CN215480963U - Device for detecting pathogen on-site nucleic acid - Google Patents
Device for detecting pathogen on-site nucleic acid Download PDFInfo
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- CN215480963U CN215480963U CN202121848296.4U CN202121848296U CN215480963U CN 215480963 U CN215480963 U CN 215480963U CN 202121848296 U CN202121848296 U CN 202121848296U CN 215480963 U CN215480963 U CN 215480963U
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Abstract
The utility model relates to a pathogen on-site nucleic acid detection device, which belongs to the technical field of laboratory equipment and comprises a base, a nucleic acid extraction part, a detection part, a display part and a control device, wherein the nucleic acid extraction part, the detection part, the display part and the control device are arranged on the base; by using the device of the utility model and combining with a matched RPA-LFD method, a more sensitive and more specific detection result can be rapidly obtained; the detection part and the display part of the device are sealed, amplification reaction and detection can be finished without opening a cover, pollution caused by high-concentration nucleic acid amplification products can be prevented, and after the experiment is finished, the detection part and the display part can be integrally discarded, so that the biological safety risk can be reduced; the device can be separated from the dependence on laboratories and valuable instruments, and can be used for on-site nucleic acid extraction and detection in places such as animal farms and the like.
Description
Technical Field
The utility model belongs to the technical field of laboratory equipment, and particularly relates to a device for detecting nucleic acid of a pathogen on site.
Background
The nucleic acid detection technology, especially real-time fluorescent PCR developed in recent years, has high sensitivity and strong specificity, and is widely accepted in clinical application at present. However, since this method is highly dependent on the laboratory environment, such as the need for a high-speed centrifuge during nucleic acid extraction, the need for a precise thermal cycler during fluorescence PCR, the need for professional technicians for reading the results, the time required for nucleic acid extraction and PCR is long, and it is difficult to meet the needs for on-site detection.
The Recombinase Polymerase Amplification (RPA) is a novel constant temperature nucleic acid amplification technology, and compared with PCR, the Recombinase polymerase amplification technology has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity and no need of precise instruments, is a nucleic acid detection technology capable of replacing PCR, and is the most potential tool for rapid molecular diagnosis at present. The combination of RPA and Lateral Flow Dipstick (LFD) (RPA-LFD) is a nucleic acid detection technology combining recombinase polymerase amplification, colloidal gold labeling (sandwich method), molecular hybridization, lateral flow chromatography and other methods, and results are visualized. In the research, nfo is used as exonuclease IV, and the detection principle is that firstly, an amplicon amplified by an upstream primer and a downstream primer containing a 3 ' end biotin (biotin) label and a specific probe containing a carboxyfluorescein (FAM) label are subjected to hybridization reaction to form a product of which the 5 ' end is the FAM label and the 3 ' end is the biotin label. The product is added into a sample hole of a lateral flow test strip and is chromatographed to a binding pad (colloidal gold particles fixed with anti-FAM antibodies) through capillary action, the 5' end FAM in the product is combined with the colloidal gold particles of the anti-FAM antibodies to form a complex, when the complex is chromatographed to a detection line (T line), because the T line is fixed with Streptavidin (SA), an amplification product containing biotin labels is captured by the streptavidin to show red, and when the complex which is not captured is continuously chromatographed upwards to a quality control line (C line), because the C line is fixed with specific antibodies, the complex is captured by the specific antibodies to show red, and the result is positive. The method can realize the visual detection of the amplified product within 15-30 minutes, and the fluorescent quantitative PCR needs 1-2 hours, so that the RPA-LFD provides a new method for realizing the on-site rapid detection of pathogens.
In situ nucleic acid detection requires three key issues to be addressed, including: sample preparation, amplification and detection, with sample preparation being among the biggest challenges. In conventional methods for extracting nucleic acid, including the centrifugal column method, the TRIzol method, and the like, a heating apparatus (including a water bath or a dry bath) and a high-speed centrifuge, and the like are not required. When nucleic acid is extracted by the conventional magnetic bead method, a heating apparatus, a rotator, a magnetic holder, and the like are required. The methods are difficult to separate from the laboratory environment and difficult to realize the field extraction of nucleic acid. Therefore, a set of simple and portable devices is needed to meet the needs of on-site nucleic acid extraction.
Because the RPA is a nucleic acid amplification method with amplification efficiency not inferior to that of fluorescence quantitative PCR, the concentration of an amplification product is very high, nucleic acid aerosol pollution is easily generated when gas rubs with the liquid surface of a sample in the processes of opening a tube cover, sampling, loading and chromatography of the sample on a lateral flow test strip, and during the next detection, due to the high sensitivity of the method, a very small amount of aerosol molecules can be amplified, so that a false positive result and potential biological safety risks are caused. Therefore, it is necessary to complete the amplification reaction and the detection reaction in a closed apparatus, and after the detection is completed, the reaction part and the detection part are disposed of as a whole to reduce false positive results and biosafety risks.
Disclosure of Invention
The utility model aims to provide a device for on-site nucleic acid extraction and detection of pathogens and a using method thereof. The device is combined with a matched RPA-LFD method, so that nucleic acid can be simply and quickly extracted, false positive and biological safety risk caused by opening a tube cover are effectively prevented, and a more sensitive and specific detection result is quickly obtained; the detection part and the display part of the device are sealed, amplification reaction and detection can be finished without opening a cover, pollution caused by high-concentration nucleic acid amplification products can be prevented, and after the experiment is finished, the reaction part and the detection part can be discarded integrally, so that the biological safety risk is reduced; the device can be separated from the dependence on laboratories and valuable instruments, and can be used for on-site nucleic acid extraction and detection in places such as animal farms and the like.
The technical scheme adopted by the utility model is as follows:
a pathogen on-site nucleic acid detection device comprises a base, a nucleic acid extraction part, a detection part, a display part and a control device, wherein the nucleic acid extraction part, the detection part, the display part and the control device are arranged on the base;
the base comprises an extraction part base and a detection part base, and the extraction part base consists of a nucleic acid extraction part and a control device; the detection part base is composed of a detection part and a display part, and the extraction part base and the detection part base can be freely closed or detached;
the nucleic acid extraction part comprises a bracket, a magnet insert box, an extraction tube, a tube sleeve and a magnet, wherein the magnet insert box is provided with a box cover capable of being opened and closed and used for placing the magnet, the tube sleeve is arranged in the middle of the side surface of the magnet insert box, the tube sleeve is vertically arranged outside the magnet insert box, the upper end of the tube sleeve is provided with a sealing cover, the tube sleeve can be opened and closed and used for sleeving the extraction tube, and the tube sleeve can be heated at constant temperature; the pipe sleeve is in signal connection with the control device; the magnet inserting box is fixed on the bracket and can be turned over on the bracket through the control device;
the detection part comprises a reaction box, and a round-mouth cutter, a Y-shaped bracket, a cup-shaped funnel, a detection tube, a fixing ring and a dilution tube which are arranged in the reaction box; the reaction box is provided with a cover capable of being opened and closed and lacks a lower bottom; the reaction box is arranged on the base of the detection part, the cup-shaped funnel comprises a cup-shaped body and a funnel neck, 2Y-shaped brackets are arranged in the cup-shaped body, a horizontally-placed round-mouth cutter is arranged above the Y-shaped brackets, the cutting edge of the round-mouth cutter is in the vertical direction, fixing rings are arranged in the reaction box and are used for fixing the detection tube and the dilution tube, when the detection tube and the dilution tube extend into the round-mouth cutter through the lower ends of the fixing rings, the detection tube and the reaction tube move downwards by pressing down the cover of the reaction box, so that the lower ends of the detection tube and the reaction tube can be vertically cut off by the round-mouth cutter, the upper ends of the detection tube and the reaction tube are prevented from falling by the fixing rings, and the cut-off parts are caught by the Y-shaped brackets; the funnel neck is S-shaped to increase liquid flow so as to fully dilute the liquid to be detected and the diluent;
the display part comprises a sample loading hole and a result display window, the sample loading hole is opposite to the outlet of the funnel neck, the result display window is arranged in a groove in the middle of the base, and the result display window is internally provided with detection test paper which is sealed in the result display window; and (4) enabling the sample added into the sample loading hole to be chromatographed to a result display window, and displaying the result.
Further, the device is also provided with an electric grinding rod and an electric rotating rod. Respectively used for grinding the sample and uniformly mixing the reaction solution.
Furthermore, the electric grinding rod and the electric rotating rod are provided with shielding plates. Preventing liquid from splashing out during grinding and stirring.
Further, the diameter of the fixing ring is larger than the diameter of the detection tube and the reaction tube, but smaller than the diameter of the detection tube and the reaction tube cover.
The using method of the device comprises the steps of firstly, extracting nucleic acid by a magnetic bead method, putting sample tissues into an extraction tube, fully grinding, adding digestive juice and proteinase K, putting the extraction tube into a tube sleeve, covering a sealing cover of the tube sleeve, opening a tube sleeve heating button on a control device, and fully digesting; adding the magnetic beads and carrierRNA, and fully mixing the magnetic beads and the nucleic acid; inserting a magnet into the magnet inserting box, adsorbing the magnetic beads on the wall of the extraction tube at the moment, removing liquid, taking out the magnet, adding a rinsing liquid to rinse the magnetic beads, then inserting the magnet into the magnet inserting box, repeating for 2 times, finally adding RNase-Free double distilled water to elute and heat in the tube sleeve, turning over the bracket and uniformly mixing, then putting the magnet into the magnet inserting box again, and taking out the solution to another extraction tube to obtain a nucleic acid sample; then using the extracted nucleic acid as a template to carry out RPA nucleic acid amplification reaction in a detection tube, wherein the whole reaction is carried out in a nucleic acid extraction part, after the reaction is finished, the detection tube and a dilution tube containing a diluent are respectively inserted into 2 round-mouth cutters in a reaction box through fixing rings, the lower ends of the dilution tube and the detection tube are just opposite to a Y-shaped bracket, a cover of the reaction box is pressed downwards with force, the lower ends of the detection tube and the dilution tube are cut off by the cutters, the cut-off parts fall on the Y-shaped bracket, the fixing rings are clamped on the covers of the detection tube and the dilution tube to prevent the dilution, the liquid in the detection tube and the dilution tube flows out and flows into an S-shaped curve of a cup-shaped funnel to promote the dilution liquid and an amplification product to be fully mixed, the mixture is vertically dripped into a sample hole to be displayed on a result display window through chromatography, after one sample is detected, a base of the detection part is detached, and the base comprising the detection part and the display part is sealed and thrown away, preventing aerosol from polluting the next batch of sample detection.
Compared with the prior art, the utility model has the beneficial effects that:
the device can be used for extracting nucleic acid on site by using a portable device, and the restriction of a plurality of devices such as a water bath kettle, a magnetic frame, a rotating instrument and the like can be removed; the nucleic acid amplification method matched with the device is RPA-LFD, because the amplification efficiency of the method is high, the concentration of the amplification product is very high, the opening of the tube cover is easy to cause aerosol pollution, the detection part of the device is finished in a closed reaction box, and the amplification reaction and the detection can be finished without opening the tube cover; the result display part is also sealed by the transparent adhesive tape, so the detection part and the display part of the device are completely sealed, false positive pollution caused by high-concentration nucleic acid amplification products can be prevented, and the detection part and the display part can be integrally discarded after the experiment is finished, so the biological safety risk can be reduced; the device can be separated from the dependence on laboratories and valuable instruments, and can be used for on-site nucleic acid extraction and detection in places such as animal farms and the like.
Drawings
FIG. 1 is a schematic view of the structure of the apparatus of the present invention: 1. the device comprises a control device, 2, a support, 3, a magnet insert box, 4, a box cover, 5, a pipe sleeve, 6, a magnet, 7, a reaction box, 8, a round-mouth cutter, 9, a Y-shaped bracket, 10, a cup-shaped funnel, 11, a detection tube, 12, a dilution tube, 13, an extraction part base, 14, a sample loading hole, 15, a result display window, 16, a fixing ring and 17.
FIG. 2 is a schematic view of a motorized grinding rod;
FIG. 3 is a schematic view of a motorized rotatable wand;
fig. 4 shows the result of the negative or positive display in the result display window.
Detailed Description
The utility model is further illustrated by the following examples, without restricting its scope in any way.
Example 1
A pathogen in-situ nucleic acid detection device is shown in figure 1, and comprises a base, a nucleic acid extraction part, a detection part, a display part and a control device 1, wherein the nucleic acid extraction part, the detection part, the display part and the control device are arranged on the base;
the base comprises an extraction part base 13 and a detection part base 17, and the extraction part base is composed of a nucleic acid extraction part and a control device; the detection part base is composed of a detection part and a display part, and the extraction part base and the detection part base can be freely closed or detached;
the nucleic acid extraction part comprises a bracket 2, a magnet insert box 3, an extraction tube, a tube sleeve 5 and a magnet 6, wherein the magnet insert box is provided with a box cover 4 which can be opened and closed, the magnet insert box is used for placing the magnet, the tube sleeve is vertically arranged in the middle of the outer side of the magnet insert box, the tube sleeve is provided with a sealing cover, the sealing cover can be opened and closed to seal the extraction tube, and the tube sleeve can be heated at constant temperature; the pipe sleeve is in signal connection with the control device; the magnet inserting box is fixed on the bracket and can be turned over on the bracket; magnet box's both sides face respectively has a bayonet socket, and one side can hang electronic grinding rod (electrified pond, figure 2), and the opposite side can hang electronic rotatory stick (electrified pond), and as shown in figure 3, the end of this rotatory stick is cross fan type for in the nucleic acid extraction process, the magnetic bead of abundant mixing. The electric grinding rod and the electric rotating rod are provided with shielding plates. Preventing liquid from splashing out during grinding and stirring.
The detection part comprises a reaction box 7, and a round-mouth cutter 8, a Y-shaped bracket 9, a cup-shaped funnel 10, a detection tube 11, a fixing ring 16 and a dilution tube 12 which are arranged in the reaction box; the reaction box is provided with a cover capable of being opened and closed and lacks a lower bottom; the reaction box is arranged on the base 17 of the detection part, the cup-shaped funnel comprises a cup-shaped body and a funnel neck, 2Y-shaped brackets are arranged in the cup-shaped body, a horizontally-placed round-mouth cutter is arranged above the Y-shaped brackets, the direction of a cutting edge of the round-mouth cutter is vertical, when the detection tube and the dilution tube are placed in the cup-shaped funnel through the round-mouth cutter, the detection tube and the reaction tube move downwards by pressing a cover of the reaction box, so that the lower ends of the detection tube and the reaction tube can be vertically cut off by the round-mouth cutter, the upper end of the detection tube and the reaction tube are provided with fixing rings to prevent the detection tube and the reaction tube from toppling over, and the cut-off parts are caught by the Y-shaped brackets; the funnel neck is S-shaped to increase liquid flow so as to fully dilute the liquid to be detected and the diluent;
the display part comprises a sample loading hole 14 and a result display window 15, the sample loading hole is opposite to the outlet of the funnel neck, and the result display window is arranged in a groove in the middle of the base; a detection test paper is arranged in the result display window and is sealed in the result display window; the aerosol is prevented from polluting the detection test paper, and the sample added into the sample loading hole is chromatographed to a result display window, so that the result is displayed.
Example 2 this example was tested using the apparatus described in example 1
The base of the detection part fixed with the test paper card, the nucleic acid extraction bracket and the bayonet of the reaction box are tightly clamped, and the device is assembled.
The first step is to extract nucleic acid:
in this example, the reagents and procedures of the magnetic bead method virus DNA/RNA extraction kit (DP438) from Tiangen corporation were used.
1) The small pieces of intestinal tissue were added to 200. mu.L of tissue digest and 20. mu.L of proteinase K and ground well with an electric grinding bar.
(1) For samples that do not require grinding, this step of grinding can be omitted, such as blood samples;
(2) for samples with macroscopic tissue masses, the samples can be placed in a pipe sleeve of a nucleic acid extraction part and digested at 65 ℃ for 30 minutes;
(3) for rat tail tissue, digestion was performed overnight at 56 ℃ in a heated centrifuge tube.
2) Transferring 200 mu L of the processed sample solution into a 1.5mL centrifuge tube;
3) adding 15 mu L of magnetic bead suspension, adding 300 mu L of LCarrier RNA working solution into a centrifuge tube, and uniformly mixing for 10 seconds by rotating an electric rotating rod;
4) standing at room temperature for 10min, and rotating and uniformly mixing the magnetic beads and the nucleic acid on a magnet insert box every 3 min for 10 s by using a control device;
5) placing the centrifuge tube in a tube sleeve, placing a magnet in a magnet insert box, standing for 1 minute, and carefully removing liquid when the magnetic beads are completely adsorbed;
6) taking the magnet out of the magnet insert box, adding 500 mu L of rinsing liquid, and uniformly mixing for 1 minute by using an electric rotating rod;
7) placing the magnet into a magnet insert box, standing for 1 minute, and carefully absorbing liquid after magnetic beads are adsorbed;
8) taking the magnet out of the magnet insert box, adding 500 mu L of rinsing liquid, and uniformly mixing for 1 minute by using an electric rotating rod;
9) the magnet was placed in a magnet cartridge and allowed to stand for 1 minute, and after the magnetic beads were completely adsorbed, the liquid was carefully aspirated.
10) Placing the magnet into a magnet insert box, and airing at 56 ℃ for 5-10 minutes;
11) taking out the magnet and inserting the magnet into a box, adding 100 mu LRNase-Free double distilled water, and uniformly mixing for 5 minutes by an electric rotating rod at 56 ℃;
12) the magnet was placed in a magnet cartridge, allowed to stand for 2 minutes, and after the beads were completely adsorbed, the nucleic acid solution was carefully transferred to a new centrifuge tube and stored under appropriate conditions.
The nucleic acid extracted here includes viral DNA and RNA, and if viral RNA is used, the DNA may be degraded by DNase, and if nucleic acid amplification reaction is performed using viral DNA, the nucleic acid may be used directly. Since rat parvovirus H1 is a DNA virus in this example, samples can be directly taken for subsequent nucleic acid amplification reactions.
The second step is to perform amplification reaction:
the procedures of the instructions for the assay of the RPA-nfo nucleic acid amplification reagent (dipstick type):
1) adding 40.9 muL of A Buffer, 4 muL of primer and 0.6 muL of probe into a detection tube filled with a detection dry powder enzyme preparation;
2) adding 2.0 mu L of DNA of a sample to be detected obtained by the first step of treatment into a detection tube;
3) adding 2.5 μ L of B Buffer on the detection tube cover, covering the tube cover, placing the detection tube into the tube sleeve, covering tightly, turning over the nucleic acid extraction support by the control device, and mixing thoroughly for 5-6 times;
4) placing the detection tube into a heating tube sleeve, and incubating for 15min at 37 ℃;
5) after the reaction, the total volume was 50. mu.L.
And step three, detection:
adding 300. mu.L of PBS to another dilution tube similar to the above-mentioned detection tube to dilute the nucleic acid amplification product;
opening the cover of the reaction box, putting the detection tube and the dilution tube into the round-mouth cutter through the fixing ring, tightly covering the cover, forcibly pressing the cover of the reaction box downwards, cutting off the bottoms of the detection tube and the dilution tube by the round-mouth cutter, lifting the cut part by the Y-shaped bracket, enabling the liquid in the detection tube and the dilution tube to flow out and flow into the S-shaped bend of the cup-shaped funnel to promote the dilution liquid and the amplification product to be fully mixed, vertically dropping the mixture into the sample hole, performing chromatography, and after 5 minutes, or simultaneously displaying red on a C line and a T line on a test strip, wherein the C line and the T line are positive, or only displaying red on the C line, not displaying color on the T line, and displaying negative results, as shown in figure 4, or no C/T line exists, and the detection is invalid.
Remarking:
primers and probes for rat parvovirus H1 were as follows:
LRMVF：AGATCTGTACACTGACTCTAGCAAGAACCAA
LRMVR：Biotin-AAATCTATCATTGCACAAGCCATAGCACAAG
LRMVP：FITC-CCTGTAGAATCTTTGCTGAGCATGGCTGGA(THF)CTATATTAAAGTCTG-C3-Spacer
the primers and probes were synthesized by Shanghai scintillation molecular Biotechnology Ltd, the test strips were purchased from Milenia-biotech HybriDecect, and the windows of the test strips of this company displaying the results were sealed with Scotch tape. Therefore, the whole device is sealed completely, and the base of the detection part, the detection part and the display part can be discarded integrally after detection is finished.
Claims (2)
1. A pathogen on-site nucleic acid detection device is characterized by comprising a base, a nucleic acid extraction part, a detection part, a display part and a control device, wherein the nucleic acid extraction part, the detection part, the display part and the control device are arranged on the base;
the base comprises an extraction part base and a detection part base, and the extraction part base consists of a nucleic acid extraction part and a control device; the detection part base is composed of a detection part and a display part, and the extraction part base and the detection part base can be freely combined or disassembled;
the nucleic acid extraction part comprises a bracket, a magnet insert box, an extraction tube, a tube sleeve and a magnet, wherein the magnet insert box is provided with a box cover capable of being opened and closed and used for placing the magnet, the tube sleeve is arranged in the middle of the side surface of the magnet insert box, the tube sleeve is vertically arranged outside the magnet insert box, the upper end of the tube sleeve is provided with a sealing cover, the tube sleeve can be opened and closed and used for sleeving the extraction tube, and the tube sleeve can be heated at constant temperature; the pipe sleeve is in signal connection with the control device; the magnet inserting box is fixed on the bracket and can be turned over on the bracket through the control device;
the detection part comprises a reaction box, and a round-mouth cutter, a Y-shaped bracket, a cup-shaped funnel, a detection tube, a fixing ring and a dilution tube which are arranged in the reaction box; the reaction box is provided with a cover capable of being opened and closed and lacks a lower bottom; the reaction box is arranged on the base of the detection part, the cup-shaped funnel comprises a cup-shaped body and a funnel neck, 2Y-shaped brackets are arranged in the cup-shaped body, a horizontally-placed round-mouth cutter is arranged above the Y-shaped brackets, the cutting edge of the round-mouth cutter is in the vertical direction, a fixing ring is arranged in the reaction box and used for fixing the detection tube and the dilution tube, the diameter of the fixing ring is larger than that of the detection tube and the reaction tube and smaller than that of the detection tube and the reaction tube cover, when the detection tube and the dilution tube extend into the round-mouth cutter through the lower end of the fixing ring, the detection tube and the reaction tube move downwards by pressing the cover of the reaction box, so that the lower ends of the detection tube and the reaction tube can be vertically cut off by the round-mouth cutter, the fixing ring is arranged at the upper end to prevent the detection tube and the reaction tube from toppling over, and the Y-shaped brackets are connected with the cut-off parts; the funnel neck is S-shaped to increase liquid flow so as to fully dilute the liquid to be detected and the diluent;
the display part comprises a sample loading hole and a result display window, the sample loading hole is opposite to the outlet of the funnel neck, the result display window is arranged in a groove in the middle of the base, and the result display window is internally provided with detection test paper which is sealed in the result display window; chromatography is carried out on the sample added into the sample loading hole to a result display window, so that a result is displayed;
the device is also provided with an electric grinding rod and an electric rotating rod.
2. The apparatus of claim 1, wherein the electric grinding rod and the electric rotating rod are provided with shielding plates.
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