CN112779359B - Integrated nucleic acid detection card box for African swine fever virus - Google Patents
Integrated nucleic acid detection card box for African swine fever virus Download PDFInfo
- Publication number
- CN112779359B CN112779359B CN202110155805.3A CN202110155805A CN112779359B CN 112779359 B CN112779359 B CN 112779359B CN 202110155805 A CN202110155805 A CN 202110155805A CN 112779359 B CN112779359 B CN 112779359B
- Authority
- CN
- China
- Prior art keywords
- cavity
- swine fever
- african swine
- fever virus
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an integrated nucleic acid detection card box for African swine fever viruses, which comprises a box body, wherein a cracking cavity, a first cleaning cavity and a second cleaning cavity are sequentially arranged in the box body from top to bottom, the cracking cavity is communicated with a box cover, and the second cleaning cavity is communicated with a reaction cavity arranged at the bottom of the outer side of the box body; plungers with plunger hole directions perpendicular to the connection direction are arranged in the connection regions of the cracking cavity, the first cleaning cavity, the second cleaning cavity and the reaction cavity, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers; amplification reaction liquid is arranged in the reaction cavity; a meltable material layer is arranged in the reaction cavity, and primer probe mixed liquor of the African swine fever virus VP72 gene is embedded in the meltable material layer. The invention can quickly and integrally carry out PCR amplification and detection on the African swine fever virus under the condition of relatively low cost, and solves the problems of poor repeatability, time-consuming detection method, complex operation and the like of the prior art.
Description
Technical Field
The invention relates to an integrated nucleic acid detection card box for African swine fever virus, and belongs to the technical field of medical detection.
Background
African Swine Fever (ASF) is an acute, thermal and highly contact animal infectious disease of pigs caused by African Swine Fever Virus (ASFV), is clinically characterized by high Fever, reticuloendothelial system bleeding and high mortality, has influence on pigs of all ages, has the disease death rate of susceptible pig groups as high as 100 percent, is seriously harmful to the Swine industry, and is classified as a type A animal epidemic disease which must be reported by the world animal health Organization (OIE).
The ASFV belongs to the only member of African swine fever virus (Asfarviridae) genus of African swine fever virus family (Asfivirus), has a double-layer envelope structure, is linear double-stranded DNA, has 1 serotype and 22 genotypes, has the genome length of about 170 kb-193 kb, can code more than 150 open reading frames, and contains more than 50 structural proteins in mature virion.
The ASF is not easy to distinguish from swine fever, highly pathogenic porcine reproduction and respiratory syndrome in clinical manifestation, so that the ASF can be used for quickly identifying and detecting suspected sick pigs. Therefore, a method for rapidly and accurately detecting the virus is established so that correct prevention and control measures can be taken in time and economic loss is reduced.
ASFV seriously threatens the healthy production of the pig industry. Not only threatens the food safety and also has a great threat to breeders and participants in other links in the supply chain, but also has a great influence on international trade due to trade restriction caused by African swine fever.
At present, the detection methods for African swine fever mainly comprise an ELISA method, a colloidal gold method and a nucleic acid detection method. The method comprises the following specific steps:
(1) ELISA method: the ELISA method has high flux, quick detection and simple operation, is a common immunological diagnosis technology at present, can be used for large-scale investigation of ASFV infection in a pig herd, but has certain hysteresis quality as a serological detection technology, and can not diagnose the early infection of the ASFV.
(2) Colloidal gold method: the colloidal gold test paper detection method is a novel immunoassay technology which takes colloidal gold as a tracer marker and utilizes a chromatography technology to realize antigen-antibody specificity detection. The method has the advantages of rapidness, sensitivity, specificity and the like, is particularly suitable for clinical rapid diagnosis, but has higher sensitivity and is easy to generate false positive.
(3) Nucleic acid detection method: by comparing the homology of the virus DNA, primers capable of specifically amplifying the African swine fever virus can be designed in a conserved region of the gene sequence (such as the VP72 protein gene), and the African swine fever virus can be identified from other viruses by PCR amplification. The method has high sensitivity, but has the limitations of easy pollution, long operation time, need of specific instruments and equipment and the like.
The method has the defects of long detection period, complex operation process, low sensitivity, expensive specific instruments and equipment and the like. Although a plurality of specific fluorescent probe PCR detection African swine fever virus nucleic acid kits are available on the market in recent years for detecting the African swine fever virus nucleic acid in a sample to be detected, the kits have some limitations for basic detection laboratories: 1) requiring professionals and secondary biosafety laboratories; 2) the specific nucleic acid extraction method and conditions are easy to pollute and prolong the operation time, so that the development of the time-saving and labor-saving African swine fever virus full-automatic nucleic acid extraction amplification typing card box is imperative to a basic layer detection mechanism.
Disclosure of Invention
The invention aims to provide an integrated nucleic acid detection card box for African swine fever virus. The method can quickly and integrally carry out PCR amplification and detection on the African swine fever virus under the condition of relatively low cost, and solves the problems of poor repeatability, time-consuming detection method, complex operation and the like of the existing technology.
The technical scheme of the invention is as follows: an integrated nucleic acid detection card box for African swine fever virus is characterized in that: the device comprises a box body with a box cover at the top, wherein a cracking cavity with cracking liquid, a first cleaning cavity with first cleaning liquid and a second cleaning cavity with second cleaning liquid are sequentially arranged in the box body from top to bottom, the cracking cavity is communicated with the box cover, and the second cleaning cavity is communicated with a reaction cavity arranged at the bottom of the outer side of the box body; plungers with plunger hole directions perpendicular to the connection direction are arranged in the connection regions of the cracking cavity, the first cleaning cavity, the second cleaning cavity and the reaction cavity, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers; amplification reaction liquid is arranged in the reaction cavity, and the amplification reaction liquid contains DNA polymerase solution; a meltable material layer is arranged in the reaction cavity, and primer probe mixed liquor of the African swine fever virus VP72 gene is embedded in the meltable material layer.
In the integrated African swine fever virus nucleic acid detection card box, magnetic beads capable of penetrating through the plunger hole of the plunger are arranged in the lysis cavity. Through promoting the plunger, can make the plunger hole aim at the intercommunication interval of two adjacent cavitys, two cavity spares rely on the downthehole hydrophobic liquid sealing material of attached to plunger to keep apart this moment, then the magnetic bead (utilize outside magnet steel to drag) alright pass the liquid sealing material of hydrophobic in plunger hole, transfer another cavity from a cavity.
In the above-mentioned African swine fever virus integrated nucleic acid detection cartridge, the DNA polymerase solution comprises Taq DNA polymerase.
In the integrated African swine fever virus nucleic acid detection card box, the primer probe mixed liquid required by the amplification reaction is in the meltable material, so that the primer is prevented from being cut by enzyme in the reaction liquid, and the stability of the card box is ensured. The meltable material is any one or combination of more of paraffin substances such as paraffin, dodecane, tetradecane, hexadecane and the like, the melting point range is 20-95 ℃, paraffin is preferred, and paraffin oil which is melted into liquid state in use can prevent PCR products forming aerosol from polluting the space between rings; the paraffin oil has weak thermal conductivity, and the temperature condition of PCR is effectively ensured; the low-temperature solidification characteristic of the paraffin can fix the position of the magnetic beads after the temperature of the magnetic bead back dragging is reduced. The paraffin is meltable so that the inclusion can consolidate the reaction solution from a separate state.
In the above integrated african swine fever virus nucleic acid detection cartridge, the primer-probe mixture of the african swine fever virus VP72 gene includes: an African swine fever virus VP72 gene upstream primer, an African swine fever virus VP72 gene downstream primer, an internal standard upstream primer, an internal standard downstream primer, an African swine fever virus VP72 gene probe and an internal standard probe, in particular to
In the integrated African swine fever virus nucleic acid detection card box, the lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-10% PEG8000 (polyethylene glycol), 0.1-10M GTC (guanidine isothiocyanate), 0.1-10% Tween-20, 0.1-5% SLS (sodium dodecyl sarcosinate), and ddH (ddH) as a solvent 2 And O. The above percentages are mass percentages.
In the integrated african swine fever virus nucleic acid detection cartridge, the first washing solution contains 1 to 500mM Tris-HCl, 10 to 500mM NaCl, 0.1 to 10% PEG 8000; the second wash solution comprises 1-500mM Tris-HCl, 10-500mM NaCl, 0.1-10% PEG 8000. The acetic acid-sodium acetate solution provides a low pH environment for DNA adsorption to the magnetic beads. The nucleic acid purification reagent (cleaning solution) and the lysis solution do not contain protease K, so that the product does not need to be stored at the temperature of-20 ℃. No alcohols are required in the cleaning solution.
In the African swine fever virus integrated nucleic acid detection card box, the diameter of a plunger hole of the plunger is 3-5mm, the diameter of a magnetic bead is 0.1-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm 3 The viscosity was 2000-. This setting can ensure that the liquid sealing material of hydrophobicity can keep apart liquid, can let the magnetic bead pass again.
In the integrated African swine fever virus nucleic acid detection card box, the hydrophobic liquid sealing material is silicone oil.
In the integrated nucleic acid detection card box for African swine fever viruses, the reaction cavity is a conical tube arranged outside the box body, one side of the conical tube is provided with a plane, external magnetic steel is convenient to pull a magnetic bead back into a meltable material above the conical tube (the magnetic bead is solidified after being cooled), and then the interference on optical detection can not be generated.
Compared with the prior art, the integrated nucleic acid detection card box for the African swine fever virus can quickly and accurately detect the African swine fever virus. By combining the primer and the probe screened by repeated research tests, the invention is superior to other molecular diagnostic kits in the aspects of detection timeliness, specificity, sensitivity, convenience, technical parameters and the like.
The integrated rapid nucleic acid detection card box for the African swine fever virus, provided by the invention, has the following advantages:
1) and (3) totally-enclosed design: 1 detection device which can drag magnetic beads and can carry out fluorescence analysis, and a small amount of laboratory basic small devices can complete complex nucleic acid detection experiments. The method integrates the processes of nucleic acid extraction, amplification, detection and the like into a totally-enclosed cassette, thereby structurally avoiding aerosol pollution, protecting the safety of detection personnel and avoiding a PCR laboratory. Greatly reducing the requirements of nucleic acid detection on the laboratory construction standard. The joint defense deployment and control of the area with limited conditions are facilitated;
2) the method comprises the following steps: the manual operation time is 2-5 minutes, and the result can be obtained 30-90 minutes after the nucleic acid is loaded into the detection equipment, so that the full automation of integration of nucleic acid extraction and amplification is conveniently realized, the requirement on the professional skill of an inspector is greatly reduced, and the method is suitable for rapid on-site bedside diagnosis;
3) and (4) refrigerating and preserving: the product is stored at 2-8 ℃ in a refrigeration way, and the conventional-20 ℃ freezing storage is not needed, so that the cold chain transportation and storage of the product are facilitated.
In addition, the invention uses the mode of combining the plunger with the hydrophobic liquid sealing material to isolate the functional cavities, so that the liquid among the cavities can not overflow, a biological sample can be respectively cracked, cleaned, purified and amplified in each cavity, and the magnetic beads can be used for bringing the nucleic acid into each cavity in sequence in an attachment mode, wherein the hydrophobic liquid sealing material can have a dispersion effect on the magnetic beads, can allow the magnetic beads to pass through the nucleic acid on the magnetic beads and can isolate other impurities, when the magnetic beads pass through, the impurities such as nonpolar large particles attached to the magnetic beads can be infiltrated into the hydrophobic liquid sealing material and can be intercepted, and the influence of the impurities on a detection result can be reduced.
FIG. 1 is a schematic view of the construction of the cartridge of the present invention;
fig. 2 is a side schematic view of fig. 1.
The symbols in the drawings are: 1-box body, 2-box cover, 3-cracking cavity, 4-first cleaning cavity, 5-second cleaning cavity, 6-reaction cavity, 7-plunger and 8-magnetic bead.
Detailed Description
The invention is further described with reference to the following figures and examples, which are not to be construed as limiting the invention.
Examples are given. An integrated nucleic acid detection cassette of African swine fever virus, as shown in figure 1: the device comprises a box body 1 with a box cover 2 at the top, wherein a cracking cavity 3 with cracking liquid, a first cleaning cavity 4 with first cleaning liquid and a second cleaning cavity 5 with second cleaning liquid are sequentially arranged in the box body 1 from top to bottom, the cracking cavity 3 is communicated with the box cover 2, and the second cleaning cavity 5 is communicated with a reaction cavity 6 arranged at the bottom of the outer side of the box body 1; plungers 7 with plunger hole directions perpendicular to the connection direction are respectively arranged in the connection areas of the cracking cavity 3, the first cleaning cavity 4, the second cleaning cavity 5 and the reaction cavity 6, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers 7; an amplification reaction solution is arranged in the reaction cavity 6, and the amplification reaction solution contains a DNA polymerase solution; a meltable material layer is arranged in the reaction cavity (6), and primer probe mixed liquor of the African swine fever virus VP72 gene is embedded in the meltable material layer. And magnetic beads 8 capable of penetrating through a plunger hole of the plunger 7 are arranged in the cracking cavity 3. The DNA polymerase solution comprises Taq DNA polymerase. The primer probe mixed solution of the African swine fever virus VP72 gene comprises: an African swine fever virus VP72 gene upstream primer, an African swine fever virus VP72 gene downstream primer, an internal standard upstream primer, an internal standard downstream primer, an African swine fever virus VP72 gene probe and an internal standard probe, in particular to
The lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-10% PEG8000, 0.1-10M GTC-, 0.1-10% Tween-20, 0.1-5% SLS. The first cleaning solution comprises 1-500mM Tris-HCl, 10-500mM NaCl, 0.1-10% PEG 8000; the second wash solution comprises 1-500mM Tris-HCl, 10-500mM NaCl, 0.1-10% PEG 8000.
The diameter of a plunger hole of the plunger 7 is 3-5mm, the diameter of the magnetic bead 8 is 0.1-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm 3 The viscosity was 2000-. The hydrophobic liquid blocking material is preferably silicone oil. The reaction chamber 6 is a taper pipe arranged at the outer side of the box body 1, and a plane is arranged at one side of the taper pipe, as shown in part A of figure 2.
Firstly, establishing an integrated nucleic acid detection card box of the African swine fever virus. 30ul of PCR amplification solution and 5. mu.l of primer probe mixture were added to each PCR tube. And 5ul of template DNA was added.
1, subpackaging a PCR buffer solution: 10mM Tris-HCl (pH8.5), 70mM KCl, 5mM MgCl 2 2.5 percent of trehalose, 0.01 percent of Bri-j23, 0.1 percent of Proclin-300, 200uM dNTP,2U Taq enzyme.
2 embedding primer probe mixed solution: mu.l of primer probe solution (40uM primer solution, 20uM probe solution) was embedded in the upper layer of PCR buffer with meltable material.
3, sealing: sealing the reaction solution with a proper amount of silicone oil;
4, subpackaging a second cleaning solution: and 140ul of second cleaning solution is dispensed into the second cleaning cavity: 100mM Tris-HCl, 100mM NaCl, 2% PEG 8000;
5, sealing: sealing the second cleaning solution by using a proper amount of silicone oil; subpackaging a first cleaning solution:
6, subpackaging 140ul of first cleaning liquid into the first cleaning cavity: 100mM Tris-HCl, 100mM NaCl, 2% PEG 8000;
7, sealing: sealing the first cleaning solution by using a proper amount of silicone oil;
8, split charging of lysate: the lysate area was aliquoted with 800ul lysate: 100mM acetic acid-sodium acetate, 1% PEG8000, 3.5M GTC, 5% Tween-20, 0.5% SLS.
Second, the method of use and rapid identification.
1 collecting and processing of samples.
Serum: blood samples were collected from the jugular vein, inferior vena cava, auricular vein, or a dissection procedure using a vacuum blood collection tube without anticoagulant. The resulting mixture was centrifuged at 3000rpm (10 mm) and the supernatant was collected and examined. In the case of an anticoagulated blood collection tube, blood was collected, mixed well with shaking, and centrifuged ((3000rpm, 10mim)) to collect the supernatant for testing.
2 detecting the use of the cartridge.
The lid of the cartridge was opened, the sealing film was opened, and 10. mu.L of magnetic beads (thoroughly suspended by shaking before use) were added. A further 200. mu.L of sample was added, the reagent was pipetted and mixed (at least 15 strokes were recommended to achieve a substantially uniform resuspension of the beads in the liquid), and the lid was closed.
3. And (3) real-time fluorescence quantitative PCR amplification and detection.
3.1 sample detection
And (3) analyzing the PCR amplification product by using a full-automatic nucleic acid detection fluorescent quantitative PCR instrument Life Ready K1000 and a full-automatic nucleic acid detection analysis system, and judging the result by integrating an amplification curve and a Tm value. The fluorescence channel settings are as follows.
| Detector | Target |
| FAM | ASFVVP72 gene |
| ROX | Internal standard |
The amplification program was set up as follows:
3.2 control set up.
The invention analyzes and researches the sequence of the African swine fever virus VP72, and designs a primer probe set shown in the following table.
Primer probes of the invention
The detection results of the reaction solution prepared by each group of primer probes are as follows:
| primer probe combination | ASFV VP72 gene CT | Internal standard CT |
| The invention | 27.62 | 25.67 |
| |
30.98 | 26.84 |
| |
31.65 | 27.32 |
| |
32.35 | 27.75 |
The results show that: compared with control groups 1-3, the primer probe provided by the invention has the best detection sensitivity and specificity.
3.3 analysis of results.
1. After the operation of the detection program is finished, the detection result is automatically reported by an applicable instrument.
2. The results showed the results of detection of ASFV VP72(FAM) gene and internal quality control (ROX).
The result of the ASFV VP72(FAM) gene should show a typical S-type amplification curve (including the S-curve before reaching the plateau), and the Ct is less than or equal to 35, and the result is judged as a corresponding positive result.
The internal quality control ROX fluorescence channel detection result should present a typical S-shaped amplification curve (including an S-curve before the plateau stage). When the target (FAM) is negative and the internal quality control Ct is less than or equal to 35, the experimental result is effective. Otherwise, it needs to be re-sampled and detected.
3.4 to test the accuracy and effectiveness of the cartridges of the invention for the detection of African swine fever nucleic acid, we tested the sensitivity and specificity of the cartridges.
1) Detecting ASFV quality control substances with different concentrations to confirm detection limit;
2) the specificity of the kit is verified by detecting swine fever, porcine reproductive and respiratory syndrome, swine erysipelas, salmonella choleraesuis, pasteurella and porcine pseudorabies virus.
As a result: we tested quality control of ASFV at different concentrations, and the following table shows the results of ASFV genes.
It was confirmed that the detection limit of ASFV by the cartridge of the present invention was 500 copies/ml.
3) The detection results of the cassette disclosed by the invention show that the cassette has no non-specific amplification to swine fever, porcine reproductive and respiratory syndrome, porcine erysipelas, salmonella choleraesuis, pasteurella and porcine pseudorabies virus.
And thirdly, detecting an example.
The test validation results from the confirmed samples from the veterinary kiosk.
Sample type: serum
Number of samples: 50 examples of
The clinical sample detection results are shown as follows:
after 41 ASFV positive samples are confirmed by the comparison method, the detection results are consistent.
And (4) conclusion: among the detection results of 50 samples, the coincidence rate of 41 ASFV positive samples is 100%, and the coincidence rate of 9 ASFV negative samples is 100%.
The above-described embodiments are not intended to limit the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and scope of the present invention should be construed as equivalents thereof.
SEQUENCE LISTING
<110> Hangzhou Zhangzhen Biotechnology Ltd
<120> integrated nucleic acid detection card box for African swine fever virus
<130> 2021012909
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
atgatgatta cctttgcttt gaa 23
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tccgtgtccc aactaatata aaa 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
acgggaggaa taccaaccca gtg 23
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gaaataaact tcctrctcaa acaagc 26
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
atccactgst ctttgggaat ttc 23
<210> 6
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
aagaccatcc acctccactt ttctaacat 29
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
gttctgcagc tcttacatac cct 23
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
ggtattcctc ccgtggcttc 20
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
tcatcatcgc acccggatcg t 21
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
gtactggaaa agcttgtaac cca 23
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
ttttcagggc cacatactct 20
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
ctcagctgct tgccgacctc a 21
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
acatttccgt aactgctcat ggta 24
<210> 14
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
catcgcaccc ggatcgtc 18
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
acggcttaca atgctctcgc tccc 24
<210> 16
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
ccactacgga ggcaatgcga tt 22
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
actaggaggc agaagaccaa 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tctgcagcta cacccaacca 20
<210> 19
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
gttctgcagc tcttacatac cctt 24
<210> 20
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
catcgcaccc ggatcgtc 18
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
ccactacgga ggcaatgcga tt 22
<210> 22
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
ctaactgagc caagtgacag c 21
<210> 23
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
agtcatccat cgttaccttt gtgc 24
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
tcacccttcc ctaggtccca a 21
Claims (7)
1. An integrated nucleic acid detection card box for African swine fever virus, which is characterized in that: the device comprises a box body (1) with a box cover (2) at the top, wherein a cracking cavity (3) with cracking liquid, a first cleaning cavity (4) with first cleaning liquid and a second cleaning cavity (5) with second cleaning liquid are sequentially arranged in the box body (1) from top to bottom, the cracking cavity (3) is communicated with the box cover (2), and the second cleaning cavity (5) is communicated with a reaction cavity (6) arranged at the bottom of the outer side of the box body (1); two connected sections of the cracking cavity (3), the first cleaning cavity (4), the second cleaning cavity (5) and the reaction cavity (6) are respectively provided with a plunger (7) with a plunger hole direction vertical to the connection direction, and a plunger hole of the plunger (7) is internally provided with a hydrophobic liquid sealing material; an amplification reaction solution is arranged in the reaction cavity (6), and the amplification reaction solution contains a DNA polymerase solution; a meltable material layer is arranged in the reaction cavity (6), and primer probe mixed liquor of the African swine fever virus VP72 gene is embedded in the soluble material layer; the primer probe mixture of the African swine fever virus VP72 gene comprises: an African swine fever virus VP72 gene upstream primer, an African swine fever virus VP72 gene downstream primer, an internal standard upstream primer, an internal standard downstream primer, an African swine fever virus VP72 gene probe and an internal standard probe, in particular to
The diameter of a plunger hole of the plunger (7) is 3-5mm, the diameter of the magnetic bead (8) is 0.1-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm 3 The viscosity was 2000-.
2. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein: magnetic beads (8) which can pass through a plunger hole of the plunger (7) are arranged in the cracking cavity (3).
3. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein: the DNA polymerase solution comprises Taq DNA polymerase.
4. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein: the lysate contains 1-1000mM acetic acid-sodium acetate, 0.1-10% PEG8000, 0.1-10M GTC, 0.1-10% Tween-20, 0.1-5% SLS.
5. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein the first wash solution comprises 1-500mM Tris-HCl, 10-500mM NaCl, 0.1-10% PEG 8000; the second wash solution comprises 1-500mM Tris-HCl, 10-500mM NaCl, 0.1-10% PEG 8000.
6. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein: the hydrophobic liquid sealing material is silicone oil.
7. The integrated African swine fever virus nucleic acid detection cartridge of claim 1, wherein: the reaction chamber (6) is a taper pipe arranged on the outer side of the box body (1), and a plane is arranged on one side of the taper pipe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110155805.3A CN112779359B (en) | 2021-02-04 | 2021-02-04 | Integrated nucleic acid detection card box for African swine fever virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110155805.3A CN112779359B (en) | 2021-02-04 | 2021-02-04 | Integrated nucleic acid detection card box for African swine fever virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112779359A CN112779359A (en) | 2021-05-11 |
| CN112779359B true CN112779359B (en) | 2022-09-06 |
Family
ID=75760894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110155805.3A Active CN112779359B (en) | 2021-02-04 | 2021-02-04 | Integrated nucleic acid detection card box for African swine fever virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112779359B (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757134B (en) * | 2014-01-13 | 2016-09-28 | 深圳澳东检验检测科技有限公司 | The fluorescence quantitative PCR detection reagent of African swine fever virus, test kit and detection method thereof |
| CN107164493A (en) * | 2017-06-08 | 2017-09-15 | 杭州遂真生物技术有限公司 | A kind of GBS kit for detecting nucleic acid |
| CN107151700B (en) * | 2017-06-08 | 2023-11-07 | 杭州遂真生物技术有限公司 | Gene detection method, gene detection kit and gene detection equipment |
| CN111020062A (en) * | 2020-01-10 | 2020-04-17 | 湖北省农业科学院畜牧兽医研究所 | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain |
| CN111876468B (en) * | 2020-09-03 | 2023-08-08 | 杭州天微基因科技有限公司 | Full-automatic nucleic acid detection method and test tube |
-
2021
- 2021-02-04 CN CN202110155805.3A patent/CN112779359B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112779359A (en) | 2021-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sandvik | Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle | |
| CN105039149B (en) | A kind of closed experimental system setup of Rapid identification nucleic acid amplification product and application | |
| CN112094948B (en) | Application of target gene combination in African swine fever virus detection and kit | |
| CN113801965B (en) | Primer group, kit and analysis method for rapid typing detection of HPV16 type and HPV18 type viruses | |
| CN108588277A (en) | A kind of canine distemper virus visualization nucleic acid detection method | |
| US20020081569A1 (en) | Rapid particle detection | |
| CN108048584A (en) | The brucellar probe of RAA Fluorometric assays and kit | |
| CN112760209B (en) | Cat coronavirus integration nucleic acid detection card box | |
| CN112760210B (en) | Novel coronavirus integrated nucleic acid rapid detection card box | |
| CN113444773B (en) | Method and kit for detecting tick pathogen nucleic acid based on liquid chip | |
| CN104388592A (en) | Pig epidemic diarrhea virus S gene RT-LAMP detection kit and detection method | |
| CN112779359B (en) | Integrated nucleic acid detection card box for African swine fever virus | |
| CN111808993A (en) | Specific primer, probe and kit for gene detection of novel coronavirus | |
| CN109457052A (en) | Detect primer, probe, kit and the detection method of human immunodeficiency virus nucleic acid | |
| US20200216870A1 (en) | Non-purified nucleic acid amplification method and device | |
| CN112779248B (en) | Chlamydia trachomatis integrated nucleic acid detection card box | |
| CN112760419B (en) | Triple detection card box for dengue, Zika and chikungunya viruses | |
| KR102274011B1 (en) | Primer set for high sensitive multiplex loop-mediated isothermal amplification reaction for detection and identification of Mycobacterium tuberculosis and Nontuberculous mycobacteria | |
| CN112553379A (en) | Method and kit for detecting respiratory infectious disease virus based on liquid-phase chip | |
| CN109457051A (en) | Detect primer, probe, kit and the detection method of hepatitis C virus nucleic acid | |
| Kirankumar et al. | Advances in protein-based diagnostic tools of plant viruses | |
| CN112899142A (en) | Integrated rapid dengue fever virus typing card box | |
| CN108977512A (en) | Primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis | |
| CN112680335A (en) | Canine adenovirus integrated nucleic acid detection card box | |
| WO2017057076A1 (en) | Method for obtaining nucleated erythrocytes and method for identifying nucleated erythrocytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |