CN111808744A - Portable full-automatic nucleic acid constant temperature amplification detector - Google Patents
Portable full-automatic nucleic acid constant temperature amplification detector Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention discloses a portable full-automatic nucleic acid constant-temperature amplification detector which comprises a shading box body, wherein a drawer type structure is designed on the front side of the shading box body, a heating panel is arranged in the drawer type structure, a micro-fluidic chip groove is processed above the heating panel, a micro-fluidic chip is arranged in the micro-fluidic chip groove, the inlet end of the micro-fluidic chip is communicated with a reagent tube and a sample tube through a micro-pipeline, the outlet end of the micro-fluidic chip is communicated with a peristaltic pump through the micro-pipeline, a temperature controller for adjusting the temperature of the heating panel is connected onto the heating panel, and a photoelectric detection module is arranged above. The automatic sample introduction is realized through the peristaltic pump; in the amplification stage, the temperature is automatically controlled through a temperature control module; the detection result is processed by the photoelectric detection module and is directly sent to a mobile phone or a computer of a detector, so that the detection result is convenient to obtain; the detection process greatly reduces the manual operation process and avoids errors caused by individual differences.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a portable full-automatic nucleic acid constant-temperature amplification detector.
Background
Nucleic acid detection is one of the important means for detecting disease pathogens in laboratories at present. Among them, the Real-time fluorescent Quantitative PCR (reverse-time PCR) is the most widely used nucleic acid detection technology in recent years, in which a fluorescent group is added to a PCR reaction system, and generates a fluorescent signal after being combined with DNA, and the intensity of the fluorescent signal is in direct proportion to the number of double-stranded DNAs, so that the PCR process can be monitored in Real time by detecting the fluorescent intensity. This method is highly accurate, but still suffers from the following disadvantages: (1) an expensive RT-PCR instrument is required; (2) easily cause non-specific amplification; (3) the temperature needs to be repeatedly increased and decreased in the amplification process, and the time is too long; (4) needs a special place and has complex operation.
The loop-mediated isothermal amplification (LAMP) technology is a novel method for in vitro isothermal amplification of nucleic acid fragments. The technology utilizes two to three pairs of specially designed specific primers and DNA polymerase with displacement activity to complete chain displacement nucleic acid amplification reaction under the condition of constant temperature, reduces the influence of temperature rise and drop and non-specific amplification on amplification effect in the traditional PCR technology, has the advantages of high specificity, high sensitivity, high reaction efficiency and the like, is simple to operate, has low requirements on instruments and equipment, can meet the requirement of basic large-scale rapid diagnosis, and is successfully applied to the detection of AIDS, avian influenza and novel coronavirus in recent years.
The microfluidic chip technology is a technology for automatically completing the whole analysis process by integrating basic unit operations such as sample preparation, reaction, separation, detection and the like in a chip with a micron scale in the biochemical analysis process, has the characteristics of less required samples, low cost, high efficiency and the like, avoids pollution and loss caused by the traditional method, and has wide prospects in various fields such as medical detection, biological medicine and the like.
The invention combines the constant temperature amplification technology, the micro-fluidic chip technology and the photoelectric detection technology, develops a portable full-automatic nucleic acid constant temperature amplification detector, integrates in-vitro isothermal amplification and detection into one instrument, realizes reaction and detection integration, realizes timely and efficient visualization, improves the accuracy and sensitivity of a detection result, and avoids the possibility of missed detection and wrong detection caused by long time and low efficiency of the traditional method.
Currently, the detection of pathogen nucleic acid by using loop-mediated isothermal amplification technology can be roughly divided into the following steps: (1) extracting and purifying nucleic acid in the crude sample; (2) preparing a constant-temperature amplification reaction system, and putting the constant-temperature amplification reaction system into a constant-temperature instrument for constant-temperature amplification reaction; (3) after the reaction is finished, the reaction solution is detected by an instrument or directly observed by eyes.
For the above steps, there are disadvantages as follows:
(1) the risk of virus leakage exists in the nucleic acid extraction process, so that the requirements on laboratory level and professional quality of operators are high, and the possibility of loss and degradation of virus nucleic acid can be caused in the extraction process, so that false negative can appear in the detection result;
(2) because the LAMP reaction sensitivity is high, aerosol diffusion pollution is easily generated in the process of preparing a reaction system by the traditional tubular loop-mediated isothermal amplification, so that the detection result has a false positive rate, RNA is easily degraded in the air, and when RNA virus is detected, the detection result is easily false negative;
(3) when a metal bath, a water bath or a common PCR instrument without a hot cover is used for carrying out constant temperature reaction, the moisture of a reaction system can be evaporated, so that the reaction efficiency and result display are influenced;
(4) the current detection methods for reaction products include the following methods: agarose electrophoresis detection, magnesium pyrophosphate turbidity detection, fluorescence quantitative detection and fluorescence visual inspection. The result of the LAMP product agarose electrophoresis detection is a continuous ladder-shaped electrophoresis strip, but because the cover opening detection is needed, the pollution is easily caused. The fluorescent quantitative detection needs specific instruments and equipment, and has the disadvantages of complex operation and high cost. The magnesium pyrophosphate turbidity detection is a result judged by observing the change of the turbidity of the solution before and after the reaction, but the detection by using a turbidity detector is undoubtedly increased in cost because the visual error is too large. The fluorescence visual method is the simplest and rapid method at present, dye is added into a reaction system, a cover is not required after reaction, the reaction result can be directly judged by naked eyes, and some tiny color changes may cause judgment errors due to individual differences.
(5) In the whole detection process, the steps of nucleic acid extraction, isothermal amplification, product detection and the like are respectively carried out in different environments or instruments, and the manual operation has individual difference, so that the complexity of the operation and the possibility of sample pollution are increased.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a portable full-automatic nucleic acid constant temperature amplification detector, which solves the problems of complex operation and easy contamination of samples in the existing nucleic acid detection process.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a portable full-automatic nucleic acid constant temperature amplification detector comprises a shading box body, wherein a drawer type structure is designed on the front side of the shading box body, a heating panel is installed in the drawer type structure, a micro-fluidic chip groove is processed above the heating panel, a micro-fluidic chip is installed in the micro-fluidic chip groove, the inlet end of the micro-fluidic chip is communicated with a reagent tube and a sample tube through a micro-pipeline, the outlet end of the micro-fluidic chip is communicated with a peristaltic pump through a micro-pipeline, solution in the reagent tube and the sample tube is transmitted to each reaction chamber of the micro-fluidic chip by the power provided by the peristaltic pump, a temperature controller for adjusting the temperature of the heating panel is connected on the heating panel, the temperature controller heats the reaction chambers to a preset temperature and maintains the temperature for isothermal amplification within a preset time, a photoelectric detection module is arranged above the micro-fluidic chip, the photoelectric detection module, the photoelectric detection module comprises a CCD camera, a light source and an image information processing module, the light source provides a photographing environment for the CCD camera to acquire picture information of the microfluidic chip and transmits the information to the image information processing module for calculation processing, and the photoelectric detection module transmits a processing result to the display end and the output end through the information transmission module.
The image information processing module is used for comparing the shot image information with the information in the standard database to obtain a corresponding calculation result.
The side wall of the box body is provided with a test tube clamp used for clamping and fixing the reagent tube and the sample tube.
The heating panel is arranged on a sliding drawer type structure.
The micro-fluidic chip adopts a multi-channel parallel structure.
The heating panel is made of any one of ceramics, metals, semiconductors and silica gel.
The light source uses single/multi-wavelength laser or single/multi-color LED light.
The information transmission module adopts a USB module, a Bluetooth module or a WiFi module.
The material of the microfluidic chip is resin, plastic, silicon rubber, glass, ceramic, silicon, metal or nitrocellulose membrane.
The multichannel microfluidic chip comprises a microfluidic chip body, wherein two independent micro reaction chambers at two ends and a plurality of micro reaction chamber groups in the middle are arranged on the microfluidic chip body, the micro reaction chambers and the micro reaction chamber groups are arranged in parallel, the micro reaction chambers at two ends are negative control grooves, each micro reaction chamber group comprises three micro reaction chambers which are arranged in parallel, three micro reaction chambers from top to bottom in each micro reaction chamber group are respectively a positive control groove, a first sample groove and a second sample groove of the microfluidic chip body are filled with a primer solution of a virus and fixed in the first sample groove and the second sample groove by using a freeze drying technology, a mixed solution of a viral nucleic acid and the primer is filled and fixed in the positive control groove by using the freeze drying technology, and a solvent is filled and fixed in the negative control groove by using the freeze drying technology, the inlet and outlet positions of the two ends of the microfluidic chip body are punched through a puncher, then the chip is bonded with a glass plate to form a closed channel and a reaction chamber, hoses are inserted into the inlet and outlet positions of the microfluidic chip body, the outlet end hose is connected with a peristaltic pump, and the inlet section end hose is connected with a sample tube and a reagent tube through a split port and a single-port plug.
The method comprises the following specific steps:
(1) the heating panel is pulled out from the interior of the box body, a microfluidic chip coated with a primer in advance is placed in a microfluidic chip groove on the heating panel, a sample tube and a reagent tube are fixed in a test tube clamp on the right side of the box body, an inlet of the microfluidic chip is connected with the sample tube and the reagent tube on the right side of the box body through a micro-pipeline, an outlet of the microfluidic chip is connected with a peristaltic pump on the left side of the interior of the box body through the micro-pipeline, and a box door is closed;
(2) starting a preset or self-defined program, starting the peristaltic pump to work, and transmitting the liquid in the sample tube and the liquid in the reagent tube to each reaction chamber of the microfluidic chip through the micro-pipeline;
(3) the temperature controller heats the reaction chamber of the microfluidic chip to a preset reaction temperature by controlling the temperature of the heating panel, and maintains the temperature until the reaction program is finished;
(4) after the reaction program is finished, automatically stopping heating, simultaneously automatically starting a detection program, starting a light source, and starting a CCD camera to acquire image information of each channel;
(5) the photoelectric detection module can output the detection result to an instrument display screen or display terminals such as a mobile phone, a computer and the like through the information transmission module by calculating and processing the fluorescence or color signal values of the reaction chambers with different channels, and can be connected with a printer to print a detection report.
The invention has the beneficial effects that:
1. full-automatic: automatic sample introduction is realized through a peristaltic pump; in the amplification stage, the temperature is automatically controlled through a temperature control module; the detection result is processed by the photoelectric detection module and is directly sent to a mobile phone or a computer of a detector, so that the detection result is convenient to obtain; the detection process greatly reduces the manual operation process and avoids errors caused by individual differences.
2. A totally-enclosed reaction system: the whole reaction system is truly totally closed, evaporation and pollution of the reaction system are avoided, accuracy of detection results is effectively improved, and infection risks of detection personnel are reduced.
3. The method is portable: the whole microfluidic nucleic acid isothermal amplification detector is small in size and convenient to carry.
4. High flux: the microfluidic chip is provided with a plurality of parallel channels, so that a plurality of samples or a plurality of viruses can be detected at one time, and the detection flux is improved.
5. The detection time is short: the nucleic acid amplification and detection can be completed within 15-30min by using the detector.
6. The cost is low: by adopting the microfluidic technology, the dosage of various reagents in the reaction process is greatly reduced, and the detection cost is reduced.
Drawings
Fig. 1 is a schematic view of the overall structure of the present invention.
Fig. 2 is a schematic diagram of the principle of the present invention.
Fig. 3 is a schematic structural diagram of a microfluidic chip according to the present invention.
Graphic notation: 1. the device comprises a display end, 2, a heating panel, 3, a microfluidic chip, 301, an outlet end, 302, a negative control groove, 303, a positive control groove, 304, a sample groove, 305, an inlet end, 4, a peristaltic pump, 5, a photoelectric detection module, 6, a micro-pipeline, 7, a reagent tube, 8, a sample tube, 9, a temperature controller, 10 and a box body.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A portable full-automatic nucleic acid constant temperature amplification detector comprises a shading box body 10, a heating panel 2 is arranged in the box body 10, a micro-fluidic chip groove is processed above the heating panel 2, a micro-fluidic chip 3 is arranged in the micro-fluidic chip groove, an inlet end 305 of the micro-fluidic chip 3 is communicated with a reagent tube 7 and a sample tube 8 through a micro-pipeline 6, an outlet end 301 of the micro-fluidic chip is communicated with a peristaltic pump 4 through a micro-pipeline, solutions in the reagent tube 7 and the sample tube 8 are transmitted to each reaction chamber of the micro-fluidic chip through power provided by the peristaltic pump 4, a temperature controller 9 for adjusting the temperature of the heating panel is connected on the heating panel 2, the reaction chamber is heated to a preset temperature by the temperature controller 9 and is maintained at the preset temperature for isothermal amplification for a preset time, a photoelectric detection module 5 is arranged above the micro-fluidic chip 3, and the photoelectric detection module 5 collects fluorescence or color, the photoelectric detection module comprises a CCD camera, a light source and an image information processing module, the light source provides a photographing environment for the CCD camera to acquire picture information of the microfluidic chip and transmits the information to the image information processing module for calculation processing, and the photoelectric detection module transmits a processing result to the display end and the output end through the information transmission module.
The image information processing module is used for comparing the shot image information with the information in the standard database to obtain a corresponding calculation result.
The side wall of the box body 10 is provided with a test tube clamp for clamping and fixing the reagent tube 7 and the sample tube 8.
The heating panel 2 is arranged on a sliding drawer type structure.
The micro-fluidic chip adopts a multi-channel parallel structure.
The heating panel is made of any one of ceramics, metals, semiconductors and silica gel.
The light source adopts monochromatic LED light.
The information transmission module adopts a WiFi module.
The material of the microfluidic chip is resin.
The multichannel microfluidic chip comprises a microfluidic chip body, wherein two independent micro reaction chambers at two ends and five groups of micro reaction chamber groups in the middle are arranged on the microfluidic chip body, the micro reaction chambers and the micro reaction chamber groups are arranged in parallel, the micro reaction chambers at two ends are negative control grooves 301, each micro reaction chamber group comprises three micro reaction chambers which are arranged in parallel, the three micro reaction chambers from top to bottom in each micro reaction chamber group are respectively a positive control groove 303 and a sample groove 304, the sample groove 304 comprises a first sample groove and a second sample groove, a virus primer solution is injected and fixed in the first sample groove and the second sample groove of the microfluidic chip body by using a freeze drying technology, a virus nucleic acid and primer mixed solution is injected and fixed in the positive control groove 303 by using the freeze drying technology, a solvent is injected and fixed in a negative control groove 301 by using a freeze drying technology, holes are punched at the inlet and outlet positions at two ends of a microfluidic chip body through a puncher, then the chip is bonded with a glass plate to form a closed channel and a reaction chamber, hoses are inserted at the inlet and outlet positions of the microfluidic chip body, an outlet end hose is connected with a peristaltic pump, an inlet section end hose is connected with a sample tube and a reagent tube through a split port and a single-port plug, and the microfluidic chip can be used for detecting five different viruses and the same virus to carry out multi-parallel detection.
The method comprises the following specific steps:
(1) the heating panel is pulled out from the interior of the box body 10, the microfluidic chip 3 coated with the primers in advance is placed in a microfluidic chip groove on the heating panel, a sample tube 8 and a reagent tube 7 are fixed in a test tube clamp on the right side of the box body 10, the inlet end of the microfluidic chip 3 is connected with the sample tube and the reagent tube on the right side of the box body through a micro-pipeline, the outlet of the microfluidic chip is connected with a peristaltic pump on the left side of the interior of the box body through the micro-pipeline, and a box door is closed;
(2) starting a preset or self-defined program, starting the peristaltic pump 4 to work, and transmitting the liquid in the sample tube 8 and the liquid in the reagent tube 7 to each reaction chamber of the microfluidic chip 3 through a micro-pipeline;
(3) the temperature controller heats the reaction chamber of the microfluidic chip 3 to a preset reaction temperature by controlling the temperature of the heating panel, and maintains the temperature until the reaction program is finished;
(4) after the reaction program is finished, automatically stopping heating, simultaneously automatically starting a detection program, starting a light source, and starting a CCD camera to acquire image information of each channel;
(5) the photoelectric detection module 5 can output the detection result to the instrument display screen or the display end 4 of a mobile phone, a computer and the like through the information transmission module by calculating and processing the fluorescence or color signal values of the reaction chambers with different channels, and can be connected with a printer to print a detection report.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. The utility model provides a portable full-automatic nucleic acid constant temperature amplifications detector which characterized in that: the device comprises a shading box body, wherein a drawer-type structure is designed on the front surface of the box body, a heating panel is installed in the drawer-type structure, a micro-fluidic chip groove is processed above the heating panel, a micro-fluidic chip is installed in the micro-fluidic chip groove, the inlet end of the micro-fluidic chip is communicated with a reagent tube and a sample tube through a micro-pipeline, the outlet end of the micro-fluidic chip is communicated with a peristaltic pump through a micro-pipeline, solutions in the reagent tube and the sample tube are transmitted to each reaction chamber of the micro-fluidic chip by the power provided by the peristaltic pump, a temperature controller for adjusting the temperature of the heating panel is connected onto the heating panel, the temperature controller heats the reaction chamber to a preset temperature and maintains the temperature for isothermal amplification in a preset time, a photoelectric detection module is arranged above the micro-fluidic chip and comprises a CCD camera, a light source and an image information processing module, the light source provides picture information of the micro-fluidic chip And calculating, wherein the photoelectric detection module transmits the processing result to the display end and the output end through the information transmission module.
2. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the image information processing module is used for comparing the shot image information with the information in the standard database to obtain a corresponding calculation result.
3. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the side wall of the box body is provided with a test tube clamp used for clamping and fixing the reagent tube and the sample tube.
4. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the heating panel is arranged on a sliding drawer type structure.
5. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the micro-fluidic chip adopts a multi-channel parallel structure.
6. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the heating panel is made of any one of ceramics, metals, semiconductors and silica gel.
7. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the light source uses single/multi-wavelength laser or single/multi-color LED light.
8. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the information transmission module adopts a USB module, a Bluetooth module or a WiFi module.
9. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the material of the microfluidic chip is resin, plastic, silicon rubber, glass, ceramic, silicon, metal or nitrocellulose membrane.
10. The portable full-automatic isothermal nucleic acid amplification detector according to claim 1, characterized in that: the multichannel microfluidic chip comprises a microfluidic chip body, wherein two independent micro reaction chambers at two ends and a plurality of micro reaction chamber groups in the middle are arranged on the microfluidic chip body, the micro reaction chambers and the micro reaction chamber groups are arranged in parallel, the micro reaction chambers at two ends are negative control grooves, each micro reaction chamber group comprises three micro reaction chambers which are arranged in parallel, three micro reaction chambers from top to bottom in each micro reaction chamber group are respectively a positive control groove, a first sample groove and a second sample groove of the microfluidic chip body are filled with a primer solution of a virus and fixed in the first sample groove and the second sample groove by using a freeze drying technology, a mixed solution of a viral nucleic acid and the primer is filled and fixed in the positive control groove by using the freeze drying technology, and a solvent is filled and fixed in the negative control groove by using the freeze drying technology, the inlet and outlet positions of the two ends of the microfluidic chip body are punched through a puncher, then the chip is bonded with a glass plate to form a closed channel and a reaction chamber, hoses are inserted into the inlet and outlet positions of the microfluidic chip body, the outlet end hose is connected with a peristaltic pump, and the inlet section end hose is connected with a sample tube and a reagent tube through a split port and a single-port plug.
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Application publication date: 20201023 |