CN220166182U - Nucleic acid detection device based on RPA (reactive power amplification) combined CRISPR/Cas12 isothermal amplification technology - Google Patents

Abstract

The utility model belongs to the field of molecular biotechnology and molecular diagnosis medical detection, and provides a nucleic acid detection device based on an RPA (reactive protein amplification) combined CRISPR/Cas12 isothermal amplification technology, which comprises an integral appearance and a three-dimensional structure, a sample acquisition inactivating device, a sample acquisition flow diagram, a centrifugal microfluidic chip disc control sample adding device, an RPA amplifying device, a CRISPR/Cas12 signal amplifying device and a visual chromatographic test strip detection device, wherein the POCT device integrates sample acquisition inactivating, nucleic acid extraction-free, microfluidic chip control RPA isothermal amplification, CRISPR/Cas12 amplification and visual chromatographic test strip detection, so that the requirement of using complex instruments for detection results is greatly simplified, the requirement of instant detection on quick result can be met, and meanwhile, the region without molecular detection conditions can be better met.

Description

Nucleic acid detection device based on RPA (reactive power amplification) combined CRISPR/Cas12 isothermal amplification technology

Technical Field

The utility model belongs to the field Of molecular biotechnology and molecular diagnosis medical detection, in particular relates to a Point-Of-Care Testing (POCT) device, and particularly relates to a nucleic acid detection device based on a constant temperature amplification technology Of RPA combined CRISPR/Cas 12.

Background

POCT refers to clinical and Bedside tests (Bedside Testing) performed beside a patient, and is not usually performed by a clinical inspector. The method is a new method for immediately analyzing the sample on the sampling site, omitting a complex processing program of the sample during laboratory test and rapidly obtaining a test result.

The on-site rapid test is uniformly named by the POCT equipment technical professional committee of the chinese medical equipment association on the basis of multiple expert demonstrations and is defined as: a detection mode which is carried out on a sampling site and rapidly obtains a detection result by using a portable analytical instrument and a matched reagent.

POCT meaning can be understood from two aspects: spatially, the test is performed at the patient's side, i.e. "bedside test"; in time, a "point-in-time test" may be performed. The main standard of POCT is that no fixed detection sites are needed, both reagents and instruments are portable and can be handled in time. POCT does not require specialized clinical testing services and specialized professionals.

A good molecular POCT product is first satisfactory for use off a PCR compartmentalized laboratory. This means that the nucleic acid extraction and nucleic acid amplification functions are integrated on the detection consumable of the molecular POCT product. More importantly, the whole implementation process is fully closed to avoid aerosol contamination of the PCR products to the maximum extent without a PCR compartmentalized laboratory.

Molecular diagnostic techniques have been recently put into great attention by virtue of their efforts, and isothermal amplification techniques have been attracting attention because they do not require special equipment during the amplification. RPA (Recombinase Polymerase Amplification) recombinase polymerase amplification technique, the key technique is 3 key enzymes or proteins (recombinase, single-chain binding protein and DNA polymerase, wherein the recombinase is derived from T4 phage), and the key points of the working principle are that:

(1) Combining a recombinase with a primer in the presence of adenosine triphosphate ATP to form a protein-nucleic acid polymer, and searching for a pairing target;

(2) When the primer finds a paired DNA template, ATP hydrolyzes to supply energy, and the recombinase leaves the primer and synthesizes a new DNA chain under the action of DNA polymerase;

(3) Meanwhile, the single-stranded DNA binding protein (Single Stranded DNA Binding, SSB) binds to the replaced DNA single strand, preventing the DNA template from forming double strands again.

The CRISPR system is used as an emerging gene editing and regulating tool, has high specificity, sensitivity and flexibility, has important functions in gene editing and transformation, and has good prospects in the aspects of infectious disease diagnosis, environment monitoring, food safety detection and the like. With the intensive research of CRISPR/Cas systems, a plurality of simple and sensitive biological sensing platforms are established through the high specificity recognition target sequence and trans-cleavage capability, and the defects of the traditional method in terms of operation, sensitivity, specificity and detection period are overcome.

The CRISPR/Cas system is expected to be a method for developing ideal diagnostic products due to the fact that excellent biological sensing performance gradually becomes a focus of next-generation diagnostic technology, detection of various target molecules has been successfully achieved based on the specific binding activity and high-efficiency trans-cleavage capability of the CRISPR/Cas system and the combination of electrochemical and fluorescent sensing technologies, detection objects are also expanded from initial nucleic acids to metal ions, proteins, bacteria, small molecules and the like, and the CRISPR/Cas system is indicated to have universality in the detection field.

The concept of microfluidic technology has been proposed for more than 30 years, with "micro total analysis system" (μ -TAS) and "lab-on-a-chip" (LOC) being the dominant forms. With the increasingly mature microfluidic technology, the method gradually enters the product commercialized research and development stage from the early-stage technical attack, has the advantages of less reagent consumption, high detection speed, easy integration and the like, and provides a huge development opportunity for the method in the field of clinical examination.

The microfluidic technology refers to a technology for accurately controlling micro-level fluid under the micrometer scale, can realize that a plurality of experimental processes are automatically completed on one chip, and has the advantages of low consumption, high efficiency, automation, integration, portability and the like. Microfluidic platforms have advanced CRISPR-based technologies to new levels in the fields of biological analysis and clinical diagnostics.

Microfluidic systems have several advantages:

(1) Due to the strict module separation and sealing, the risk of cross contamination is minimal;

(2) The reagent consumption is low, and the cost is reduced;

(3) Sample injection and sample discharge can be realized without any additional manual operation;

(4) A universal detection platform is less affected by sample characteristics (such as density, pH, conductivity, etc.);

(5) Multiple detection can be realized by reasonably designing the micro-channel;

(6) In one chip assay, multiple cascade reactions can be performed in sequence without affecting each other.

In order to solve the problem of multifunctional integrated full sealing of the molecular POCT product, the utility model designs a nucleic acid detection device based on the RPA-combined CRISPR/Cas12 isothermal amplification technology, and the device integrates the functions of sample collection and inactivation, nucleic acid extraction-free, RPA+CRISPR/Cas 12 amplification and visual detection.

Disclosure of Invention

The utility model aims to provide a nucleic acid detection device based on the RPA combined CRISPR/Cas12 isothermal amplification technology, which solves the problems in the background technology.

In order to achieve the above purpose, the present utility model provides the following technical solutions: a nucleic acid detection device based on RPA-binding CRISPR/Cas12 isothermal amplification technology, comprising: sample collection inactivating device, nucleic acid exempt from extraction device, micro-fluidic chip dish control application of sample device, RPA isothermal amplification device, CRISPR/Cas12 signal amplification device and visual chromatography test paper strip detection device, in addition, whole device uses centrifugal micro-fluidic chip dish to carry out fine control and accurate control, guarantees that whole testing process can carry out with inclosed automation and relatively independent mode.

As an alternative to the technical solution of the present utility model, the sample collection and inactivation device: the biological sample is collected and inactivated, and the biological sample mainly comprises an oral swab and a nasopharyngeal swab. Furthermore, the biological sample is put into an inactivated sample preservation solution free of nucleic acid extraction for cracking and inactivating, and further, 2 layers of embedded clamping plate devices are arranged in the sample preservation tube, the upper part is wide, the lower part is narrow, and when the disposable collection swab is unscrewed from the preservation tube, the collection swab can be effectively extruded and scraped, so that cells can be effectively transferred into the sample preservation solution for cracking and inactivating. Furthermore, the sample preservation solution is an inactivation type preservation solution free of nucleic acid extraction, and the needle at the bottom of the sample preservation tube can penetrate through the bottom of the preservation tube with different force by adjusting the gear of the sample injection button, so that different amounts of inactivated nucleic acid samples are transferred to the microfluidic chip disc to control the sample adding device.

As an alternative of the technical scheme of the utility model, the microfluidic chip disc controls the sample adding device: based on a microfluidic chip disc control technology, injecting a sufficient amount of nucleic acid extraction products into freeze-dried balls of a pre-placed RPA amplification system;

as an alternative to the technical solution of the present utility model, the RPA isothermal amplification device: performing RPA isothermal amplification reaction based on a microfluidic chip disc control technology;

as an alternative to the technical solution of the present utility model, the CRISPR/Cas12 amplification device: based on a microfluidic chip disc control technology, injecting a sufficient amount of amplification products into freeze-dried balls of a pre-placed CRISPR/Cas12 amplification system for reaction;

as an alternative to the technical solution of the present utility model, the visual chromatographic test strip device: based on the micro-fluidic chip disc control technology, a sufficient amount of amplified products are injected into a sample-adding hole of the visual chromatographic test strip for chromatographic operation and presentation of a final detection result.

Compared with the prior art, the utility model has the following beneficial effects:

1. the utility model can obtain the result (RPA: 25-30 minutes, CRISPR/Cas12:5-10 minutes) by benefiting the whole reaction very speed of the RPA isothermal amplification technology about 25-40 minutes; the sensitivity can reach 10 Copies/mu L; the length of the amplification primer is 30-35bp, and no complex instrument and equipment are needed during the amplification; the utility model discloses a nucleic acid detection device based on an RPA combined CRISPR/Cas12 isothermal amplification technology, which is a POCT device integrating sample collection and inactivation, nucleic acid extraction-free, centrifugal microfluidic chip control and sample addition, RPA isothermal amplification, CRISPR/Cas12 signal amplification and visual chromatographic test strip detection. In order to facilitate bedside test application, RPA and CRISPR/Cas12a reactions are implemented in a centrifugal microfluidic chip disc nucleic acid detection device so as to realize automatic and integrated detection of various viruses, the whole process rotates in an instrument, and a customized compact machine of a heating and optical detection assembly operates, so that complex instruments for detecting results are greatly simplified, the requirement of quick result output can be met, and meanwhile, the device can be better used in areas without molecular detection conditions.

Drawings

Other features, objects and advantages of the present utility model will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a schematic view of the overall structure of the appearance 000 of the present utility model;

FIG. 2 is a schematic view of a 000-appearance partial structure according to the present utility model;

FIG. 3 is a schematic top view of 000 structures of the present utility model;

FIG. 4 is a schematic view of the internal structure of the present utility model at 000;

FIG. 5 is a schematic view showing the three-dimensional structure of a 000-heater film according to the present utility model;

FIG. 6 is a schematic diagram of a 100 sample collection and inactivation device according to the present utility model;

FIG. 7 is a schematic diagram of a 200 sample collection flow in the present utility model;

FIG. 8 is a schematic diagram of a sample loading device with a control of a 300-micro fluidic chip disc according to the present utility model;

FIG. 9 is a schematic representation of 400RPA amplification and 500CRISPR/Cas12 signal amplification structures in the present utility model;

FIG. 10 is a schematic diagram of a reaction of a 600 visual chromatographic test strip device in the present utility model;

FIG. 11 is a diagram illustrating a structure of a visual inspection window 600 according to the present utility model.

In the figure: 000 overall structure schematic: 001. a knob; 002. a cover is arranged; 003. a detection window; 004. a fixed base; 005. a shock pad; 006. a motor; 007. a turntable; 008. a control board; 009. a power switch; 010. heating the film;

100 sample collection and inactivation device: 101. a disposable medical sampling swab; 102. a sample collection tube; 103. an inactivated sample preservation solution free of nucleic acid extraction; 104. a double-layer splint; 105. a rubber plug structure at the bottom of the centrifuge tube;

200 sample collection flow diagram: specifically, the steps 201-207 are collected;

300 micro-fluidic chip disc control sample adding device; 301 first sample application chamber and 302 second sample application chamber, 303 one diluent application chamber, 304 one mixing chamber, 305 eight separate mini RPA reaction chambers, 306 eight separate detection chambers and 307 first waste chamber and 308 second waste chamber;

400RPA amplification device; 500. CRISPR/Cas12 signal amplification means;

600 visual chromatographic test strip detection device; 601. a sample adding hole; 602. a visualization window; 603. a quality control line; 604. and (5) detecting a line.

Detailed Description

Referring to fig. 1-10, the present utility model provides a technical solution: a nucleic acid detection device based on RPA-binding CRISPR/Ca12 isothermal amplification technology, comprising: the whole appearance 000 and the three-dimensional structure, the sample collection and inactivation device 100, the sample collection flow diagram 200, the microfluidic chip disc control sample adding device 300, the RPA amplification device 400, the CRISPR/Cas12 signal amplification device 500 and the visualized chromatographic test strip detection device 600.

Wherein the overall appearance 000 and the perspective structure of the utility model are shown in figures 1-5, and specifically comprise: knob 001, upper cover 002, detection window 003, unable adjustment base 004, shock pad 005, motor 006, carousel 007, control panel 008, switch 009, heating film 010, wherein the constitution of instrument: the motor 006 is fixed on unable adjustment base 004, and unable adjustment base 004 below is pasted and is had shock pad 005, is equipped with transparent detection window 003 on the unable adjustment base 004, and carousel 007 is fixed in the pivot of motor 006 and is rotated together with motor 006, and the carousel 007 back has heating film 010 to provide the heating function, and microfluidic chip dish is controlled the loading device 300 and is placed on carousel 007 and push down with last lid 002, screws up knob 001 locking again. The instrument also has a control board 008 for controlling the motor 006 and heating film 010, and a power switch 009 for connecting to a power source.

The schematic structure of the sample collection and inactivation device 100 of the present utility model is shown in fig. 6: the sample collection and inactivation device comprises a disposable medical sampling swab 101, a sample collection tube 102 and an inactivation sample preservation solution 103 free of nucleic acid extraction, wherein the inactivation device is characterized in that a double-layer clamping plate 104 is embedded in the collection and inactivation device, a rubber plug structure 105 is arranged at the bottom of a centrifuge tube, in order to reduce interference in the sample as much as possible, the sample does not need to eat any food within 30 minutes before sampling, smoking and drinking are prohibited, a chewing gum can be chewed appropriately, and the specific collection method is as shown in fig. 7:

201: unscrewing 102 the sample collection tube cap, not allowing the swab to contact the sample preservation fluid 103 in the tube;

202: peeling off an outer packaging bag of the 101 disposable sampling swab, and taking out the sampling swab;

203: wiping the inner wall of one side of the oral cavity by using a sampling swab for at least 30S;

204: gently insert 102 the sampling swab into the sample collection tube in a spiral motion, with little effort, possibly with some resistance;

205: shaking the tip 20S of the collection swab up and down in 103 sample preservation fluid, but not beyond the insertion site;

206: rotating the sampling swab upward in an unscrewing action until the swab is completely removed from the tube, discarding the used sampling swab;

207: repeating steps (202) to (206), and wiping the inner wall of the other side of the oral cavity with a clean sampling swab.

The microfluidic chip tray controlled sample loading device 300 of the present utility model is schematically shown in fig. 8, and is manufactured by precision injection molding using polyethersulfone, and comprises a first sample loading chamber 301 and a second sample loading chamber 302 (serving as a plurality of RT-RPA reaction chambers 302 for pre-storing related lyophilized reagents and dried primers), a diluent loading chamber 303, a mixing chamber 304 (serving as a pre-storing chamber for RPA related lyophilized reagents), eight independent small RPA reaction chambers 305, eight independent detection chambers 306 (CRISPR/Cas 12 system for pre-storing lyophilized) and a first waste liquid chamber 307 and a second waste liquid chamber 308 (neither dried primers nor preloaded lyophilized reagents are operated as NTCs of the reaction itself), and two stainless steel balls subjected to hydrophobic surface treatment (diameter of 1.5 mm) are pre-loaded in the mixing chamber to improve the mixing effect. Prior to assay on microfluidic chip trays, the reagent loading procedure was as follows: first, the primers were dried (1.6. Mu.M after re-dissolution of RT-RPA, 0.8. Mu.M for RPA), then the lyophilized pellet was placed in the corresponding chamber (final concentration of reagents per step was the same as in the dissolved tube), finally the microfluidic chip disc was sealed with a single-sided tape, and during the assay on the microfluidic chip disc, 40. Mu.L of extraction-free nucleic acid was added to both sample chambers 301 and 302 (20. Mu.L in each chamber), and the sample inlet of the microfluidic chip disc was sealed with an adhesive aluminum foil.

The schematic of the structure of the RPA amplification device 400 and CRISPR/Cas12 amplification device 500 of the present utility model is shown in fig. 9, comprising injection of an extraction-free nucleic acid sample into a chip to solubilize pre-stored reagents, first multiplex RT-RPA at 39.5 ℃ for 10-15 minutes, then mixing a small fraction of the amplicons with reagents for the second RPA and partitioning into a first row of amplification chambers, pre-stored with primer pairs for each target, after re-incubation of the second RPA at 39.5 ℃ for 15 minutes, the product is transferred to a corresponding second row of detection chambers to solubilize lyophilized reagents for CRISPR/Cas12a based detection. The entire detection of multiple targets in a fully sealed and automated manner, with less than 40 minutes of overall process, eliminates the risk of contamination of the amplicon during manual handling. Eight samples can also be tested simultaneously, which greatly improves efficiency during large-scale screening, and the design of the eight individual units works in a more flexible manner, avoiding wasting reagents during sporadic sample testing.

The chromatographic test strip detects nucleic acid, namely, the immunochromatography technology is used for detecting nucleic acid products after nucleic acid amplification or signal products of a reaction system in a sample, and the CRISPR product detection test strip is used for detecting the principle of sandwich immunochromatography in the visualized chromatographic test strip device 600 disclosed by the utility model, as shown in FIG. 10:

(1) Specific FITC monoclonal antibodies are marked on the colloidal gold, streptavidin (SA) is fixed on a detection line (T line) of the NC membrane, and secondary antibodies are fixed on a quality control line (C line) of the NC membrane;

(2) When a certain amount of FAM-biotin (or FITC-biotin) double-labeled probes exist in the sample, a polymer of colloidal gold-FITC monoclonal antibody-FAM-biotin is formed by the double-labeled probes and colloidal gold, the polymer is captured on a T line, so that the T line is developed, and the C line captures the colloidal gold marked with the FITC monoclonal antibody and develops the color;

(3) When the target molecule exists, the Cas12 protein is activated to generate nonspecific ssDNA cleavage, the double-labeled probe is degraded, the polymer of the colloidal gold-FITC monoclonal antibody-FAM can not be captured by a T line, the T line does not develop color, and the C line can still capture the colloidal gold labeled with the FITC monoclonal antibody to develop color.

The schematic diagram of the visual chromatographic test strip detection device 600 in the utility model is shown in fig. 11, wherein the lower end is provided with a sample adding hole 601, and the upper end is provided with a visual window 602, and the visual chromatographic test strip detection device comprises a quality control line 603 and a detection line 604.

The working principle and the using flow of the utility model are as follows: after the utility model is installed, a disposable medical collection swab is used for collecting a sample, the sample is transferred to a sample preservation solution for cracking and enrichment, when a sample transfer button is pressed, the non-extracted nucleic acid enters an RPA freeze-drying ball for reaction, CRISPR/Cas12a signals are amplified, amplified nucleic acid products enter a sample adding hole, a preservation chromatography test strip is vertically placed, only the button is twisted left and right to ensure that the amplified products and an electrophoresis buffer solution are fully mixed, the test strip is kept at the vertical position for 2-5 minutes, the reaction mixture carries out immunochromatography reaction on the test strip, and when an independent quality control line band appears on the test strip, the function of the test strip is normal, and the amplified products are positive; two strips appear on the test strip, a quality control line strip and a detection line strip, and the amplification product is a negative result; and when the test strip is not provided with a strip, the detection result is out of control and is not credible.

Claims (5)

1. The utility model provides a nucleic acid detection device based on RPA combines CRISPR/Cas12 isothermal amplification technique, includes whole outward appearance and spatial structure, sample collection inactivation device, sample collection flow schematic diagram, centrifugal micro-fluidic chip dish to control application of sample device, RPA amplification device, CRISPR/Cas12 signal amplification device, visual chromatography test paper strip detection device, its characterized in that:

the whole outward appearance and three-dimensional structure include, knob, upper cover, detection window, unable adjustment base, shock pad, motor, carousel, control panel, switch, heating film, and wherein the constitution of instrument: the motor is fixed on the fixed base, a shock pad is attached below the fixed base, and a transparent detection window is arranged on the fixed base;

the sample collection and inactivation device structurally comprises a disposable medical sampling swab, a sample collection tube and an inactivation type sample preservation solution free of nucleic acid extraction;

the centrifugal microfluidic chip disc control sample adding device structure comprises a microfluidic chip disc;

the RPA amplification device and the CRISPR/Cas12 amplification device structurally comprise an extraction-free nucleic acid sample injection chip for dissolving a pre-storage reagent;

the visual window structure is characterized in that a sample adding hole is formed in the lower end of the visual window structure, and a visual window is formed in the upper end of the visual window structure and comprises a quality control line and a detection line.

2. The nucleic acid detection device based on the RPA-binding CRISPR/Cas12 isothermal amplification technology according to claim 1, wherein: the rotary table is fixed on a rotary shaft of the motor and rotates together with the motor, and a heating film is arranged on the back of the rotary table to provide a heating function;

the microfluidic chip disc control sample adding device is placed on the rotary disc and pressed by the upper cover, and then the knob is screwed for locking;

the apparatus also has a control board for controlling the motor and the heating film, and a power switch connected to a power supply.

3. The nucleic acid detection device based on the RPA-binding CRISPR/Cas12 isothermal amplification technology according to claim 1, wherein: the sample collection inactivating device is embedded with a double-layer clamping plate and a rubber plug structure is arranged at the bottom of the centrifuge tube.

4. The nucleic acid detection device based on the RPA-binding CRISPR/Cas12 isothermal amplification technology according to claim 1, wherein: the microfluidic chip disc control sample adding device is manufactured by using polyethersulfone through precise injection molding.

5. The nucleic acid detection device based on the RPA-binding CRISPR/Cas12 isothermal amplification technology according to claim 1, wherein: the microfluidic chip tray comprises two sample loading chambers.