CN112816701A - Method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit - Google Patents
Method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit Download PDFInfo
- Publication number
- CN112816701A CN112816701A CN202011585426.XA CN202011585426A CN112816701A CN 112816701 A CN112816701 A CN 112816701A CN 202011585426 A CN202011585426 A CN 202011585426A CN 112816701 A CN112816701 A CN 112816701A
- Authority
- CN
- China
- Prior art keywords
- lateral flow
- line
- detection
- ricin
- flow chromatography
- 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.)
- Granted
Links
- 108010039491 Ricin Proteins 0.000 title claims abstract description 76
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 50
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 77
- 238000012360 testing method Methods 0.000 claims abstract description 36
- 238000011534 incubation Methods 0.000 claims abstract description 11
- 239000010931 gold Substances 0.000 claims abstract description 10
- 229910052737 gold Inorganic materials 0.000 claims abstract description 10
- 239000002245 particle Substances 0.000 claims abstract description 9
- 238000009877 rendering Methods 0.000 claims abstract description 7
- 238000010521 absorption reaction Methods 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims abstract description 6
- 239000002105 nanoparticle Substances 0.000 claims abstract description 6
- 229920003023 plastic Polymers 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 108010090804 Streptavidin Proteins 0.000 claims description 10
- 230000000007 visual effect Effects 0.000 claims description 6
- 238000003384 imaging method Methods 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 239000003053 toxin Substances 0.000 abstract description 6
- 231100000765 toxin Toxicity 0.000 abstract description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 18
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 16
- 239000000523 sample Substances 0.000 description 14
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 10
- 229930024421 Adenine Natural products 0.000 description 10
- 229960000643 adenine Drugs 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 8
- 229960002685 biotin Drugs 0.000 description 8
- 235000020958 biotin Nutrition 0.000 description 8
- 239000011616 biotin Substances 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 108010058846 Ovalbumin Proteins 0.000 description 7
- 229940098773 bovine serum albumin Drugs 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 229940092253 ovalbumin Drugs 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 235000004443 Ricinus communis Nutrition 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 108010066676 Abrin Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000013076 target substance Substances 0.000 description 4
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 150000001615 biotins Chemical class 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003008 fumonisin Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000012125 lateral flow test Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- ZJAOAACCNHFJAH-UHFFFAOYSA-N phosphonoformic acid Chemical compound OC(=O)P(O)(O)=O ZJAOAACCNHFJAH-UHFFFAOYSA-N 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- PETSAYFQSGAEQY-UHFFFAOYSA-N ricinine Chemical compound COC=1C=CN(C)C(=O)C=1C#N PETSAYFQSGAEQY-UHFFFAOYSA-N 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101710186708 Agglutinin Proteins 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000006721 cell death pathway Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 229960005102 foscarnet Drugs 0.000 description 1
- 230000008073 immune recognition Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- -1 mercury ions Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
-
- 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
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of toxin detection, and relates to a method for rapidly detecting ricin by using a colloidal gold lateral flow chromatography, and colloidal gold lateral flow chromatography test paper and a kit. The method comprises the following steps: (1) incubating H1-AuNPs-FAM antibody, FAM antibody-AuNPs and a sample to be detected containing ricin; the sequence of H1 is shown in SEQ ID NO: 1 is shown in the specification; the particle size of AuNPs is 2-10 nm; (2) dropwise adding the mixed solution obtained by incubation in the step (1) onto lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC membrane with a plastic back card; the detection zone comprises a T line binding zone and a C line binding zone; the T line combination area is provided with a detection line, and the C line combination area is provided with a control line; (3) and judging and reading the detection result according to the color rendering values of the gold nanoparticles on the detection line and the control line. The invention establishes the colloidal gold lateral flow chromatography method for detecting ricin, the method does not need to prepare corresponding antibodies, is simple to operate, and can meet the requirements of field detection.
Description
Technical Field
The invention belongs to the field of toxin detection, and particularly relates to a method for rapidly detecting ricin by using a colloidal gold lateral flow chromatography, and a colloidal gold lateral flow chromatography kit for rapidly detecting ricin.
Background
Castor, the latin school name Ricinus communis l, originally produced in somali and kenya in northeast africa, is widely distributed in tropical regions of the world. The stem, leaf or castor seed of castor plant contains toxic substances, mainly including ricin, ricinine, castor reactogen, etc. Ricin (RT) can be dissolved in weak acid or water after chemical purification, and has stable physicochemical properties. Chemically, ricin is a highly toxic plant protein consisting of A, B two peptide chains. Active ricin enters eukaryotic cells through B-chain mediated endocytosis, and then a-chain purifies specific adenine from 28S rRNA, thereby inducing protein synthesis failure and finally activating cell death pathways.
Ricin is used as a highly toxic protein, and the toxicity of ricin is higher than that of cyanide 6000 times. Ricin, due to its strong toxicity and ready availability, has attracted the interest of researchers and has become an interest for terrorists, the well-known markov virus case being an example. Development of sensitive and efficient detection means is a main measure for dealing with threats. The traditional detection technology mainly comprises a radioimmunoassay, an enzyme-linked immunosorbent assay (ELISA), a high performance liquid chromatography, an immuno-PCR, a colloidal gold labeling technology and the like. However, these detection methods need to be carried out by means of expensive instruments and professional technicians, which is not favorable for field use and limits further application and popularization.
The Gold Immunochromatography (GICA) is a field and rapid detection method which is developed at the end of the 20 th century and combines an immune recognition technology and a chromatography technology by using gold nanoparticles as a color development element. The basic principle is that the color of the colloidal gold appears on a lateral chromatography test strip by utilizing the specific recognition and combination of antigen and antibody. By utilizing the lateral chromatography test strip technology, the rapid determination of mercury ions in water and the hypersensitive detection of the foscarnet and the like in the aquatic products through signal amplification are realized.
Ricin is strong in toxicity and easy to obtain, and is a potential bioterrorism agent, so that a method for rapidly detecting ricin is urgently needed to be established so as to guarantee national public safety.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting ricin by using a colloidal gold lateral flow chromatography and a colloidal gold lateral flow chromatography kit.
In order to achieve the above object, a first aspect of the present invention provides a method for rapidly detecting ricin by colloidal gold lateral flow chromatography, comprising the steps of:
(1) incubating the H1-AuNPs-FAM antibody with a sample to be tested containing ricin; the sequence of H1 is shown in SEQ ID NO: 1 is shown in the specification; the particle size of AuNPs is 2-10 nm;
5’-FAM-GAC CTA CTT ATG AAA AAA AAA AAA TTA TAT CTA TCT- Biotin-3’(SEQ ID NO：1)
(2) dropwise adding the mixed solution obtained by incubation in the step (1) onto lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC membrane with a plastic back card; the detection zone comprises a T line binding zone and a C line binding zone; the T line combination area is provided with a detection line, and the C line combination area is provided with a control line; secondary antibodies of streptavidin and an anti-FAM antibody are respectively fixed on the detection line and the control line;
(3) and judging and reading the detection result according to the color rendering values of the gold nanoparticles on the detection line and the control line.
According to the invention, the H1-AuNPs-FAM antibody can be obtained by incubating H1 with AuNPs-FAM antibody.
The lateral flow test strip of the present invention may be obtained commercially or may be obtained by self-manufacture.
According to the invention, the manner of interpreting the detection result in step (3) may be either visual observation or instrumental imaging, such as Image J imaging. Visual observation is usually a qualitative judgment, and instrumental imaging is usually a quantitative test.
According to the present invention, preferably, the method further comprises the step of preparing a standard curve: according to the methods in the steps (1) and (2), detecting a series of samples to be detected containing ricin with known concentration, measuring a plurality of color rendering values, and respectively drawing by taking the concentration and the color rendering values as horizontal and vertical coordinates to obtain a standard curve. At this time, the concentration of ricin in the test sample containing ricin can be calculated based on the standard curve.
The detection principle of the ricin lateral flow chromatography test paper is shown in figure 1. It comprises two steps: (1) H1-AuNPs-FAM antibody was incubated with ricin; (2) the test was performed with Lateral Flow Assay (LFA) test paper and the results were read. The present invention designs an oligonucleotide sequence H1 containing consecutive adenine based on the principle that ricin is a target substance and adenine can be purified by active ricin a chain to cleave the adenine-containing oligonucleotide sequence, and as shown in fig. 1, when ricin is a target substance, consecutive adenine is cleaved in the H1 sequence, resulting in cleavage of the H1 sequence. For the LFA strip, as shown in fig. 1, it includes four sections: sample pad, detection zone (C-line binding zone, T-line binding zone), absorbent pad and NC film with plastic back card. And secondary antibodies of streptavidin and an anti-FAM antibody are respectively fixed on a detection line and a control line of the NC membrane. The surface of AuNPs is modified with FAM antibody, when no target exists, the mixed liquid of H1 sequence and FAM antibody-AuNPs is dripped on a sample pad, the mixed liquid migrates to an absorption pad under the action of capillary force, H1 marked with biotin and FAM is specifically combined with gold nanoparticles of the modified FAM antibody to form a compound. When these complexes pass through the detection line, they are captured by streptavidin immobilized on the detection line, producing a visible signal on the detection line. Excess complex continues to migrate on the NC membrane and is captured by a secondary antibody against the FAM antibody immobilized on the control line, thereby developing color. If ricin exists in the sample, after mixed incubation, the H1 sequence is broken and dissolved, the sandwich structure on the detection line is reduced or disappeared, and the color of the detection line is lightened or disappeared. In short, in the absence of the target ricin, a negative result was observed with two red lines ("on-off"). As the concentration of the target substance increases, the color of the detection line gradually becomes lighter, and when the concentration reaches a certain level, only one red line ("off") is observed on the control line, and as a positive result, the red color on the control line is used for proving that the LFA test paper works normally.
The second aspect of the invention provides a colloidal gold lateral flow chromatography kit for rapidly detecting ricin, which comprises the following components:
(1) colloidal gold lateral flow chromatography test paper; the test paper comprises: the device comprises a sample pad, a detection area, an absorption pad and an NC (numerical control) film with a plastic back card; the detection zone comprises a T line binding zone and a C line binding zone; the T line combination area is provided with a detection line, and the C line combination area is provided with a control line; secondary antibodies of streptavidin and an anti-FAM antibody are respectively fixed on the detection line and the control line;
(2) FAM antibodies-AuNPs; wherein the particle size of AuNPs is 2-10 nm;
(3) an H1 nucleic acid strand; the sequence of H1 is shown in SEQ ID NO: 1 is shown.
In the kit of the present invention, FAM antibody-AuNPs and H1 chains may be provided separately, or may be provided in the form of H1-AuNPs-FAM antibody.
Based on a colloidal gold immunochromatographic method, the invention designs an oligonucleotide sequence H1 modified by biotin and FAM together as an identification probe according to the principle that a ricin A chain can purify adenine so as to break an oligonucleotide sequence containing adenine, develops a colloidal gold lateral flow chromatography test strip taking the H1 sequence modified by FAM and biotin as an identification element, establishes a method for rapidly and semi-quantitatively detecting ricin, realizes the visual semi-quantitative detection of ricin, and investigates the sensitivity and specificity of the detection method. The ricin colloidal gold lateral flow chromatography test strip provided by the invention has the minimum detection value lower than 10ng/mL, and can meet the detection requirement. The detection method provided by the invention is simple to operate, does not need large-scale instruments and equipment, and can meet the requirements of field detection. In addition, the invention adopts the oligonucleotide chain as the identification element, and the ricin antibody does not need to be prepared, thereby reducing the cost.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a schematic diagram of the flow and principle of qualitative detection of ricin by the colloidal gold lateral flow test strip of the present invention.
Fig. 2 shows the screening results of oligonucleotide sequences, wherein a is polyacrylamide gel electrophoresis pattern, and b is fluorescence spectrum of oligonucleotide sequences, wherein Lane 1: p1; lane 2: p2; lane 3: p3; lane 4: p3+ RT; lane 5: a2; lane 6: a2+ RT; lane 7: h1; lane 8: h2+ RT.
FIG. 3 shows the concentration optimization result of the H1 sequence, wherein 1-5 sequentially represent the concentrations of H1 as follows: 100nM, 50nM, 25nM, 10nM and 1 nM.
FIG. 4 shows the optimization result of the mixing incubation time of ricin and H1-AuNPs-FAM antibody, wherein 1-4 are added with 1 mug/mL ricin in sequence for incubation for 30min, 60min, 90min and 120 min.
Figure 5 shows the visual sensitivity of the ricin colloidal gold lateral flow chromatography test strip. Wherein, 1-5 are ricin 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL and 1ng/mL in sequence; 6: blank control.
Fig. 6 shows a linear relationship between relative intensity and log concentration of ricin.
Fig. 7 is a specific detection result of a ricin colloidal gold lateral flow chromatography test strip, wherein, 1: ricin; 2: abrin; 3: SEB; 4: BSA; 5: OVA; 6: FB (full Fall Back)1(ii) a 7: blank control.
FIG. 8 is a 20nm AuNPs-FAM antibody TEM characterization result chart.
FIG. 9 shows the results of H1-AuNPs-FAM (20nm) detection of ricin. a: detecting ricin by 5 mu L of 25pM H1, wherein 1-3 are ricin 10 mu g/mL, 500ng/mL and 10ng/mL in sequence; 4 is blank control; b: detecting ricin by using 5 mu L of 25pM H1, wherein 1-3 are ricin 10 mu g/mL, 100ng/mL and 10ng/mL in sequence; and 4 is blank control.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
An LX-200 palm centrifuge, an ST70-2 micropore constant temperature oscillator, an OSE-DB-02 refrigeration type five-section program control metal bath, a constant temperature incubator, a ricin standard substance (Beijing Han spectral pharmaceutical and biological research institute), staphylococcus aureus enterotoxin B (SEB) (self-made), a fumonisin standard substance (Shandong blue all biotechnology limited), Bovine Serum Albumin (BSA) and Ovalbumin (OVA) (Sigma-Aldrich), abrin agglutinin (poison drug research institute of military medical research institute, Beijing), and a colloidal gold lateral flow chromatography test strip (R ((R))
Hybrid detect). All nucleotides (ODN) were purchased from shanghai bio-technology ltd (shanghai, china) and purified by High Performance Liquid Chromatography (HPLC) as follows:
TABLE 1 oligonucleotide sequences
The invention takes ricin as a target object, designs an oligonucleotide P3 sequence containing continuous adenine, a modified biotin P1 sequence and a modified FAM P2 sequence, and uses NUPACK software to fit and optimize analysis; designs A1 sequence of modified biotin and A2 sequence of modified FAM; an H1 sequence which simultaneously modifies biotin and FAM is designed, and a DNA sequence is screened through a polyacrylamide gel electrophoresis experiment and a SYBR Green1 fluorescence experiment.
Screening of sequences
The designed oligonucleotide sequences containing the sequence A are listed in Table 1, wherein the sequences P1 and P2 are complementary and paired with the two ends of the sequence P3 respectively, so that a P1-P3-P2 complex is formed, the 5 'end of P1 is modified with biotin, and the 3' end of P2 is modified with FAM, so that the complex can be captured by streptavidin on a detection line and can be developed. The A1 sequence can be complementary and matched with the A2 sequence, biotin is modified at the 3 'end of A1, FAM is modified at the 3' end of A2, and the biotin and the FAM can be captured by streptavidin on a detection line for color development. According to the designed sequence, ricin with the same concentration is added into different oligonucleotide sequences respectively under the same volume and concentration, and polyacrylamide gel electrophoresis is used for representing the cleavage and dissolution capacity of the toxin on the oligonucleotide sequences. And (3) observing the complementary pairing condition of the sequences by using the principle that SYBR Green1 only stains double-stranded DNA, and finally screening out a proper oligonucleotide sequence.
And (3) using polyacrylamide gel electrophoresis to characterize the cleavage and dissolution capacity of the target substance on the oligonucleotide sequence. As shown in a of FIG. 2, the same concentration of the target was added to different oligonucleotide sequences at the same volume and concentration, respectively, and a diffuse band appeared after the addition of the target, and the brightness was significantly reduced. Indicating that the target can cleave and dissolve the oligonucleotide sequence containing consecutive adenine. When the P1-P3-P2 composite system is used for detection, the detection limit is too high, so the system is not used. A principle that SYBR Green1 only stains double-stranded DNA is utilized, the sequences A1 and A2 are mixed and incubated and then stained by SYBR Green1, and a fluorescence result (b in figure 2) shows that after the sequences A1 and A2 are mixed and incubated, an A1-A2 binding chain and A1 and A2 self-folding are generated to form a double-stranded structure, so that the cutting capability of toxin on the sequence A2 is influenced, and the color change of a detection line is interfered. The H1 sequence was therefore finally selected as the recognition element.
AuNPs particle size optimization
At the early stage, gold nanoparticles (20nm gold transmission electron microscopy picture is shown in figure 8) of 20nm modified FAM antibody are placed in a micropore constant temperature oscillator to be mixed and incubated with 25pM of H1 sequence, then a target is added for detection, 50 mu L of reaction solution is dropwise added in a test strip sample area, and the test result is observed after standing for 10 min. The results are shown in FIG. 9. As can be seen from the results, the color of 20nm gold particles was not lightened even at a concentration of 10. mu.g/mL of toxin. Therefore, experiments were subsequently performed with 5nm gold particles.
H1 sequence incubation concentration optimization
mu.L of oligonucleotide sequence H1 of 100nM, 50nM, 25nM, 10nM and 1nM is placed in a metal bath to be incubated for 1H (4 stages are set: 95 ℃ for 5min, 80 ℃ for 10min, 50 ℃ for 10min, 37 ℃ for 10min), 10. mu.L of 50ng/mL FAM-AuNPs and 85. mu.L of Tris-HCl buffer solution are added into a reaction tube to prepare a reaction system of 100. mu.L, 50. mu.L of reaction solution is dropped into a test strip sample area, and the experiment result is observed after being stood for 10 min.
The H1 sequence which modifies FAM and biotin is a recognition element in a lateral flow strip, and can be specifically combined with streptavidin fixed on a test strip detection line, and a sandwich structure formed on the detection line can generate obvious visible signals. Therefore, the color of the detection line is greatly influenced by the addition of the H1 sequence concentration. In the present invention, color changes of the detection line and the control line were observed with the addition of H1 sequences (100nM, 50nM, 25nM, 10nM and 1nM) at different concentrations, and the results are shown in FIG. 3, from which it can be seen that the optimal concentration of H1 added is 10 nM.
Target addition incubation time optimization
Adding ricin standard substances with different concentrations into reaction tubes containing 5 mu L H1-AuNPs-FAM antibodies respectively, placing the ricin standard substances into a micropore constant temperature oscillator, incubating the ricin standard substances for 30min, 60min, 90min and 120min at 37 ℃ and 700rpm respectively, after the reaction is finished, dropwise adding 50 mu L of reaction liquid into a test strip sample area, standing for 10min, and observing the experimental result.
When the target ricin exists, the cutting dissolves an H1 sequence containing adenine, so that the H1 sequence is broken, and the color of the detection line is lightened or disappears. Therefore, the incubation time of the added target and the H1 sequence has a great influence on the detection effect. In the present invention, the color changes of the detection line and the control line were observed under the conditions of different incubation times (30min, 60min, 90min and 120min) when the same concentration of H1 sequence and the target were added, and the results are shown in FIG. 4. As can be seen from FIG. 4, the optimal time for mixing and incubating the sequence H1 with the target was 120 min.
Determination of detection range
Ricin was diluted with Tris-HCl buffer to 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL, 1ng/mL, and each concentration was assayed 3 times. The sample treatment solution was used as a negative control. And adding 95 mu L of ricin with different concentrations into a 5 mu L H1-AuNPs-FAM antibody mixture for mixed incubation for 120min, after the reaction is finished, dropwise adding 50 mu L of reaction liquid into a test strip sample area, standing for 10min to observe an experimental result, and performing result analysis by using image J software.
Ricin was diluted to 100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL and 1ng/mL with Tris-HCl buffer and detected with a colloidal gold lateral flow strip. As a result, as shown in FIG. 5, the sensitivity was 10ng/mL by direct visual observation.
Fitting of standard curve
Based on the above results, analysis was performed using image J software, using the logarithm of the ricin concentration (100ng/mL, 80ng/mL, 40ng/mL, 10ng/mL, 1ng/mL) as the abscissa, the relative intensity detected at each concentration as the ordinate, the relative intensity representing the integrated density ratio of the detection line and the control line, and the error bar representing the standard deviation of three measurements. As shown in fig. 6, the linear equation is-0.1563 x + 0.9521; r20.98647. The linear relationship is good within the range of 1-100 ng/mL.
Specificity detection
The prepared colloidal gold lateral flow chromatography test strip is used for detecting abrin, fetal Bovine Serum Albumin (BSA), Ovalbumin (OVA), staphylococcus aureus enterotoxin B and fumonisin under the same experimental conditions. Repeating for 3 times to judge the specificity of the test strip.
Mixing Ovalbumin (OVA), fetal Bovine Serum Albumin (BSA), abrin, Staphylococcus aureus enterotoxin B (SEB), and Fumonisin (FB) with Tris-HCl buffer solution1) Diluted to 100ng/mL, and detected by a colloidal gold lateral flow chromatography test strip. As shown in fig. 7, it is clear from fig. 7 that the color of the test line band of ricin alone is relatively lighter, while the bands of other proteins and toxins are not significantly changed from the control group.
Repeatability detection
In the linear detection range, high, medium and low 3 concentrations (100ng/mL, 60ng/mL, 20ng/mL) were detected 6 times, concentrations were calculated from a fitted curve, and the repetition rate (CV) was calculated as the ratio of the variance (STDEVA) to the mean (AVERAGE). The results are shown in Table 2.
TABLE 2 repeated detection of ricin colloidal gold lateral flow chromatography test paper strip
| Actual concentration (ng/mL) | AVERAGE(ng/mL) | STDEVA | CV(％) |
| 100 | 103.1609 | 17.52 | 16.98 |
| 60 | 65.391 | 7.185 | 10.99 |
| 20 | 22.446 | 2.73 | 12.17 |
The invention uses the oligonucleotide sequences of modified biotin and FAM as recognition probes to combine with colloidal gold particles for marking FAM antibodies; coating streptavidin on a nitrocellulose membrane as a detection line, coating a secondary antibody of an anti-FAM antibody as a control line, establishing a ricin naked eye detection method, evaluating the specificity and repeatability of the ricin naked eye detection method, performing data processing analysis by using image J software, and drawing a detection curve. The colloidal gold lateral flow chromatography test strip completes detection within 120min, can detect 1ng/mL at least, and has good specificity and repeatability. The invention establishes the colloidal gold lateral flow chromatography method for detecting ricin, the method does not need to prepare corresponding antibodies, is simple to operate, and can meet the requirements of field detection.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> institute of environmental and operational medicine of military medical research institute of military science institute
<120> method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit
<130> BJI2002405PY
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gacctactta tgaaaaaaaa aaaattatat ctatct 36
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttttttttta tatatata 18
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taacataatt aggtcttttt t 21
<210> 4
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gacctaatta tgaaaaaaaa aaaattatat atatat 36
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
taacataatt aggtcttttt t 21
<210> 6
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gacctaatta tgaaaaaaaa aaaattatat atatat 36
Claims (7)
1. A method for rapidly detecting ricin by colloidal gold lateral flow chromatography is characterized by comprising the following steps:
(1) incubating the H1-AuNPs-FAM antibody with a sample to be tested containing ricin; the sequence of H1 is shown in SEQ ID NO: 1 is shown in the specification; the particle size of AuNPs is 2-10 nm;
(2) dropwise adding the mixed solution obtained by incubation in the step (1) onto lateral flow chromatography test paper; the lateral flow chromatography test paper comprises a sample pad, a detection area, an absorption pad and an NC membrane with a plastic back card; the detection zone comprises a T line binding zone and a C line binding zone; the T line combination area is provided with a detection line, and the C line combination area is provided with a control line; secondary antibodies of streptavidin and an anti-FAM antibody are respectively fixed on the detection line and the control line;
(3) and judging and reading the detection result according to the color rendering values of the gold nanoparticles on the detection line and the control line.
2. The method for rapidly detecting ricin by colloidal gold lateral flow chromatography as claimed in claim 1, wherein the H1-AuNPs-FAM antibody is obtained by incubating H1 with AuNPs-FAM antibody.
3. The method for rapidly detecting ricin by colloidal gold lateral flow chromatography as claimed in claim 1, wherein the manner of interpreting the detection result in step (3) is visual observation or instrumental imaging.
4. The method for rapidly detecting ricin by colloidal gold lateral flow chromatography as claimed in claim 1, wherein the method further comprises the step of preparing a standard curve: according to the methods in the steps (1) and (2), detecting a series of samples to be detected containing ricin with known concentration, measuring a plurality of color rendering values, and respectively drawing by taking the concentration and the color rendering values as horizontal and vertical coordinates to obtain a standard curve.
5. The method for rapidly detecting ricin by colloidal gold lateral flow chromatography as claimed in claim 4, further comprising: based on the standard curve, the concentration of ricin in the test sample containing ricin was calculated.
6. A colloidal gold lateral flow chromatography kit for rapidly detecting ricin comprises the following components:
(1) colloidal gold lateral flow chromatography test paper; the test paper comprises: the device comprises a sample pad, a detection area, an absorption pad and an NC (numerical control) film with a plastic back card; the detection zone comprises a T line binding zone and a C line binding zone; the T line combination area is provided with a detection line, and the C line combination area is provided with a control line; secondary antibodies of streptavidin and an anti-FAM antibody are respectively fixed on the detection line and the control line;
(2) FAM antibodies-AuNPs; wherein the particle size of AuNPs is 2-10 nm;
(3) an H1 nucleic acid strand; the sequence of H1 is shown in SEQ ID NO: 1 is shown.
7. The kit for rapidly detecting ricin by colloidal gold lateral flow chromatography as claimed in claim 6, wherein FAM antibody-AuNPs and H1 chains are provided in the form of H1-AuNPs-FAM antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011585426.XA CN112816701B (en) | 2020-12-28 | 2020-12-28 | Method for rapidly detecting ricin by colloid Jin Celiu chromatography and colloid Jin Celiu chromatography kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011585426.XA CN112816701B (en) | 2020-12-28 | 2020-12-28 | Method for rapidly detecting ricin by colloid Jin Celiu chromatography and colloid Jin Celiu chromatography kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112816701A true CN112816701A (en) | 2021-05-18 |
| CN112816701B CN112816701B (en) | 2023-12-26 |
Family
ID=75854432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011585426.XA Active CN112816701B (en) | 2020-12-28 | 2020-12-28 | Method for rapidly detecting ricin by colloid Jin Celiu chromatography and colloid Jin Celiu chromatography kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112816701B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113640522A (en) * | 2021-09-02 | 2021-11-12 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip for detecting T-2 toxin, kit and detection method |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997001758A1 (en) * | 1995-06-29 | 1997-01-16 | Dako A/S | Method for the simultaneous detection of different antibodies and/or antigens |
| WO2008001372A2 (en) * | 2006-06-29 | 2008-01-03 | Zaid Orniv Ltd. | System and method for management of property data and processes |
| CN101206224A (en) * | 2006-12-21 | 2008-06-25 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting ricin and preparation method thereof |
| CN101609091A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of new fast qualitative and the colloidal gold immunochromatographimethod method of detection by quantitative ricin (WA) |
| CN108486258A (en) * | 2018-01-29 | 2018-09-04 | 南昌大学 | A kind of kit and its method of visual quickly detection food-borne pathogens |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| CN111621598A (en) * | 2020-06-12 | 2020-09-04 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper by adopting wire-free method and application thereof in CRISPR nucleic acid detection |
-
2020
- 2020-12-28 CN CN202011585426.XA patent/CN112816701B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997001758A1 (en) * | 1995-06-29 | 1997-01-16 | Dako A/S | Method for the simultaneous detection of different antibodies and/or antigens |
| WO2008001372A2 (en) * | 2006-06-29 | 2008-01-03 | Zaid Orniv Ltd. | System and method for management of property data and processes |
| CN101206224A (en) * | 2006-12-21 | 2008-06-25 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immunity chromatography test paper for detecting ricin and preparation method thereof |
| CN101609091A (en) * | 2009-07-29 | 2009-12-23 | 中国检验检疫科学研究院 | A kind of new fast qualitative and the colloidal gold immunochromatographimethod method of detection by quantitative ricin (WA) |
| CN108486258A (en) * | 2018-01-29 | 2018-09-04 | 南昌大学 | A kind of kit and its method of visual quickly detection food-borne pathogens |
| CN109142704A (en) * | 2018-07-06 | 2019-01-04 | 军事科学院军事医学研究院环境医学与作业医学研究所 | A kind of kit and method based on bio-barcode and rolling circle amplification detection T-2 toxin |
| CN109765375A (en) * | 2019-01-10 | 2019-05-17 | 南京农业大学 | Three mycotoxin colloidal gold immune chromatography tests and preparation method |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| CN111621598A (en) * | 2020-06-12 | 2020-09-04 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper by adopting wire-free method and application thereof in CRISPR nucleic acid detection |
Non-Patent Citations (5)
| Title |
|---|
| YUAN SHUAI 等: "Dual Signal Amplification Strategy for Detection of Ricin Toxin through Bio-Barcode Assay Combined with Hybridization Chain Reaction", CHINESE JOURNAL OF ANALYTICAL CHEMISTRY, vol. 47, no. 1, pages 147 - 154 * |
| 王宗义;贺平丽;张丽英;杨文军;李德发;金曙光;: "夹心ELISA快速检测蓖麻粕中残留的蓖麻毒素", 饲料工业, no. 17, pages 30 - 32 * |
| 王玉霞;乔虹;刘子侨;: "蓖麻毒素毒性作用机制及防治研究进展", 中国药理学与毒理学杂志, no. 12, pages 1385 - 1395 * |
| 胡金强;魏向珂;黄润娜;孙新城;景建洲;高辉;耿尧;董彩文;姜春鹏;: "食源性致病菌RPA检测技术研究进展", 食品工业科技, no. 07, pages 335 - 340 * |
| 赵旭 等: "蓖麻毒素及其解毒剂研究进展", 国际药学研究杂志, vol. 44, no. 01, pages 35 - 39 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113640522A (en) * | 2021-09-02 | 2021-11-12 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colloidal gold test strip for detecting T-2 toxin, kit and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112816701B (en) | 2023-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2002506200A (en) | Biosensor array containing cell populations confined in micropores | |
| Kondratov et al. | Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting | |
| CN111440793B (en) | Novel coronavirus nucleic acid detection kit | |
| US20230081589A1 (en) | Universal Aptamer-based Colloidal Gold Lateral Flow Test Strip for Detecting Small-molecule Substances | |
| AU2021100190A4 (en) | CRISPR-Cas12a-based MOF-DNA Hydrogel Colorimetric Assay Kit and Method | |
| WO1989010566A1 (en) | Process for forming and using microdroplets | |
| CN109142710B (en) | Method for rapidly and sensitively detecting tetrodotoxin TTX | |
| JP2009537816A (en) | Sample control for correction of sample matrix effects in analytical detection methods | |
| CN108192948A (en) | A kind of method using alpha hemolysin nano-pore detection DNA glycosylase activity | |
| WO2020206828A1 (en) | Alternariol aptamer affinity column and preparation method and application therefor | |
| CN108728554A (en) | Fluorescence method test strips and the quick discriminating mutton adulteration method for using the fluorescence method test strips | |
| CN110106233A (en) | A kind of digital pcr detection method of extracellular vesica/excretion body | |
| CN112816701A (en) | Method for rapidly detecting ricin by colloidal gold lateral flow chromatography and colloidal gold lateral flow chromatography kit | |
| CN110296961A (en) | The building and application of controllable Nano-Au probe based on double block D NA | |
| WO2020014883A1 (en) | Single-stranded dna aptamer specifically recognizing tobramycin and application thereof | |
| CN108950078A (en) | A kind of primed probe group and kit based on double fluorescent PCR method joint-detection batai virus and Turner virus | |
| CN110734990A (en) | Detection reagent, kit and application thereof | |
| CN112608983B (en) | Paper-based detection method for exosome | |
| Yalçin et al. | Is There an Association Between Extreme Levels of Boron Exposure and Decrease in Y: X Sperm Ratio in Men? Results of an Epidemiological Study | |
| EP1564556A1 (en) | Method and apparatus for assaying several analytes simultaneously with an internal control | |
| EP3929307A1 (en) | Methods for detecting the presence of sepsis | |
| CN110095422B (en) | Aptamer for rapidly detecting bisphenol A, aptamer nanogold material, kit and application thereof | |
| CN114113576B (en) | Colloidal gold test strip for detecting cocaine, preparation method and method for testing cocaine | |
| CN110412024B (en) | Hg2+ detection method based on aptamer and nanogold | |
| CN109142751B (en) | Method for sensitively detecting tetrodotoxin TTX based on nucleic acid cleavage enzyme I immune marker |
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 |