CN109991414A - A kind of preparation of detection method and its test strips that quickly making a definite diagnosis feline panleukopenia - Google Patents
A kind of preparation of detection method and its test strips that quickly making a definite diagnosis feline panleukopenia Download PDFInfo
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- CN109991414A CN109991414A CN201711496446.8A CN201711496446A CN109991414A CN 109991414 A CN109991414 A CN 109991414A CN 201711496446 A CN201711496446 A CN 201711496446A CN 109991414 A CN109991414 A CN 109991414A
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- feline panleukopenia
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
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Abstract
The present invention relates to field of biological detection, it provides a kind of for the quantitative detecting method of feline panleukopenia quickly made a definite diagnosis and its preparation of test strips, the test strips are successively made of bottom plate, bonding pad, reaction film, blotting paper, sample pad, Feline Panleukopenia Virus antibody fluorescence microballoon marker is coated on the bonding pad, it include detection line and nature controlling line on the reaction film, detection line is coated with Feline Panleukopenia Virus antibody, and nature controlling line is coated with sheep anti-mouse antibody.Test strips needs described in the invention are used in combination with matched fluorescent quantitation instrument, it is possible to provide quantitative testing result.Determination method provided by the invention can judge coincident with severity degree of condition and prognosis according to quantitative value inspection result.Provided by the present invention for the test strips that feline panleukopenia quickly detects, easy to operate, high sensitivity and high stability.
Description
Technical field
The present invention relates to a kind of for the detection method of feline panleukopenia quickly made a definite diagnosis and its system of test strips
It is standby, belong to field of medical examination.
Background technique
Feline Panleukopenia Virus (Feline panleukopenia virus, FPLV) also known as feline panleucopenia virus
(FPV), feline panleukopenia virus, feline infectious enteritis virus are the cause of diseases for causing feline panleukopenia.Feline panleukopenia
Viral distribu-tion is all over the world, be infection the most important virus of felid, thus virus caused by infectious disease with high fever, vomit
It spits, be dehydrated, leucocyte is seriously reduced and hemorrhagic enteritis is main feature.Study on etiology shows feline panleukopenia
Virus can infect felid such as tiger, leopard, lion and Mustelidae, Procyonidae many animals, the lesser cat of figure under field conditions (factors)
Section animal and mink most easy infection.
Feline panleukopenia is a kind of highly pathogenic infectious disease for seriously endangering felid, currently, cat whiting is thin
Born of the same parents reduce the common laboratory diagnostic method of disease include Electronic Speculum observation, virus purification, micro HA-HI test, agar gel diffusion test,
Micro SN test, PCR detection, enzyme-linked immunosorbent assay (ELISA) etc..But these methods have it is cumbersome, to instrument and
The features such as operator is more demanding cannot popularize use.
Summary of the invention
The shortcomings that in view of product described above, the purpose of the present invention is to provide a kind of easy to operate, time-consuming short, sensitivity
High, the method for quantitative detection Feline Panleukopenia Virus and its test strips preparation.
To achieve the goals above and other related purposes, this invention takes following technical schemes:
A kind of fluorogenic quantitative detection test strips for quickly making a definite diagnosis feline panleukopenia, by bottom plate, bonding pad, sample
Pad, reaction film, blotting paper successively form.
The bonding pad is coated with fluorescence polystyrene microsphere marker.
It include detection line T line and nature controlling line C line on the reaction film, the detection line is coated with cat whiting Leukopenia
Syndrome virus antibody, the nature controlling line are coated with sheep anti-mouse antibody.
It is a further object to provide a kind of methods for preparing above-mentioned test strips, method includes the following steps:
Step 1: the preparation of bonding pad:
(1) Feline Panleukopenia Virus monoclonal antibody is marked with fluorescent latex microballoon；
(2) the fluorescent latex microballoon marker of step (1) preparation is drawn into film instrument with metal spraying and is sprayed on pretreated glass fibers
It ties up on film or polyester film, is placed in spare after baking oven is dried；
Step 2: the preparation of reaction film:
Feline Panleukopenia Virus antibody and sheep anti-mouse antibody are sprayed respectively and drawn in detection line T line and nature controlling line C line；
Step 3: the preparation of sample pad:
Polyester film is put in baking oven after buffer impregnates and is dried for standby；
Step 4:
It is got stuck after the material of completion made by step 1-3 is completed with the small item loading that machine is cut into 3.95mm
In.
It is a further object to provide a kind of fluorescent quantitation methods for quickly making a definite diagnosis feline panleukopenia, should
Method the following steps are included:
1) it sample pre-treatments: quantitatively draws sample and is placed in dilution；
2) it is detected with above-mentioned feline panleukopenia test strips of making a definite diagnosis；
3) test strips after addition sample are placed in mating fluorescent quantitation instrument and are examined, read testing result.
The present invention provides a kind of judgment method for judging the feline panleukopenia state of an illness and prognosis based on quantitative result,
It is characterized by:
0-20AU: negative
20-50AU: doubtful carrying
50-100AU: weak sun
100-300AU: moderate positive
> 300AU: Qiang Yang
Advantages of the present invention are as follows:
It is provided by the present invention that quickly making a definite diagnosis feline panleukopenia fluorescent quantitation test strips can provide quantitative value knot
Fruit；
The present invention provides a kind of state of an illness based on quantitative result and Index for diagnosis methods；
This method has the advantages that step sample-adding can be read as a result, easy to operate.
Figure of description
Fig. 1 shows the structural schematic diagram for quickly making a definite diagnosis feline panleukopenia test strips of the invention；Wherein, 1 is
Bottom plate；2 be loading slot；3 be sample pad；4 be bonding pad；5 be detection line；6 be nature controlling line.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example
Embodiment 1, the composition for quickly making a definite diagnosis feline panleukopenia fluorescent quantitation test strips
Quickly make a definite diagnosis feline panleukopenia test strips by bottom plate, bonding pad, reaction film, sample pad, blotting paper successively
Composition.Reaction film is first attached to the centre of PVC bottom plate, then pastes blotting paper, the beginning of blotting paper is connected with the end of reaction film, inhales
The end of water paper is aligned with the end of PVC bottom plate, and the end of bonding pad is connected with the beginning of reaction film, and bonding pad has 1/ from beginning
3 regions are covered by sample pad, and the beginning of sample pad is aligned with the beginning of PVC bottom plate.There are detection line T line and matter on the reaction film
Line C line is controlled, detection line T line is located at the side close to the end of bonding pad；Nature controlling line C line is located remotely from the one of the end of bonding pad
Side；Detection line T line is coated with Feline Panleukopenia Virus antibody, and nature controlling line C line is coated with sheep anti-mouse antibody.
Test strips are cut into the small item of wide 3.95mm after being completed with machine, are fitted into during plastics get stuck, then the dress that will get stuck
Enter in aluminium foil bag, and sealed after being packed into desiccant, the shelf-life was up to 24 months.
The preparation method of test strips described in embodiment 2, embodiment 1
The preparation of test strips:
The preparation method of the test strips mainly comprises the steps that
Step 1: the preparation of bonding pad:
(1) Feline Panleukopenia Virus monoclonal antibody is marked with fluorescent latex microballoon；
(2) the fluorescent latex microballoon marker of step (1) preparation is drawn into film instrument with metal spraying and is sprayed on pretreated glass fibers
It ties up on film or polyester film, is placed in spare after baking oven is dried；
Step 2: the preparation of reaction film:
Feline Panleukopenia Virus antibody and sheep anti-mouse antibody are sprayed respectively and drawn in detection line T line and nature controlling line C line；
Step 3: the preparation of sample pad:
Glass film or polyester film are put in baking oven after buffer impregnates and are dried for standby；
Step 4:
It is got stuck after the material of completion made by step 1-3 is completed with the small item loading that machine is cut into 3.95mm
In.
Narration in detail separately below:
1. the preparation of Feline Panleukopenia Virus-Fluorescent microsphere marker
(1) 500 μ L buffer solutions are added in the fluorescence polystyrene microsphere of 50 μ L, obtain fluorescence polystyrene after ultrasound
Microballoon dispersion liquid.
(2) vortex will be quickly placed into after in fluorescent latex microballoon dispersion liquid that EDC&NHS activator is quickly adding into (1)
On oscillator after low speed concussion 6min, it is fixed on vortex oscillator, at room temperature concussion activation.
(3) after activating, the fluorescent latex microballoon dispersion liquid in step (2) is centrifuged, parameter of noncentricity 16000rpm,
20min removes supernatant after centrifugation, is centrifuged after being cleaned with the buffer solution in step (1) with the same terms, removes supernatant
It is redissolved after liquid with buffer solution in step (4).
(4) Feline Panleukopenia Virus antibody is taken, buffer solution is added and is diluted to 0.06mg/ml.
(5) antibody fluorescence polystyrene microsphere particle dispersion being resuspended in step (3) being added in step (4) is dilute
It is shaken on turbula shaker immediately after releasing in liquid, then ultrasound, it is anti-to be then attached on vortex oscillator concussion at room temperature
Answer 6h.
(6) after reaction, it will be centrifuged after solution ultrasound 6min in step (5), parameter of noncentricity 16000rpm, 20min,
Supernatant is removed after centrifugation, is fixed on turbula shaker concussion reaction under room temperature after confining liquid is added.
(7) solution is centrifuged by step (6) after reaction, and confining liquid cleaning, at 16000rpm, centrifugation are added after centrifugation
Supernatant is removed after 20min, and the antibody of fluorescent microsphere label can be obtained.
(8) buffer solution of the buffer solution and step (4) that redissolve in step (3) is borate buffer solution, pH
Value is 7.8.
2. the preparation of bonding pad
(1) appropriate microballoon re-suspension liquid is added in the antibody of fluorescent microsphere label, is blown and beaten repeatedly up to no visible particle is deposited
Subsequent ultrasound 5min to precipitating is completely disappeared.
(2) antibody of the fluorescent microsphere label in step (1) is drawn into film instrument with metal spraying and is sprayed at pretreated glass fibre
It on film or polyester film, is subsequently dried, taking-up, which is placed in dry environment, to be saved backup.
3. the preparation of reaction film
It will test line and nature controlling line need coated antibody, respectively with after suitable diluted, draw film instrument with metal spraying
Machine sprays it respectively to be drawn on reaction film.The coated antibody of nature controlling line is sheep anti-mouse antibody, and coated weight is 2 μ L/cm；Detection line packet
The antibody of quilt is Feline Panleukopenia Virus antibody, and coated weight is 2 μ L/cm.
4. the preparation of sample pad
Sample pad is impregnated into 2h, drying for standby with phosphate buffer containing 0.02mol/L (pH7.2).
5. the assembling of test strips
Reaction film is first attached to the centre of PVC bottom plate, then pastes blotting paper, the end phase at the beginning and reaction film of blotting paper
Even, the end of blotting paper is aligned with the end of PVC bottom plate, and the end of bonding pad is connected with the beginning of reaction film, and bonding pad is from the beginning
End has 1/3 region to be covered by sample pad, and the beginning of sample pad is aligned with the beginning of PVC bottom plate.
The application of embodiment 3, test strips
General detection method is as follows:
1. the preparation of sample: dipping appropriate animal wastes with cotton swab and be added in Sample dilution, mix well rear to be measured.
2. pattern detection: drawing 120 μ L of prepare liquid with pipettor and vertically drip in well, by test paper after timing 15min
Item is inserted into matched fluorescent quantitative detector.
3. Analysis of test results: instrument provides survey report automatically, can obtain negative/positive tie according to examining report result
Fruit, while the content of Feline Panleukopenia Virus can be obtained.
Experiment and verification result:
1. measuring 200 parts of clinical samples, section using present case Feline Panleukopenia Virus Rapid detection test strip
The coincidence rate for determining result and sample value is 97.8%.Feline panleukopenia Rapid detection test strip of the present invention can
With the Feline Panleukopenia Virus in delicately quantitative detection sample.
2. the stability using present case Feline Panleukopenia Virus Rapid detection test strip is verified: the examination after packaging
Paper slip is respectively after January, two months, March with same sample detection, and CV shows that this test strips has less than 8% as the result is shown
Preferable stability.
Claims (5)
1. a kind of fluorogenic quantitative detection test strips for quickly making a definite diagnosis feline panleukopenia, by bottom plate, bonding pad, sample pad,
Reaction film, blotting paper successively form.
2. according to claim 1 quickly make a definite diagnosis feline panleukopenia test strip, it is characterised in that described
Bonding pad is coated with fluorescence polystyrene microsphere marker.
3. the fluorogenic quantitative detection test strips according to claim 1 for quickly making a definite diagnosis feline panleukopenia, feature
It is to include detection line T line and nature controlling line C line on the reaction film, the detection line is coated with feline panleukopenia disease
Malicious antibody, the nature controlling line are coated with sheep anti-mouse antibody；
A kind of preparation method for quickly making a definite diagnosis feline panleukopenia test strip, includes the following steps:
Step 1: the preparation of bonding pad:
(1) Feline Panleukopenia Virus monoclonal antibody is marked with fluorescent latex microballoon；
(2) the fluorescent latex microballoon marker of step (1) preparation is drawn into film instrument with metal spraying and is sprayed on pretreated glass fibre membrane
Or on polyester fiber film；
Step 2: the preparation of reaction film:
Feline Panleukopenia Virus antibody and sheep anti-mouse antibody are sprayed respectively and drawn in detection line T line and nature controlling line C line；
Step 3: the preparation of sample pad:
Glass fibre membrane or polyester film is spare after buffer pre-processes；
Step 4:
It is fitted into after the material of completion made by step 1-3 is completed with the single that machine is cut into 3.95mm in getting stuck.
4. a kind of fluorescent quantitation method of inspection for making a definite diagnosis feline panleukopenia, it is characterised in that this method includes following step
It is rapid:
1) sample pre-treatments；
2) it is detected with the described in any item test strips of claim 1-3；
3) mating with lateral chromatography fluorescent quantitation instrument, after sample is added, reacts 3-15 minutes, be put into fluorescent quantitation instrument,
Show quantitative testing result.
5. a kind of foundation quantitative testing outcome evaluation feline panleukopenia state of an illness and the method for prognosis, it is characterised in that foundation
Inspection result numerical value judges the corresponding state of an illness and provides prognosis guiding opinion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711496446.8A CN109991414A (en) | 2017-12-31 | 2017-12-31 | A kind of preparation of detection method and its test strips that quickly making a definite diagnosis feline panleukopenia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711496446.8A CN109991414A (en) | 2017-12-31 | 2017-12-31 | A kind of preparation of detection method and its test strips that quickly making a definite diagnosis feline panleukopenia |
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| Publication Number | Publication Date |
|---|---|
| CN109991414A true CN109991414A (en) | 2019-07-09 |
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| CN201711496446.8A Pending CN109991414A (en) | 2017-12-31 | 2017-12-31 | A kind of preparation of detection method and its test strips that quickly making a definite diagnosis feline panleukopenia |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN116735866A (en) * | 2023-02-01 | 2023-09-12 | 深圳粒影生物科技有限公司 | Test strip for triple detection of feline panleukopenia virus, feline herpesvirus and feline calicivirus antigen and preparation method thereof |
-
2017
- 2017-12-31 CN CN201711496446.8A patent/CN109991414A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112194709A (en) * | 2020-12-07 | 2021-01-08 | 北京纳百生物科技有限公司 | Immunochromatographic test strip for detecting feline panleukopenia syndrome virus antibody, and preparation method and application thereof |
| CN116735866A (en) * | 2023-02-01 | 2023-09-12 | 深圳粒影生物科技有限公司 | Test strip for triple detection of feline panleukopenia virus, feline herpesvirus and feline calicivirus antigen and preparation method thereof |
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Application publication date: 20190709 |