CN105759029A - Detection kit for measles virus and application method of detection kit - Google Patents
Detection kit for measles virus and application method of detection kit Download PDFInfo
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- CN105759029A CN105759029A CN201610100829.8A CN201610100829A CN105759029A CN 105759029 A CN105759029 A CN 105759029A CN 201610100829 A CN201610100829 A CN 201610100829A CN 105759029 A CN105759029 A CN 105759029A
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- measles virus
- phosphate buffer
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- 241000712079 Measles morbillivirus Species 0.000 title claims abstract description 24
- 238000001514 detection method Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 19
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 16
- 201000005505 Measles Diseases 0.000 claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 12
- 241000700605 Viruses Species 0.000 claims abstract description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 10
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 10
- 230000000149 penetrating effect Effects 0.000 claims abstract description 9
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract description 7
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 4
- 238000012360 testing method Methods 0.000 claims abstract description 4
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- 229960002685 biotin Drugs 0.000 claims abstract 3
- 239000011616 biotin Substances 0.000 claims abstract 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 14
- 238000010166 immunofluorescence Methods 0.000 claims description 13
- 239000008363 phosphate buffer Substances 0.000 claims description 13
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- 238000010168 coupling process Methods 0.000 claims description 7
- 238000005859 coupling reaction Methods 0.000 claims description 7
- 238000003149 assay kit Methods 0.000 claims description 6
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- 230000003834 intracellular effect Effects 0.000 claims description 4
- -1 polyoxyethylene Polymers 0.000 claims description 4
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 3
- 210000004700 fetal blood Anatomy 0.000 claims description 3
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- 238000000703 high-speed centrifugation Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
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- 230000002101 lytic effect Effects 0.000 claims 1
- 239000003172 expectorant agent Substances 0.000 abstract description 6
- 229940066491 mucolytics Drugs 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
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- 239000000463 material Substances 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 abstract 3
- 108060003951 Immunoglobulin Proteins 0.000 abstract 1
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- 102000018358 immunoglobulin Human genes 0.000 abstract 1
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- 208000035473 Communicable disease Diseases 0.000 description 2
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- 102000011931 Nucleoproteins Human genes 0.000 description 2
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- 239000010452 phosphate Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
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- 206010018910 Haemolysis Diseases 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 102000001621 Mucoproteins Human genes 0.000 description 1
- 108010093825 Mucoproteins Proteins 0.000 description 1
- NMOJAXCSURVGEY-UHFFFAOYSA-N N#CC#N.[S] Chemical compound N#CC#N.[S] NMOJAXCSURVGEY-UHFFFAOYSA-N 0.000 description 1
- 206010033733 Papule Diseases 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 206010049590 Upper respiratory tract inflammation Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 239000003228 hemolysin Substances 0.000 description 1
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- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
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- 208000026435 phlegm Diseases 0.000 description 1
- 108091005981 phosphorylated proteins Proteins 0.000 description 1
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- 229960005486 vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Virology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
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Abstract
The invention discloses an immumofluorescence method detection kit for measles virus and a detection method of the immumofluorescence method detection kit, belonging to a method for testing materials by generating color change according to results of a test reaction through visible light. The technical scheme provided by the invention is that the kit comprises mucolytics, a cytomembrane penetrating agent, a biotinylated anti-measles virus antibody IgM (Immunoglobulin M), streptavidin coupled fluorescein isothiocyanate, a mounting medium and a biological slide. Mucus on the surface of a pharyngeal epithelial cell is dissolved by N-acetyl-L-cysteine; a pharyngeal epithelial membrane is enabled to be penetrated by using a nonionic surfactant; the antibody is subjected to biological operation; the streptavidin coupled fluorescein isothiocyanate and glycerinum enter cells; the measles virus is detected by using a streptavidin-biotin coupled fluorescein isothiocyanate method, so that the sensitivity can be remarkably improved and the background is reduced; the detection kit and the application method are suitable for early infection detection of the measles virus.
Description
Technical field
A kind of measles virus immuno-fluorescence assay kit and using method thereof, belong to technical field of medical examination.
Background technology
Measles virus (measles virus) is a kind of acute infectious disease that children are common, and its infectiousness is very strong, Yi Piqiu
Rash, heating and respiratory symptom are characterized, since China applies attenuated live vaccine from the beginning of the sixties, under the incidence of disease of children is notable
Fall, measles is one of modal Acute respiratory infectious disease of children, and its infectiousness is very strong, clinically with heating, the upper respiratory tract
Inflammation, eye conjunctivitis etc. and occur having in red grouper papule and buccal mucosa spots,Filatov's and rash to step back with skin and leave over pigment and sink
Companion's bran sample furfur to be characterized.China, from nineteen sixty-five, has controlled after starting general kind of attenuvax and has been very popular, measles
Virus is spherical or filate, diameter about 120nm～250nm, and core is single strand RNA, and non-segmented negative, full-length genome is about
16kb, genome has 6 genes of N, P, M, F, H, L, is separately encoded 6 26S Proteasome Structure and Function albumen: nucleoprotein
(nucleoprotein, NP), phosphorylated protein (phosphoprotein, P), M albumen (membrane protein, M), melt
Hop protein (fusion protein, F), hemagglutinin (hemagglutinin, H) and the RNA polymerase of dependenc RNA
(large polymerase, L), nucleocapsid is the most symmetrical, has outward coating, surface to have two kinds of furcellas, i.e. HA and hemolysin
(haemolyxin, HL), their composition is all glycoprotein, but distinct, and HA can only aggegation monkey red blood cells, moreover it is possible to host
Cell receptor adsorbs, and HL has haemolysis and makes cell occur to merge the effect forming multinucleate giant cell, HA and HL all has antigenicity,
The corresponding antibodies produced has protective effect.Clinical labororatory's detection method has, take morbidity's blood in early days, throat wash or
Throat swab, after antibiotic treatment, is inoculated in human embryo kidney (HEK), monkey kidney or human amniotic cell cultivation, with indirect fluorescent antibody technique or
ELISA detects IgM antibody, and the mucomembranous cell checked in patient's catarrhal period Pharyngeal aspirate with FLA resists with or without measles virus
Former, intracellular viral nucleic acid also can be detected with hybridization.
Summary of the invention
The present invention is to solve measles virus early diagnosis, easy, quickly, result is prone to interpretation and provides a kind of measles
Poison immunofluorescence detection agent box.Immunofluorescence technique is easy, quickly, highly sensitive, but current detection method positive rate is low, knot
Fruit is difficult to interpretation, and sample disposal is improper, false positive and false negative easily occurs, needs to carry out bigger improvement, and the pharyngeal epithelium of patient is thin
Cellular surface is stained with substantial amounts of mucus, not uncomplicated laundering, is difficult to separate, and N-acetyl-L-cysteine medicine is mucolytic agent, faces
Being usually used in division of respiratory disease patient on bed to reduce the viscosity of sputum and be prone to discharge, the sulfydryl (-SH) structure of this product can make mucoprotein
The structural break of double sulphur (-S-S-), reduce phlegm viscosity, in the present invention, make pharyngeal epithelium through N-acetyl-L-cysteine effect
Cell is separated from mucus, and epithelial cell membrane penetrating agent is nonionic surfactant, consists of polyoxyethylene surfactants
Agent 0.18%, organic quaternary ammonium salt 0.08%, in the present invention, it can make bioid antibody, Streptavidin even by penetration cell film
In connection fluorescein isothiocynate and mountant, glycerol medium enters intracellular, carries out determination of immunofluorescence method, and mountant glycerine is not
It is only capable of making cell moisten, keeps permanent fluorescence, and enter cell and intracellular structure can be made limpid, increase contrast, more can increase
Add the contrast of fluorescence and background, biotin-Streptavidin system is introduced fluorescence detecting system, sensitiveness can be significantly improved
And specific, the method can significantly improve the sensitivity of detection method and specific.
The present invention is realized by techniques below scheme:
A kind of measles virus immuno-fluorescence assay kit, it includes box body and lid, and in long limit, box body side
It is formed in the reagent rack of jack, accommodates mucolytic agent, cell membrane penetrating agent, biotinylation anti-measles virus antibody respectively
IgM, Streptavidin coupling fluorescein isothiocynate, mountant, the longitudinal divider side in box body is formed and houses phosphate
(PBS) space of buffer solution bottle, and demarcation strip opposite side forms the space of the biological slide of storage, by certain on described biological slide
Spacing is formed with reaction groove.
So reagent used when present invention have the advantage that detection used of design, biological slide etc. all concentrates on
Together, it is simple to detection, simple to operate, save manpower and time, the detection time is about 2.5 hours, it is provided that operating efficiency.
Accompanying drawing explanation
Fig. 1 kit of the present invention perspective view.
Biological slide structural representation in Fig. 2 present invention.
Fig. 3 is to apply assay method Positive fluorescence figure of the present invention.
Fig. 4 is to apply original assay method Positive fluorescence figure.
In figure: 1. box body 2. lid
3. jack 4. reagent rack
5. mucolytic agent reagent bottle 6. cell membrane penetrates reagent bottle
7. biotinylation anti-measles virus antibody reagent bottle
8. Streptavidin coupling fluorescein isothiocynate reagent bottle
9. mountant bottle 10. phosphate buffer reagent bottle
11. biological slide 11a.11b.11C bio-reaction zones
Below in conjunction with the accompanying drawings and embodiment the present invention will be described in detail
A kind of measles virus immunofluorescence detection agent box, including box body 1 and lid 2, and in long limit, box body 1 side shape
Become to have in the reagent rack 4 of jack 3, accommodate A reagent bottle mucolytic agent (5), B reagent bottle cell membrane penetrating agent (6) respectively,
C reagent bottle biotinylation anti-measles virus antibody IgM (7), D reagent bottle Streptavidin coupling fluorescein isothiocynate tries
Agent bottle (8), E reagent bottle mountant (9), F reagent bottle phosphate buffer (10), biological slide (11) composition, such as Fig. 1.
Described biological slide, its groove formed at a certain distance, is 3 square grooves 11a, 11b, 11c respectively, side
The specification of connected in star is 3 × 3 × 1mm, and correspondence is labeled with the word marking of a, b, c, such as Fig. 2.
Described measles virus detection kit, its box body 1, the material of lid 2 are papery or rigid plastics.
In order to vibration damping protects bottle, box body 1 bottom liner corrugated paper or foamed plastics (not shown).
According to the kit of said structure design, in reagent bottle 5, pre-install 100ml mucolytic agent reagent bottle, reagent respectively
Prepackage 100ml cell membrane penetrating agent in bottle 6, prepackage 2ml biotinylation anti-measles virus antibody IgM, reagent bottle 8 in reagent bottle 7
Interior prepackage 2ml Streptavidin coupling fluorescein isothiocynate reagent, prepackage 10ml mountant in reagent bottle 9, in reagent bottle 10
Prepackage 100ml 0.01mmol/L phosphate buffer reagent bottle.
It is as follows that the application present invention carries out measles virus Immunofluorescence test step:
Embodiment 1
1. preparation of reagents method
The configuration NaCL 8.5 grams of 1.1 0.01mol/L PBS (pH7.4), Na2HPO4.12H2O 30.8 grams,
NaH2PO4.2H22.8 grams of distilled waters of O are settled to 1000ml.
1.2 10mg/L N-acetyl-L-cysteine phosphate buffer 30mg N-acetyl-L-cysteines are dissolved into
1L 0.01mol/L PBS.
The configuration polyoxyethylene surfactant 0.18% of 1.3 penetrating agents, organic quaternary ammonium salt 0.08%.
The configuration biotinylation anti-measles antibody IgM (commercially available) of 1.4 biotinylated antibodies and fluorescein uses 0.01mol/L
PBS is diluted to working solution by 1:100；The Streptavidin (commercially available) of fluorescein isothiocynate presses 1:100 with 0.01mol/L PBS
It is diluted to working solution.
Configuration 0.5mol/L carbonate buffer solution (pH8.5) of 1.5 mountant, lOml；Unstressed configuration glycerine (AR level)
90ml, fully mixes after mixing on agitator.
2. detection method
2.1 assay methods of the present invention
With throat swab, wiping patient is pharyngeal, joins 2ml and contains 10mg/L N-acetyl-L-cysteine phosphate-buffered
Acutely shaking in liquid, quiet to 30min, high speed centrifugation 10000g, 10min, by PBS adjustment cell number to 100/μ l, spot film arrives
Containing on poly-D-lysine slide, 56 DEG C of drying are killed virus and fix, and seal, and-70 DEG C of Cord blood make biological slide.
During experiment, after biology slide is placed room temperature, dropping cell membrane penetrating agent 50 μ l stands 1min, 0.01mol/L PBS and washes 3 times,
50 μ l biotinylation anti-measles virus antibody IgMs of dropping 0.01mol/L PBS1:100 dilution, 37 DEG C of 30min, 0.01mol/L
PBS washes 2min 3 times；The Streptavidin of the fluorescein isothiocynate of dropping 0.01mol/L PBS1:100 dilution, 37 DEG C
60min, 0.01mol/L PBS washes 2min 4 times；Mountant mounting, observes under 10 40 times of visuals field of fluorescence microscope；Different sulphur cyanogen
Acid fluorescein excites 490～495nm, launches fluorescence 520～530nm, in yellow green, i.e. can determine whether that patient infects measles
Poison.
2.2 original assay methods
Pharyngeal with throat swab wiping infant, join in 2ml phosphate buffer and acutely shake, quiet to 60min, at a high speed from
The heart 10000g, 10min, adjust cell number to 100/μ l with PBS, spot film to containing on poly-D-lysine slide, 56 DEG C of bakings
Dry killing virus and fix, sealing ,-70 DEG C of Cord blood make biological slide.During experiment, after biology slide is placed room temperature,
50 μ l biotinylation anti-measles virus antibody IgMs of dropping 0.01mol/LPBS1:100 dilution, 37 DEG C of 30min, 0.01mol/L
PBS washes 2min 3 times；The SABC-FITC, 37 DEG C of 60min, 0.01mol/L PBS of dropping 0.01mol/L PBS1:100 dilution wash
2min 4 times；Mountant mounting, observes under 10 40 times of visuals field of fluorescence microscope；Fluorescein isothiocynate is 490～495nm
Excite, launch fluorescence 520～530nm, in yellow green, i.e. can determine whether that patient infects measles virus.
3. interpretation of result
Fig. 3 is measles virus immunofluorescence technique positive sample detection figure of the present invention, and Fig. 4 is original measles virus immunofluorescence
Method positive sample detection figure, on result judges, there is larger difference in two kinds of methods, the easy interpretation of the present invention, fluorescence intensity is big；
And original method less fluorescence is difficult to interpretation, easily cause false positive and false negative, therefore, though the inventive method is through simple place
Reason, but in methodology, reached more preferable effect.
Claims (4)
1. a measles virus immuno-fluorescence assay kit, it is characterised in that pharyngeal with throat swab wiping patient, by viscous
Liquid lytic agent makes pharyngeal surface epithelial cell mucolysis, makes pharyngeal epithelial cell membrane penetrate with cell membrane penetrating agent, makes biology
Change antibody, Streptavidin coupling fluorescein isothiocynate and glycerine and enter intracellular, apply Streptavidin-biotin coupling
Fluorescein isothiocynate method detection measles virus.
Measles virus immuno-fluorescence assay kit the most according to claim 1, it is characterised in that described mucolysis
Agent is containing 10mg/L N-acetyl-L-cysteine phosphate buffer.
Measles virus immuno-fluorescence assay kit the most according to claim 1, it is characterised in that described cell membrane is worn
Agent is nonionic surfactant thoroughly, consists of polyoxyethylene surfactant 0.18%, organic quaternary ammonium salt 0.08%.
Measles virus immuno-fluorescence assay kit the most according to claim 1, it is characterised in that detection method is: use
Wiping patient is pharyngeal for throat swab, joins 2ml and contains in 10mg/L N-acetyl-L-cysteine phosphate buffer and acutely shake
Swinging, quiet to 30min, high speed centrifugation 10000g, 10min, by phosphate buffer adjustment cell number to 100/μ l, spot film is to containing
Having on poly-D-lysine slide, 56 DEG C of drying are killed virus and fix, and seal, and-70 DEG C of Cord blood make biological slide.Real
When testing, after biology slide is placed room temperature, dropping cell membrane penetrating agent 50 μ l stands 1min, 0.01mol/L phosphate buffer
Wash 3 times, dropping 0.01mol/L phosphate buffer 1: 50 μ l biotinylation anti-measles virus antibody IgMs of 100 dilutions, 37 DEG C
30min, 0.01mol/L phosphate buffer washes 2min 3 times；Dropping 0.01mol/L phosphate buffer 1: the chain of 100 dilutions
Mould Avidin-Biotin coupling fluorescein isothiocynate, 37 DEG C of 60min, 0.01mol/L phosphate buffers wash 2min 4 times；
Mountant mounting, observes under 10 40 times of visuals field of fluorescence microscope；Fluorescein isothiocynate excites 490～495nm, 520
～530nm launches fluorescence, in yellow green, i.e. can determine whether that patient infects measles virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610100829.8A CN105759029A (en) | 2016-02-24 | 2016-02-24 | Detection kit for measles virus and application method of detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610100829.8A CN105759029A (en) | 2016-02-24 | 2016-02-24 | Detection kit for measles virus and application method of detection kit |
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| CN105759029A true CN105759029A (en) | 2016-07-13 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546570A (en) * | 2016-11-03 | 2017-03-29 | 天津市宝坻区人民医院 | A kind of detection method of ABCG2 antibody |
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| CN106908607A (en) * | 2017-03-01 | 2017-06-30 | 天津市宝坻区人民医院 | The detection method of thyrotrophin receptor antibody |
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