excitation light assembly and the emission light detection assembly to be started simultaneously, the excitation light emitted by the excitation light source sequentially passes through the first narrow-band filter and the dichroic mirror, the dichroic mirror is used for reflecting the excitation light into the sample tube, irradiating for 10-30 minutes, and closing the excitation light assembly and the emission light detection assembly;
step S3: the control component controls the light-operated light source to start, the light-operated light source directly irradiates the sample tube for 25-35 seconds, and the light-operated light source is closed;
step S4: the control component controls the excitation light component and the emission light detection component to start simultaneously, and the temperature is adjusted to the temperature required by the detection reaction through the temperature control component; after the exciting light irradiates the sample, fluorescent molecules in the sample are excited to emit fluorescence, the fluorescence is detected by the emitted light detection assembly, the emitted light detection assembly transmits a detection signal to the control assembly, and the control assembly analyzes the detection signal and displays the detection signal to the display terminal.
9. The detection method of the light-operated nucleic acid detection device according to claim 8, wherein 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-initiated nucleic acid isothermal amplification, or strand exchange amplification technology.
10. The method for detecting the light-operated nucleic acid detecting device according to claim 8, wherein the sample is DNA or RNA.
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| CN202210451446.0A CN114672411A (en) | 2022-04-27 | 2022-04-27 | Light-operated nucleic acid detection device and detection method |
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| CN202210451446.0A CN114672411A (en) | 2022-04-27 | 2022-04-27 | Light-operated nucleic acid detection device and detection method |
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