CN111197110A - RPA-FLD method for detecting feline panleukopenia virus - Google Patents
RPA-FLD method for detecting feline panleukopenia virus Download PDFInfo
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Abstract
The invention discloses an RPA-FLD method for detecting feline panleukopenia virus, which comprises the following steps: s1: synthesizing an RPA specific primer group and a probe; s2: extracting DNA in a sample to be detected; s3: RPA amplification reaction: using the extracted DNA as a template, a probe, the RPA specific primer group in the step S1 as a primer, a buffer solution, a magnesium acetate solution and ddH2Performing RPA amplification reaction in an RPA reaction tube; s4: analyzing the amplification product obtained in the step S3 by using a quantum dot fluorescence immunochromatography method; the method has good specificity, can directly observe and judge the result by naked eyes, only needs 5-10min, shortens the detection time by 15-20min compared with the detection product of agarose gel electrophoresis, avoids contacting with toxic nucleic acid dye, lightens the pollution to the environment, and ensures that the detection is more convenient and more environment-friendly.
Description
Technical Field
The invention belongs to the technical field of molecular biology and immunology, and particularly relates to an RPA-FLD method for detecting feline panleukopenia viruses.
Background
The traditional feline panleukopenia virus is mainly identified by a hemagglutination experiment, a hemagglutination inhibition experiment and a traditional enzyme-linked immunosorbent assay method. For hemagglutination or hemagglutination inhibition experiments, because the hemagglutination activity of the feline panleukopenia virus is weak, the method has higher requirements on operating conditions and the related experimental conditions are more rigorous, such as the agglutination generation temperature (4 ℃), the agglutination cell types (pig, monkey red blood cells) and the like, false negative results are easy to appear in clinical detection, so that misjudgment is easy to be caused on the detection results; the enzyme linked immunosorbent assay can detect animal serum, tissue suspension or virus culture, but the assay is time-consuming and requires special instruments, such as: the support of the enzyme-labeling instrument, the constant-temperature incubator and other instruments is difficult to popularize and use in the field of rapid detection.
With the development of molecular biology detection technology, the established PCR technology has been successfully applied to laboratory diagnosis of feline panleukopenia virus by amplifying the conserved sequence of the feline panleukopenia virus, and has strong specificity and high sensitivity, but similar to the enzyme-linked immunosorbent assay, related instruments and equipment must be equipped, and related detection personnel also have corresponding technical capability, so that the applicability of the method on the basic level is limited.
As a novel nucleic acid isothermal amplification technology, Recombinase Polymerase Amplification (RPA) can complete amplification within 20min at 37-42 ℃ by simulating the DNA replication process in organisms. The technique relies mainly on 3 enzymes: recombinase (recombinase) binding to the oligonucleotide primer, single-stranded DNA binding protein (SSB), and strand-displacing DNA polymerase (polymerase). Since these three enzymes can maintain their activities at room temperature, amplification products can be obtained at detectable levels within ten minutes. The method only needs a simple water bath device, and the amplification product can be detected by gel electrophoresis, real-time fluorescence, a test strip and other ways. Simple operation and is suitable for use in base course and site.
At present, an RPA-LFD detection method based on an RPA and transverse flow test strip technology (LFD) is widely and successfully used for detecting pathogens such as adenovirus, chicken infectious laryngotracheitis virus, foot and mouth disease virus, bovine viral diarrhea virus, bovine infectious rhinotracheitis virus and the like; wherein: in the technology of the lateral flow test strip, the colloidal gold-based immunochromatography is mainly adopted at present, in the colloidal gold-labeled RPA-LFD detection reaction, an amplicon containing Biotin (Biotin) labels can be hybridized and reacted with a specific probe containing carboxyfluorescein (FAM) labels, and the amplicon is dripped on the test strip to be combined with an anti-FAM antibody labeled by the colloidal gold to form a ternary complex, and when the ternary complex is diffused to a detection line, the Biotin ligand is captured to form the detection line; the non-hybridized FAM labeled probe is combined with the colloidal gold labeled anti-FAM antibody to form a binary complex without biotin, and the binary complex is combined on a quality control line. And judging the amplification result by observing whether the detection line is developed. The detection method mainly depends on the specific amplification of a target sequence, avoids the false positive caused by nonspecific amplification in agarose gel electrophoresis or fluorescent dye dyeing visual observation, and improves the specificity and the sensitivity of the reaction. And an electrophoresis device and a gel imaging system are not needed, and toxic reagents such as nucleic acid dye and the like are not needed. The RPA-LFD detection method is safe, rapid and efficient, and has incomparable advantages compared with other methods. Although the cost of the colloidal gold immunochromatography is low, the defect of low sensitivity is increasingly revealed, and the future detection requirements cannot be met.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an RPA-FLD method for detecting feline panleukopenia virus, which is based on quantum dots as fluorescent markers, can generate fluorescence under the action of excitation light and has high sensitivity.
The technical scheme adopted by the invention is as follows: a RPA-FLD method for detecting feline panleukopenia virus comprising the steps of:
s1: synthesizing an RPA specific primer group and a probe;
s2: extracting DNA in a sample to be detected;
s3: RPA amplification reaction: carrying out RPA amplification reaction in an RPA reaction tube by using the extracted DNA as a template, a probe, the RPA specific primer group in the step S1 as a primer, a buffer solution, a magnesium acetate solution and ddH 2O;
s4: analyzing the amplification product obtained in the step S3 by using a quantum dot fluorescence immunochromatography method;
wherein: the RPA specific primer group comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are as follows:
FPV-RPA-VP2-F：5’-AGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAA-3’,
FPV-RPA-VP2-R：5’-TTCTTGTTCAAAACTAACTAAATGCAACTCACTCA-3’,
the 5' end of FPV-RPA-VP2-R is labeled by biotin;
the nucleotide sequence of the probe is formed by connecting two parts, namely a first part and a second part, wherein the nucleotide sequence of the first part is as follows: 5'-AGGAAACATGGCTTTAGATGATATTCATGTACA-3', the nucleotide sequence of the second part is: 5'-AATTGTAACACCTTGGT-3' the flow of the air in the air conditioner,
the 5 'end of the first part is connected with carboxyfluorescein, the 3' end of the second part is connected with phosphate, and the 3 'end of the first part and the 5' end of the second part are connected through tetrahydrofuran.
The working principle of the invention is as follows: the cat leukopenia virus gene segment is amplified through a designed primer, the 5 'end of a downstream primer is provided with biotin, the 5' end of the first part of the probe is provided with carboxyl Fluorescein (FAM), a double-labeled product can be captured by an anti-FAM antibody labeled by a fluorescent quantum dot, the double-labeled product is driven by the flowing force of liquid to be analyzed to the positions of an avidin detection line and a rabbit anti-mouse quality control line, and a strip is gradually formed through accumulation, so that the aim of detecting the cat leukopenia virus is fulfilled by judging the existence of the detection line and the rabbit anti-mouse quality control line.
Further limiting, in the step S3, the amplification temperature is 33-42 ℃, and the reaction time is 20-30 min.
Further, the step S4 is specifically: and (5) adding lateral chromatography buffer solution into the amplification product obtained in the step (S3) for dilution, and adding the diluted lateral chromatography buffer solution into a sample injection hole of the quantum dot fluorescence immune layer test strip.
Further, the test strip used in the quantum dot fluorescence immunochromatography in step S4 is provided with a quantum dot-labeled feline carboxyfluorescein antibody, an avidin detection line, and a rabbit anti-mouse antibody quality control line.
Further limited, the quantum dot-labeled feline carboxyfluorescein antibody is prepared by the following steps:
a1: preparing a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution;
a2: mixing the carboxyl modified water-soluble quantum dot, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride solution and the N-hydroxysuccinimide solution, sequentially activating and centrifuging at room temperature, removing the supernatant, adding a MES buffer solution containing carboxyl fluorescein, sequentially activating and centrifuging at room temperature, removing the supernatant, adding the buffer solution, repeatedly centrifuging, removing the supernatant, and finally adding a preserving solution for preservation.
Further, the step S2 specifically includes the following steps:
s2-1: adding Carrier RNA working solution into a sample to be detected, and uniformly mixing by vortex oscillation;
s2-2: centrifuging after incubation, and collecting liquid on the tube wall;
s2-3: adding absolute ethyl alcohol into the collected liquid, and standing at room temperature after vortex oscillation;
s2-4: centrifuging the liquid after standing at room temperature and collecting the liquid on the tube wall;
s2-5: transferring the liquid collected in the step S2-4 into an adsorption column for centrifugation;
s2-6: adding a buffer solution into the adsorption column and centrifuging;
s2-7: adding rinsing liquid into the adsorption column after centrifugation in the step S2-6, standing, centrifuging, and repeating the step after centrifugation;
s2-8: adding absolute ethyl alcohol into the adsorption column after the step S2-7, centrifuging, and standing;
s2-9: suspending the middle part of the adsorption film of the adsorption column after the step S2-8, dropwise adding ddH2O, standing at room temperature for 5-10min, and centrifuging to collect a solution on the tube wall, wherein the solution is the extracted DNA.
The storage solution is further defined as borate buffer solution with 0.05mol/L and pH 8.
Further defined, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution are both 10 mg/mL.
Further limiting, the preservation temperature in step A2 is 4 ℃.
Further defined, the adsorption column contains rnase.
Compared with the prior art, the invention has the beneficial effects that: a quantum dot fluorescence immunochromatography test strip is fixed with a quantum dot labeled mouse carboxyl fluorescein antibody (QDs-Anti FAM), an avidin detection line and a rabbit Anti-mouse antibody quality control line; if the rabbit anti-mouse antibody quality control line is normal while the quantum dot immunochromatography test strip avidin detection line has a strip, the sample is indicated to contain the feline panleukopenia virus, and if the rabbit anti-mouse antibody quality control line only has a strip, the sample is indicated to contain no the feline panleukopenia virus; the enzyme and other related components required by amplification are freeze-dried and stored at normal temperature, only hydrolysis buffer solution, primers, probes and templates need to be added when the components are to be detected, and magnesium ions are added for initial reaction, so that the requirement on operators is low, only a constant-temperature water bath kettle is needed in the detection process, expensive equipment such as a PCR instrument is not needed, and the basic layer applicability is good.
In addition, the RPA technology is combined with the transverse flow test strip technology, and the fluorescent quantum dots are used as markers, so that the result can be directly observed and judged by naked eyes, the time is only 5-10min, the detection time is shortened by 15-20min compared with that of an agarose gel electrophoresis detection product, the toxic nucleic acid dye is avoided being contacted, the pollution to the environment is reduced, and the detection is more convenient and more environment-friendly.
Drawings
FIG. 1 is a graph showing the results of fluorescence detection of feline panleukopenia virus after gene amplification;
FIG. 2 is a graph showing the results of fluorescence detection after gene amplification of a control virus;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The RPA kit used in the examples below was a Twist Amp Basic kit, purchased from Twist dxinc, england; all related components exist in a dry powder state in a provided RPA reaction tube and are diluted by a reaction buffer carried by the kit.
Example 1
Primer for detecting feline panleukopenia virus
According to the gene sequence of feline panleukopenia virus VP2 (accession number: M38246) published in GenBank, FPV-RPA-VP2-F was synthesized: 5'-AGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAA-3' as upstream primer, FPV-RPA-VP 2-R: 5'-TTCTTGTTCAAAACTAACTAAATGCAACTCACTCA-3' as downstream primer, the 5 ' end of the downstream primer is labeled with Biotin (Biotin) to form Biotin-TTCTTGTTCAAAACTAACTAAATGCAACTCACTCA.
The nucleotide sequence of the probe is formed by connecting two parts, namely a first part and a second part, wherein the nucleotide sequence of the first part is as follows: 5'-AGGAAACATGGCTTTAGATGATATTCATGTACA-3', the nucleotide sequence of the second part is: 5'-AATTGTAACACCTTGGT-3' the flow of the air in the air conditioner,
the 5' end of the first part is connected with carboxyfluorescein (FAM); the 3' end of the second part is connected with a phosphate (P) for preventing the extension of the chain; the linkage between the 3 'end of the first moiety and the 5' end of the second moiety was via Tetrahydrofuran (THF) with the following results: FPV-RPA nfo: [ FAM ] -AGGAAACATGGCTTTAGATGATATTCATGTACA [ THF ] AATTGTAACACCTTGGT [ P ].
Preparation of quantum dot marked mouse source carboxyl fluorescein antibody
Adopting borate buffer solution (0.01mol/L, pH 7.4) as a reagent to prepare a coupling agent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and an N-hydroxysuccinimide (NHS) solution, wherein the concentrations are both 10mg/mL, taking 50 muL of carboxyl modified water-soluble quantum dots (obtained from QD618-C-S001, the company Limited in the Biotechnology of royal Engton dragon, Shenzhen), adding 200 muL 0.05mol/L of 2- (N-morpholino) ethanesulfonic acid buffer solution (MES, pH 6), adding EDC and NHS, activating by a shaking table at room temperature for 30min, then centrifuging at 15000rpm for 30min, discarding the supernatant, adding 1mL MES buffer solution (pH 6) containing 5 mug FAM monoclonal antibody, centrifuging at 15000rpm for 30min after reacting by a shaking table at room temperature for 2h, discarding the supernatant, the suspension was resuspended in 1ml of 0.01mol/L phosphate buffer (0.01mol/L, pH 7.4), centrifuged, and the remaining antibody was washed off, finally resuspended in 100 μ L of a preservative solution (0.05mol/L borate buffer, pH 8.0), and then left at 4 ℃ for further use.
Detection of feline panleukopenia Virus
A RPA-FLD method for detecting feline panleukopenia virus comprising the steps of:
s1: synthesizing an RPA specific primer group and a probe;
s2: extracting DNA in a sample to be detected, and specifically comprising the following steps;
(1) adding 20 mu L of protease K into a clean 1.5mL centrifuge tube;
(2) adding 200 mu L of sample to be detected into the centrifuge tube (balancing at room temperature); if the sample volume is less than 200 mu L, 0.9% NaCl solution can be added for supplement;
(3) adding 200 mu L of Carrier RNA working solution, covering a tube cover, carrying out vortex oscillation for 15 seconds, and mixing uniformly, wherein in order to ensure that the cracking is sufficient, the sample and the Carrier RNA working solution need to be thoroughly mixed;
(4) incubating at 56 deg.C for 15min, centrifuging, and collecting liquid on tube wall;
(5) adding 250 μ L of anhydrous ethanol, vortex and oscillating for 15 s, and standing at room temperature for 5 min;
(6) centrifuging for 2min, and collecting liquid;
(7) transferring the solution in the centrifuge tube to RNase-Free adsorption column CR2, centrifuging at 8,000rpm (6000 Xg) for 1min, discarding the waste liquid, and placing the adsorption column back into the collection tube;
(8) opening the cover of the adsorption column, adding 500 mu L of buffer GD, covering the tube cover, centrifuging for 1min at 8,000rpm (6000 Xg), discarding the waste liquid, and placing the adsorption column back to the collection tube;
(9) opening the cover of the adsorption column, adding 600 μ L of rinsing liquid PW, covering the tube cover, standing for 2min, centrifuging at 8,000rpm (about 6,000 Xg) for 1min, discarding the waste liquid, and returning the adsorption column to the collection tube;
(10) repeating the step (9);
(11) the cover is opened, 500 mu L of absolute ethyl alcohol is added, centrifugation is carried out for 1min at 8,000rpm (about 6,000 Xg), and waste liquid is discarded;
(12) the adsorption column was returned, centrifuged at 12,000rpm (-13,400 Xg) for 3min, and the waste liquid was discarded;
(13) placing the adsorption column in RNase-Free centrifuge tube with volume of 1.5mL, standing at room temperature for 3min, and dripping 20-150 μ L of RNase-Free dd H into the middle part of the adsorption membrane2O, standing at room temperature for 5 min; centrifuging at 12,000rpm (-13,400 Xg) for 1min to collect the product;
s3: RPA amplification reaction: using the extracted DNA as a template, a probe, the RPA specific primer group in the step S1 as a primer, a buffer solution, a magnesium acetate solution and ddH2Performing an RPA amplification reaction in an RPA reaction tube by using O, wherein the following steps are performed:
the RPA reaction system was 50. mu.L, containing 2. mu. L, RehydrationBuffer 30. mu.L each of FPV-RPA-VP2-F and FPV-RPA-VP2-R and 2.7. mu.L of MgAC (280mmol/L), template and ddH2Total 11.3. mu.L of O; performing amplification reaction in water bath at 33-42 deg.C for 20 min;
s4: analyzing the amplification product obtained in the step S3 by using a quantum dot fluorescence immunochromatography method, which specifically comprises the following steps:
after mixing 10 μ L of RPA amplification product with 90 μ L of lateral chromatography buffer (30mmol/L Tris-HCL, 170mmol/L NaCl and 0.05% Tween-20), the mixture is dropped into a sample adding hole of a test strip containing the mouse-derived carboxyl fluorescein antibody marked by quantum dots, and after 10min, the result is observed, photographed and recorded, and the result is shown in figure 1.
And (4) judging a result: the positive sample, the quality control line and the detection line of the test strip all have clearly visible fluorescent strips; in the negative sample, only the control line shows a clearly visible fluorescence band.
As can be seen from FIG. 1, the RPA-FLD method can be used to detect feline panleukopenia virus.
Fourth, specificity test of feline panleukopenia Virus RPA-LFD method
Control viruses included: feline rhinotracheitis virus (FHV), calicivirus (FCV), Feline Infectious Peritonitis Virus (FIPV), feline coronavirus (FCoV);
extracting virus genes: adopting a cell/tissue RNA or DNA extraction kit of Tiangen biology company, and referring to the instruction;
the extracted DNA of each control virus is respectively subjected to the following amplification reaction and is dripped on a quantum dot fluorescence transverse flow test strip for detection,
and (3) amplification reaction: reagent: twist Amp Basic Kit instructions; feline panleukopenia virus RPA primers; the total volume of the amplification reaction system is 50 mu L, and the components and the final concentration are respectively as follows: FPV-RPA-VP2-F and FPV-RPA-VP2-R each 2. mu.L, Rehydration Buffer 30. mu.L and MgAC (280mM) 2.7. mu.L, template and ddH2Total 11.3. mu.L of O; and (3) amplification reaction process: at 40 ℃ for 20 min.
After mixing 10 mul of RPA amplification product with 90 mul of lateral chromatography buffer solution (30mmol/L Tris-HCL, 170mmol/L NaCl and 0.05% Tween-20), dripping the mixture into a quantum dot fluorescence transverse flow test strip in the embodiment, and observing, photographing and recording the result after 10 min; the results are shown in FIG. 2.
As can be seen from fig. 2, only the control line of the quantum dot fluorescence lateral flow test strip in this embodiment shows a clearly visible fluorescence band, thus indicating that the quantum dot fluorescence lateral flow test strip has specificity.
The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.
Sequence listing
<110> Zhejiang agriculture and forestry university
Zhejiang Guo Zhengzhi detection technology Co Ltd
<120> an RPA-FLD method for detecting feline panleukopenia virus
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Claims (10)
1. A method of detecting RPA-FLD for feline panleukopenia virus comprising the steps of:
s1: synthesizing an RPA specific primer group and a probe;
s2: extracting DNA in a sample to be detected;
s3: RPA amplification reaction: using the extracted DNA as a template, a probe, the RPA specific primer group in the step S1 as a primer, a buffer solution, a magnesium acetate solution and ddH2Performing RPA amplification reaction in an RPA reaction tube;
s4: analyzing the amplification product obtained in the step S3 by using a quantum dot fluorescence immunochromatography method;
wherein: the RPA specific primer group comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are as follows:
FPV-RPA-VP2-F：5’-AGCAGACTTGTACATTTAAATATGCCAGAAAGTGAAA-3’,
FPV-RPA-VP2-R：5’-TTCTTGTTCAAAACTAACTAAATGCAACTCACTCA-3’,
the 5' end of FPV-RPA-VP2-R is labeled by biotin;
the nucleotide sequence of the probe is formed by connecting two parts, namely a first part and a second part, wherein the nucleotide sequence of the first part is as follows: 5'-AGGAAACATGGCTTTAGATGATATTCATGTACA-3', the nucleotide sequence of the second part is: 5'-AATTGTAACACCTTGGT-3' the flow of the air in the air conditioner,
the 5 'end of the first part is connected with carboxyfluorescein, the 3' end of the second part is connected with phosphate, and the 3 'end of the first part and the 5' end of the second part are connected through tetrahydrofuran.
2. The RPA-FLD method according to claim 1, wherein the amplification temperature in step S3 is 33-42 ℃ and the reaction time is 20-30 min.
3. The RPA-FLD method according to claim 1, wherein step S4 specifically is:
and (5) adding lateral chromatography buffer solution into the amplification product obtained in the step (S3) for dilution, and adding the diluted lateral chromatography buffer solution into a sample injection hole of the quantum dot fluorescence immune layer test strip.
4. The RPA-FLD method according to claim 1, wherein the test strip used in the quantum dot fluorescence immunochromatography in step S4 is provided with a quantum dot-labeled murine carboxyfluorescein antibody, an avidin detection line, and a rabbit anti-mouse antibody quality control line.
5. The RPA-FLD method according to claim 4, wherein said quantum dot-labeled murine carboxyfluorescein antibody is prepared by:
a1: preparing a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and an N-hydroxysuccinimide solution;
a2: mixing the carboxyl modified water-soluble quantum dot, the 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride solution and the N-hydroxysuccinimide solution, sequentially activating and centrifuging at room temperature, removing the supernatant, adding a MES buffer solution containing carboxyl fluorescein, sequentially activating and centrifuging at room temperature, removing the supernatant, adding the buffer solution, repeatedly centrifuging, removing the supernatant, and finally adding a preserving solution for preservation.
6. The RPA-FLD method according to any of claims 1-5, wherein step S2 specifically comprises the steps of:
s2-1: adding Carrier RNA working solution into a sample to be detected, and uniformly mixing by vortex oscillation;
s2-2: centrifuging after incubation, and collecting liquid on the tube wall;
s2-3: adding absolute ethyl alcohol into the collected liquid, and standing at room temperature after vortex oscillation;
s2-4: centrifuging the liquid after standing at room temperature and collecting the liquid on the tube wall;
s2-5: transferring the liquid collected in the step S2-4 into an adsorption column for centrifugation;
s2-6: adding a buffer solution into the adsorption column and centrifuging;
s2-7: adding rinsing liquid into the adsorption column after centrifugation in the step S2-6, standing, centrifuging, and repeating the step after centrifugation;
s2-8: adding absolute ethyl alcohol into the adsorption column after the step S2-7, centrifuging, and standing;
s2-9: suspending the middle part of the adsorption film of the adsorption column after the step S2-8 and dropwise adding ddH2And O, standing at room temperature for 5-10min, and centrifuging to collect a solution on the tube wall, wherein the solution is the extracted DNA.
7. The RPA-FLD method according to claim 5, wherein said preservation solution is 0.05mol/L borate buffer at PH 8.
8. The RPA-FLD process according to claim 5, wherein the concentration of each of said 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and N-hydroxysuccinimide solution is 10 mg/mL.
9. The RPA-FLD process according to claim 5, wherein the preservation temperature in step A2 is 4 ℃.
10. The RPA-FLD method according to claim 6, wherein said adsorption column contains rnase.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391500A (en) * | 2020-12-09 | 2021-02-23 | 杭州奥泰生物技术股份有限公司 | Fluorescent quantitative PCR detection primer, probe and kit for simultaneously detecting cat parvovirus and cat HIV |
| CN112458205A (en) * | 2020-11-18 | 2021-03-09 | 东莞源和生物科技有限公司 | Reaction solution, primer pair and kit for detecting nucleic acid of feline pestivirus |
| CN117051173A (en) * | 2023-10-12 | 2023-11-14 | 上海基灵生物科技有限公司 | Cat digestive tract virus PCR detection method and application |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002053768A2 (en) * | 2001-01-05 | 2002-07-11 | Diagnosticart Llc | Dna rapid immune detection methods and apparatus |
| WO2005118853A2 (en) * | 2004-06-01 | 2005-12-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
| CN101907625A (en) * | 2010-07-08 | 2010-12-08 | 浙江大学 | Method for preparing quantum dot immune fluorescent probe |
| CN105296674A (en) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | Detecting kit for severe fever with thrombocytopenia syndrome viruses (SFTSV) and preparation method of detecting kit |
| CN107435080A (en) * | 2017-04-20 | 2017-12-05 | 华南农业大学 | A kind of recombinase normal temperature amplification of nucleic acid for detecting CSFV（RT‑RPA）Test strips kit and application |
| CN109136410A (en) * | 2018-10-16 | 2019-01-04 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Feline Panleukopenia Virus LAMP detection primer group, kit and detection method |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
| CN110257368A (en) * | 2019-05-16 | 2019-09-20 | 上海臻迪基因科技有限公司 | The method and system of free nucleic acid is separated from the sample containing free nucleic acid |
-
2020
- 2020-01-16 CN CN202010049723.6A patent/CN111197110A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002053768A2 (en) * | 2001-01-05 | 2002-07-11 | Diagnosticart Llc | Dna rapid immune detection methods and apparatus |
| WO2005118853A2 (en) * | 2004-06-01 | 2005-12-15 | Asm Scientific, Inc. | Recombinase polymerase amplification |
| CN101907625A (en) * | 2010-07-08 | 2010-12-08 | 浙江大学 | Method for preparing quantum dot immune fluorescent probe |
| CN105296674A (en) * | 2015-12-03 | 2016-02-03 | 江苏省疾病预防控制中心 | Detecting kit for severe fever with thrombocytopenia syndrome viruses (SFTSV) and preparation method of detecting kit |
| CN107435080A (en) * | 2017-04-20 | 2017-12-05 | 华南农业大学 | A kind of recombinase normal temperature amplification of nucleic acid for detecting CSFV（RT‑RPA）Test strips kit and application |
| CN109136410A (en) * | 2018-10-16 | 2019-01-04 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Feline Panleukopenia Virus LAMP detection primer group, kit and detection method |
| CN109652597A (en) * | 2019-01-26 | 2019-04-19 | 沈阳农业大学 | It is a kind of for detect dog, cat, ermine parvovirus general RPA primer, RPA probe, kit and nucleic acid detection method |
| CN110257368A (en) * | 2019-05-16 | 2019-09-20 | 上海臻迪基因科技有限公司 | The method and system of free nucleic acid is separated from the sample containing free nucleic acid |
Non-Patent Citations (4)
| Title |
|---|
| 刘冬虹等: "重组酶聚合酶扩增技术的研究进展", 《检验检疫学刊》 * |
| 周宏专等: "犬细小病毒研究进展", 《动物医学进展》 * |
| 樊晓旭等: "重组酶聚合酶扩增技术在疾病快速检测中的研究进展", 《中国动物检疫》 * |
| 高威芳等: "重组酶聚合酶扩增技术:一种新的核酸扩增策略", 《中国生物化学与分子生物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458205A (en) * | 2020-11-18 | 2021-03-09 | 东莞源和生物科技有限公司 | Reaction solution, primer pair and kit for detecting nucleic acid of feline pestivirus |
| CN112391500A (en) * | 2020-12-09 | 2021-02-23 | 杭州奥泰生物技术股份有限公司 | Fluorescent quantitative PCR detection primer, probe and kit for simultaneously detecting cat parvovirus and cat HIV |
| CN117051173A (en) * | 2023-10-12 | 2023-11-14 | 上海基灵生物科技有限公司 | Cat digestive tract virus PCR detection method and application |
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