CN109406780A - A kind of rotavirus Test paper card and its preparation method and application - Google Patents
A kind of rotavirus Test paper card and its preparation method and application Download PDFInfo
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- CN109406780A CN109406780A CN201811574593.7A CN201811574593A CN109406780A CN 109406780 A CN109406780 A CN 109406780A CN 201811574593 A CN201811574593 A CN 201811574593A CN 109406780 A CN109406780 A CN 109406780A
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- Prior art keywords
- rotavirus
- antibody
- test paper
- paper card
- quality control
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
Abstract
A kind of test card and its preparation method and application detecting rotavirus, the test card includes support base and the sample pad being arranged successively thereon, bonding pad, nitrocellulose filter and water absorption pad；The bonding pad contains the first anti-rotavirus antibody and Quality Control antigen of near-infrared fluorescent label substance markers；Detection line and nature controlling line are set gradually on the nitrocellulose filter, the detection line includes the second anti-rotavirus antibody, and the nature controlling line includes the antibody of anti-Quality Control antigen.Method provided by the invention has the characteristics that low background fluorescence, detection sensitivity are high compared with colloidal gold method；It is easy to operate compared to the methods of electron microscopy, enzyme-linked immunosorbent assay, inverse transcription polymerase chain reaction；Cooperate Portable fluorescence scanner to use, is suitable for rotavirus field quick detection.
Description
Technical field
The present invention relates to a kind of test cards for detecting rotavirus, in particular to a kind of to be based on near-infrared immunochromatography technique
Rotavirus Test paper card and its preparation method and application, the invention belongs to fluorescence immune chromatography fields.
Background technique
Rotavirus (rotavirus, abbreviation RV) is a kind of diplornavirus, belongs to Reoviridae.It
It is the single main cause of baby and infantile diarrhea, almost each about five years old child once infected rotavirus at least in the world
Once.The symptoms such as anorexia, vomiting, diarrhea, dehydration are mainly shown as after infection rotavirus, and serious person can lead to death.Colyliform
A total of seven monoids of virus, are A, B, C, D, E, F and G with English alphabet number.Wherein, A groups are most commonly seen one kind, and
The case that human rotavirus's infection is more than 90% is also all caused by this kind.
Currently, China is electron microscopy, enzyme-linked immunosorbent assay to the classical detection method of rotavirus illness
(ELISA), inverse transcription polymerase chain reaction (RT-PCR), colloidal gold method etc..Electron microscopy, enzyme-linked immunosorbent assay
(ELISA), the methods of inverse transcription polymerase chain reaction (RT-PCR) requires have special installation and professional technician, base
The universal difficulty of unit using being restricted, and is only applicable to laboratory testing, is unsuitable for on-site test.Common fluorescence mark
Object light spectral limit is remembered mostly at visible region (400-750nm), since the albumen of organism, nucleic acid etc. are under the excitation of ultraviolet light
Fluorescence can be generated, therefore, stronger background fluorescence, Interference Detection signal, drop can be generated using the fluorescent marker of visible region
The sensitivity of low detection.Test paper can be made to show certain color using colloidal gold developing technology, sample is judged by color
Product are negative or the positive, but its sensitivity is lower, can not develop the color if sample concentration is too low, while without quantitative analysis.
The launch wavelength range of near-infrared fluorescent material is in 650-1000nm, and within this range, organism autofluorescence is weaker, uses
Near-infrared fluorescent material has low background fluorescence, detection signal-to-noise ratio height, the high spy of detection sensitivity as detection fluorescent marker
Point.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of simple and quick, suitable for on-site test rotavirus inspections
Paper card and its preparation method and application is tested, to reduce detection error, improves the accuracy of testing result.
In order to achieve the above object, present invention employs following technological means:
The present invention utilizes double-antibody sandwich principle, constructs a kind of rotavirus Test paper card, including support base and its
On the sample pad, bonding pad, nitrocellulose filter and the water absorption pad that are arranged successively；The bonding pad contains near-infrared fluorescent marker
The the first anti-rotavirus antibody and Quality Control antigen of label；Detection line and nature controlling line are set gradually on the nitrocellulose filter,
The detection line includes the second anti-rotavirus antibody, and the nature controlling line includes the antibody of anti-Quality Control antigen.
Quality Control antigen of the present invention can be any known antigen, and the antibody of the anti-Quality Control antigen is anti-for corresponding Quality Control
Former monoclonal or polyclonal antibody.In some embodiments of the present invention, Quality Control antigen is chicken IgY, anti-Quality Control antigen
Antibody is goat-anti chicken IgY polyclonal antibody.
First anti-rotavirus antibody of the present invention and the second anti-rotavirus antibody are different antibody, preferably
For different monoclonal antibodies.
Near-infrared fluorescent marker material of the present invention is launch wavelength in the dyestuff of 650-l000nm, and preferably NHS is living
The near infrared fluorescent dye Dylight800 of change.
Fluorescent scanning instrument of the present invention can be commercially available any recognizable near-infrared wavelength light wave
Scanner, portable highly sensitive near-infrared point Fluorescence Scanner disclosed in preferably Chinese patent CN201220372662.8.
Further, the present invention provides a kind of methods for preparing the rotavirus Test paper card, including following step
It is rapid:
(1) near infrared fluorescent dye label the first anti-rotavirus antibody and Quality Control antigen；
(2) 1~5%BSA will be contained, the PBS buffer solution of 0.1~0.5%Tween 20 is added on sample pad, is air-dried at room temperature；
(3) the first anti-rotavirus antibody of the near infrared fluorescent dye label of step (1) preparation and Quality Control antigen are added on knot
Pad is closed, is air-dried at room temperature；
(4) the second anti-rotavirus antibody and anti-Quality Control antigen-antibody are taken, is respectively prepared on nitrocellulose filter with film instrument is drawn
Nature controlling line and detection line, are air-dried at room temperature；
(5) by step (2) preparation sample pad, step (3) preparation bonding pad, step (4) preparation nitrocellulose filter, with
And water absorption pad is sequentially placed into support base, is assembled into rotavirus Test paper card.
Further, the present invention also provides the near-infrared fluorescent immuno-chromatographic test paper strips to prepare rotavirus inspection
Application in test agent.
Further, the present invention also provides a kind of methods for detecting rotavirus, including, use is prepared by the present invention
Rotavirus Test paper card follows the steps below:
(1) 50~150 μ L test samples are added dropwise in sample pad, after being placed at room temperature for 5~15min, with Portable fluorescence scanner
Reading；
(2) result judgement:
All there is fluorescence emission peak in the nature controlling line and the detection line, are determined as positive findings；
Only there is fluorescence emission peak in the nature controlling line, is determined as negative findings；
The nature controlling line unstressed configuration emission peak occurs, no matter whether the detection line fluorescence emission peak occurs, determines that result is invalid.
Compared with prior art, the invention has the following beneficial effects: compared with colloidal gold method, have low background fluorescence,
The high feature of detection sensitivity；Compared to electron microscopy, enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction
It answers the methods of (RT-PCR), it is easy to operate；Cooperate Portable fluorescence scanner to use, is suitable for the quickly inspection of rotavirus scene
It surveys.
Detailed description of the invention
Fig. 1 is the side schematic view of Test paper card；
1: water absorption pad；
2: nitrocellulose membrane；
3: bonding pad；
4: PVC bottom plate is supported in reaction；
5: detection line；
6: nature controlling line；
7: sample pad.
Fig. 2 rotavirus qualitative detection result.
Fig. 3 rotavirus specific detection result.
Specific embodiment
The preparation of 1 rotavirus Test paper card of embodiment
1 material and reagent
1.1 material
Nitrocellulose filter (M135) is purchased from U.S. Millipore, glass fibre membrane, sample pad, blotting paper, PVC board, fast
Speed detection plastic clip is purchased from Beijing Ai Dekangrui Science and Technology Ltd..
1.2 viruses and cell
Rotavirus, adenovirus, norovirus, enterovirus EV 71 are saved by this laboratory.MA-104 cell is by this
Laboratory saves.
1.3 main agents
Fluorescent dye Dylight800 is purchased from U.S. Thermo, and rotavirus monoclonal antibody is purchased from Zhuhai biological section of rich U.S.
Skill Co., Ltd, chicken Ig Y and goat-anti chicken Ig Y polyclonal antibody are purchased from Britain Abcam.
2 experimental methods
The preparation of 2.1 near infrared fluorescent dye labelled antibodies
Fluorescent dye Dylight800 stoste PBS buffer solution 1:10 is diluted, takes 7uL and 100uL 1mg/mL the respectively
Primary antibody rotavirus antibody and 1mg/mL chicken Ig Y are protected from light 1h in room temperature after mixing.After completion of the reaction, metastatic marker
Solution is into bag filter, and for 4 DEG C of dialysed overnights to remove unbonded dyestuff, the antibody addition marked is dense eventually in PBS solution
Degree is 1%BSA, 0.1%Tween 20 and 0.1 ‰, 4 DEG C of sodium azide preservations.
The preparation of 2.2 rotavirus immuno-chromatographic test paper strips
2.2.1 prepared by sample pad
Cellulose membrane band is chosen as sample pad, spraying contains 5%BSA, the PBS buffer solution of 0.1%Tween 20, room temperature wind
It is dry.
2.2.2 prepared by bonding pad
It chooses glass fibre element band and is used as bonding pad solid phase material, in article take that spraying near infrared fluorescent dye marks the
Primary antibody rotavirus antibody and Quality Control antigen, are air-dried at room temperature band.
2.2.3 nitrocellulose film preparation
By goat-anti chicken Ig Y polyclonal antibody and the second anti-rotavirus antibody, made on nitrocellulose filter with film instrument is drawn
At nature controlling line and detection line, the spacing of nature controlling line and detection line is 0.5cm, is air-dried at room temperature.
2.2.4 assembling Test paper card
Sample pad, bonding pad, nitrocellulose filter, water absorption pad and reaction are supported into PVC bottom plate such as Fig. 1 mode in sequence
It installs, is then cut into test strips with cutting machine, be put into plastics card slot, assemble finished product test card.
2.3 qualitative detection
Under aseptic condition, take respectively thin in the rotavirus stoste and rotavirus infection infant excrement of laboratory preservation
Rear each 50 μ l is released, adds the mixing of 50 μ l chromatographic solutions to be added drop-wise in sample pad, after being placed at room temperature for 15min, with Portable fluorescence scanner
Reading.Same operation is carried out by blank control group of chromatographic solution.
2.4 specific detection
Under aseptic condition, rotavirus, adenovirus, norovirus, the enterovirus EV 71 for taking laboratory to save respectively are former
Each 50 μ l after liquid dilution adds the mixing of 50 μ l chromatographic solutions to be added drop-wise in sample pad, after being placed at room temperature for 15min, is swept with Portable fluorescence
Retouch instrument reading.Same operation is carried out by blank control group of chromatographic solution.
3 results
3.1 rotavirus qualitative detection results
Experiment detects rotavirus using the double-antibody sandwich immuno-chromatographic test paper strip of near infrared fluorescent dye label.Experiment
It was found that near infrared fluorescent dye used in detection is the organic molecule that launch wavelength is located at 777nm, preferably NHS activation
Near infrared fluorescent dye Dylight 800.The rotavirus immunity-chromatography test based on low noise excitation formula fluorescent marker
Paper slip, it is 650~1000nm that low noise excitation formula fluorescent dye, which is launch wavelength,.Detection line and nature controlling line region are measured respectively
Fluorescence intensity shows that the test strips are effective if fluorescence emission peak occurs in nature controlling line region, on the contrary then invalid；If detection line area
It is then positive findings that domain, which fluorescence emission peak occurs simultaneously, it is on the contrary then for feminine gender.
Testing result is shown in Fig. 2.
3.2 specific detection results
After taking rotavirus, adenovirus, norovirus, the dilution of enterovirus EV 71 stoste respectively, it is added drop-wise to rotavirus
Immuno-chromatographic test paper strip is detected.In addition to rotavirus test positive, adenovirus, norovirus, enterovirus EV 71 are equal
For feminine gender.Thus the double antibody sandwich method immuno-chromatographic test paper strip specificity for proving that the present invention establishes is good, not viral with other
Cross reaction occurs.Testing result is shown in Fig. 3.
Above embodiments be only it is exemplary, it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Claims (12)
1. a kind of rotavirus Test paper card, wherein the test card includes support base and the sample being arranged successively thereon
Pad, bonding pad, nitrocellulose filter and water absorption pad；The bonding pad contains the first anti-colyliform of near-infrared fluorescent label substance markers
Antiviral antibody and Quality Control antigen；Set gradually detection line and nature controlling line on the nitrocellulose filter, the detection line includes the
Secondary antibody rotavirus antibody, the nature controlling line include the antibody of anti-Quality Control antigen.
2. rotavirus Test paper card as described in claim 1, wherein the support base is PVC board.
3. rotavirus Test paper card as claimed in claim 1 or 2, wherein the bonding pad is glass fibre membrane.
4. the rotavirus Test paper card as described in claims 1 to 3 is any, wherein the near-infrared fluorescent marker is hair
Ejected wave grows the dyestuff in 650-l000nm.
5. rotavirus Test paper card as claimed in claim 4, wherein the near-infrared fluorescent marker is NHS activation
Near infrared fluorescent dye Dylight800.
6. the rotavirus Test paper card as described in claim 1 to 5 is any, wherein the Quality Control antigen is chicken IgY, described
The antibody of anti-Quality Control antigen is goat-anti chicken IgY polyclonal antibody.
7. rotavirus Test paper card as claimed in claim 6, wherein the goat-anti chicken IgY polyclonal antibody using dense
Degree is 0.5~2mg/mL.
8. the rotavirus Test paper card as described in claim 1 to 7 is any, wherein the first anti-rotavirus antibody and
The second anti-rotavirus antibody is different antibody.
9. rotavirus Test paper card as claimed in claim 8, wherein the use concentration of the second anti-rotavirus antibody
For 0.5~2mg/mL.
10. a kind of method for detecting rotavirus, including, it is detected and is tried using rotavirus as described in any one of claim 1 to 9
Paper card follows the steps below,
(1) 50~150 μ L test samples are added dropwise in sample pad, after being placed at room temperature for 5~15min, with Portable fluorescence scanner
Reading；
(2) result judgement:
All there is fluorescence emission peak in the nature controlling line and the detection line, are determined as positive findings；
Only there is fluorescence emission peak in the nature controlling line, is determined as negative findings；
The nature controlling line unstressed configuration emission peak occurs, no matter whether the detection line fluorescence emission peak occurs, determines that result is invalid.
11. a kind of method for preparing rotavirus Test paper card as described in any one of claim 1 to 9, includes the following steps,
(1) near infrared fluorescent dye label the first anti-rotavirus antibody and Quality Control antigen；
(2) 1~5%BSA will be contained, the PBS buffer solution of 0.1~0.5%Tween 20 is added on sample pad, is air-dried at room temperature；
(3) the first anti-rotavirus antibody of the near infrared fluorescent dye label of step (1) preparation and Quality Control antigen are added on knot
Pad is closed, is air-dried at room temperature；
(4) the second anti-rotavirus antibody and anti-Quality Control antigen-antibody are taken, is respectively prepared on nitrocellulose filter with film instrument is drawn
Nature controlling line and detection line, are air-dried at room temperature；
(5) by step (2) preparation sample pad, step (3) preparation bonding pad, step (4) preparation nitrocellulose filter, with
And water absorption pad is sequentially placed into support base, is assembled into rotavirus Test paper card.
12. rotavirus detection reagent as described in any one of claim 1 to 9 is stuck in the application in detection rotavirus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811574593.7A CN109406780A (en) | 2018-12-21 | 2018-12-21 | A kind of rotavirus Test paper card and its preparation method and application |
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|---|---|---|---|
| CN201811574593.7A CN109406780A (en) | 2018-12-21 | 2018-12-21 | A kind of rotavirus Test paper card and its preparation method and application |
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| Publication Number | Publication Date |
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| CN109406780A true CN109406780A (en) | 2019-03-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110455766A (en) * | 2019-09-03 | 2019-11-15 | 莎穆(上海)生物科技有限公司 | Detection method based on two area's fluorescence imaging of near-infrared for the strip of lateral flow immune detection, detection device and non-diagnostic purpose |
-
2018
- 2018-12-21 CN CN201811574593.7A patent/CN109406780A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110455766A (en) * | 2019-09-03 | 2019-11-15 | 莎穆(上海)生物科技有限公司 | Detection method based on two area's fluorescence imaging of near-infrared for the strip of lateral flow immune detection, detection device and non-diagnostic purpose |
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