CN206725585U - A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper - Google Patents
A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper Download PDFInfo
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- CN206725585U CN206725585U CN201720584578.5U CN201720584578U CN206725585U CN 206725585 U CN206725585 U CN 206725585U CN 201720584578 U CN201720584578 U CN 201720584578U CN 206725585 U CN206725585 U CN 206725585U
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- test paper
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- epidemic diarrhea
- diarrhea virus
- porcine epidemic
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Abstract
The utility model discloses a kind of Porcine epidemic diarrhea virus quickly to early diagnose test paper, and it includes supporting layer, diaphragm, sample pad, pad, nitrocellulose layer and adsorptive pads is sequentially provided with above supporting layer；It is located at using the recombinant N protein mark collaurum after Ni NTA affinity chromatographys and gel filtration chromatography as detection probe on pad；Nitrocellulose filter is coated with as detection line and nature controlling line, the common assembling for completing test paper by the use of staphylococcal protein A and His monoclonal antibodies respectively.The utility model is easy to operate, quickly；Have a wide range of application, be easy to popularity application.
Description
Technical field
Test paper is the utility model is related to, particularly a kind of early stage being applied to for Porcine epidemic diarrhea virus infection
Diagnose test paper.
Background technology
Pig epidemic diarrhea（Porcine epidemic diarrhea, PED）It is by Porcine epidemic diarrhea virus
（Porcine epidemic diarrhea virus, PEDV）One kind of caused pig is mainly to face to vomit, suffer from diarrhoea and be dehydrated
The enteric infectious disease of bed symptom.Since 2010, the disease is caused huge to live pig industry in the multiple province outbreak of epidemic in China
Economic loss.Currently, the disease has turned into restricts one of important swine disease that China's pig industry develops in a healthy way.
Fast and accurately disease diagnosis is the important prerequisite for preventing and treating pig epidemic diarrhea, is worked as in particular with virus in swinery
In progressively adaptation, PED occurs new epidemic characteristic on many pig farms, as the death rate decreases than before, age in days of falling ill
Delay and clinical symptoms be not true to type increasingly, from clinical manifestation be difficult with other cause of diseases caused by diarrhoeal diseases reflected
Not.Virus purification and identification are diagnosis PEDV infection the most intuitively methods, however, separation PEDV need specific condition and
It is more time-consuming, it is impossible to which that quick diagnosis is carried out to it.In serological diagnostic method, such as neutralization test, fluorescent antibody test and enzyme-linked
Immunosorbent adsorption test etc., due to its higher non-specificity and specific instrument is needed, limit it and apply model in clinic
Enclose, particularly the application in basic unit.In addition, as molecule gives birth to the development of technology, although many laboratories are established for examining
Disconnected PEDV RT-PCR and fluorescence quantifying PCR method, but because above method needs the people of special instrument and equipment and specialty
Member's operation, is only applicable to laboratory diagnosis at present.Therefore, need to establish at present a kind of detection accurate quick, it is easy to operate, be more easy to
In the on-site diagnosis PEDV diagnostic methods of basic unit.
The content of the invention
The purpose of this utility model be to provide it is a kind of can be with the quick early diagnosis of quick detection Porcine epidemic diarrhea virus
Test paper.
The technical solution of the utility model is that a kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper, and it includes branch
Support layer, diaphragm, it is characterised in that：Sample pad, pad, nitrocellulose layer and water suction are sequentially provided with above supporting layer
Pad；Visited using the recombinant N protein mark collaurum after Ni-NTA affinity chromatographys and gel filtration chromatography as detection
Pin is on pad；Staphylococcal protein A is used respectively（SPA）With His monoclonal antibodies coating nitrocellulose filter as inspection
Survey line and nature controlling line, the common assembling for completing test paper.The supporting layer is made up of the hard plastic piece or cardboard bar not absorbing water；Institute
Adsorptive pads are stated to be made of blotting paper.The sample pad is made up of mineral wool, nylon fiber or polyester fiber.The pad（Gold
Mark azelon layer）It is made up of mineral wool, nylon fiber or polyester fiber.The cellulose film layer is by nitrocellulose filter, pure
Cellulose membrane, carboxylated cellulose film or PVDF membrane are made.It is laid with the sample pad, pad and adsorptive pads
Diaphragm.The structure of the test paper includes handle end（Adsorptive pads one end）, test section（It is made up of nature controlling line, detection line）, sample end
（It is made up of pad and sample pad）Three parts, assembling process are：First NC films are pasted and are fixed among supporting plate, respectively in NC
The upper end of film is stained with adsorptive pads, and lower end is stained with pad（Glue gold pad）, then it is stained with sample pad, each layer again in one end of pad
Between overlapping about 1-2 mm.The test paper semi-finished product assembled pass through cutting（Cutting width is 4.08 mm）Load test paper plastics afterwards
Complete to assemble in getting stuck, after compression, kept dry under room temperature condition.
The recombinant N protein of the colloid gold label is prepared by the following method：
Aurosol is prepared with reduction of sodium citrate method：In the 0.01-0.05wt% aqueous solution of chloraurate of 50-100 mL boilings
Middle addition 2-4 mL 0.5-2 wt % citric acid three sodium solutions, a diameter of 15-20nm colloid aurosol is obtained after reaction；
With 0.1 mol/L K2CO3The pH value of colloid aurosol is adjusted to 8.5-9.5.By every milliliter of μ of colloidal gold solution mark 115.2
G recombinant N protein, after 10 min, add final concentration of the 0.05% of 20 wt % PEG-10000 to PEG -10000,4 DEG C
Under, 1500-3000 r/min centrifuge 20 min, uncombined colloid gold particle is removed, at 4 DEG C, 15000 r/min centrifugations 1
H, supernatant is abandoned, obtain the gold mark protein mixture of preliminary purification.
The test paper diagnosis principle and flow are as follows：
Dripped with the measuring samples after normal saline dilution in sample pad, due to capillarity, sample will be from sample pad one end
Chromatograph and spread to adsorptive pads one end, if containing PEDV IgG in measuring samples, the gold mark N protein being coated on pad will
It is in combination, when diffusing to the detection line on NC films（T lines）During position, IgG Fc fragments are combined with SPA, here due to gold
Mark the aggregation of N protein-IgG-SPA compounds and form a red line；When diffusing to nature controlling line（C lines）It is anti-on C lines during position
His monoclonal antibody IgGs can then be combined with gold mark N protein, form another red line, now result can be judged to the positive.When in testing sample
During without PEDV IgG antibodies, gold mark antigen can not formation Ag-Ab-colloidal gold composite in combination, in detection line
SPA cannot also intercept gold mark antigen, and now redfree band at detection line, is as a result judged to feminine gender.Regardless of whether sample is the positive
Or negative, the His monoclonal antibody IgGs on nature controlling line can mark the N protein reaction in antigen all the time with gold and show a red line, if not
Display then represents test paper failure, therefore the reliability of the test paper can be judged by nature controlling line.
The beneficial effects of the utility model are：
（1）The present apparatus is that one kind is based on PEDV recombinant nucleocapsid proteins（N protein）The diagnosis test paper developed, for PEDV
Rapid＆Early diagnosis.First by building recombinant expression plasmid pET28a (+)-N, optimized expression condition, realize its
Solubility expression in Escherichia coli, is respectively adopted Ni-NTA affinity chromatographys and gel permeation chromatography row purifies to it, collaurum
Pad is fixed on after mark as detection probe, respectively with 1.0 mg/mL staphylococcal protein A（SPA）With 2.9
Mg/mL His monoclonal antibodies coating nitrocellulose filter is assembled into immunity chromatography detection test paper as detection line and nature controlling line, preparation.
（2）The present apparatus detects high specificity, and sensitiveness is high.With the test paper positive serum to PEDV and negative blood respectively
Clearly and TGEV, CSFV, PRRSV, PRV and FMDV positive serum is detected.As a result show, except PEDV positive serums pass through
Occur outside 2 macroscopic red stripes after test paper detection（As a result present positive）, only observe after remaining Virus monitory
To a clearly nature controlling line, the detection line being visible by naked eyes occurs, and testing result is feminine gender, and it is preferable to show that the test paper has
Specificity；By carrying out doubling dilution to PEDV positive serums, the sensitiveness of the test paper is identified with this.As a result show, work as blood
It is diluted to 1 clearly:After 12800, detection line is barely perceivable colour developing, only observes a red line in nature controlling line, shows the test paper
Detection is limited to serum dilution 1:12800, there is higher sensitiveness.
（3）It is easy to operate, quickly.Using any other instrument and reagent need not be added during test strips of the present invention, as long as will
30 seconds or so in its test lead insertion measuring samples liquid, then testing result can determine that at 5 minutes or so.
（4）As a result intuitive display, accurate.The test strips are used as detection using the detection trace and control trace for showing brownish red
Positive and negative trace, i.e., on cellulose membrane show two brownish red traces, represent be detected sample in there is PEDV to resist
Physical examination goes out, and is as a result the positive；Only one brownish red of display compares trace C on cellulose membrane, represents in sample liquid is detected
Antiviral antibody is not detected, is as a result feminine gender.Result judgement is directly perceived, accurate, simple and clear, is less prone to false negative and false positive is missed
Sentence.
（5）Reduce investment and testing cost.Using the detection method, it is not required to separately match somebody with somebody Other Instruments, equipment and reagent, saves
Big measuring appratus, equipment and additive reagent expense；Specialty and layman can carry out Site Detection whenever and wherever possible, save detection
Cost, testing cost are low.
（6）Have a wide range of application, be easy to popularity application.The present invention is simple to operate, and is convenient for carrying and preserves, can be full
The needs of sufficient different levels personnel, including professional chemical examination, customs quarantine control, health and epidemic prevention, quality-monitoring, livestock products are processed, are intensive
Change cultivation and arrive individual cultivation etc., there are wide market prospects and preferable economical, societal benefits.
Brief description of the drawings
Fig. 1 is sectional view of the present utility model, and Fig. 2 is top view of the present utility model.
Embodiment
Embodiment is described in detail with reference to the figures above,
A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper, and it includes supporting layer 1, diaphragm 8-1 and 8-2,
Sample pad 2, pad 3, nitrocellulose layer 4 and adsorptive pads 5 are sequentially provided with above supporting layer；Using by the affine layers of Ni-NTA
Recombinant N protein mark collaurum after analysis and gel filtration chromatography is as detection probe on pad；Respectively with gold
Staphylococcus aureus A albumen（SPA）It is common to complete with His monoclonal antibodies coating nitrocellulose filter as detection line 6 and nature controlling line 7
The assembling of test paper；Supporting layer described in the present embodiment is made up of the hard plastic piece or cardboard bar not absorbing water；Described in the present embodiment
Adsorptive pads are made of blotting paper；The sample pad is made up of mineral wool, nylon fiber or polyester fiber；Knot described in the present embodiment
Close pad（Gold mark azelon layer）It is made up of mineral wool, nylon fiber or polyester fiber；Cellulose film layer described in the present embodiment
It is made up of nitrocellulose filter, pure cellulose film, carboxylated cellulose film or PVDF membrane；Sample described in the present embodiment
Diaphragm is laid with pad, pad and adsorptive pads.
Above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.For institute
For the those of ordinary skill in category field, other various forms of changes or change can also be made on the basis of the above description
It is dynamic.There is no necessity and possibility to exhaust all the enbodiments, and the obvious change or change thus extended out
Within the dynamic protection domain created still in the utility model.
Claims (6)
1. a kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper, it includes supporting layer, diaphragm, it is characterised in that：
Sample pad, pad, nitrocellulose layer and adsorptive pads are sequentially provided with above supporting layer；Using by Ni-NTA affinity chromatographys and
Recombinant N protein mark collaurum after gel filtration chromatography is as detection probe on pad；Respectively with golden yellow
Staphylococcal protein A and His monoclonal antibodies coating nitrocellulose filter are as detection line and nature controlling line, the common assembling for completing test paper.
2. a kind of Porcine epidemic diarrhea virus as claimed in claim 1 quickly early diagnoses test paper, it is characterised in that：The branch
Support layer is made up of the hard plastic piece or cardboard bar not absorbing water；The adsorptive pads are made of blotting paper.
3. a kind of Porcine epidemic diarrhea virus as claimed in claim 1 quickly early diagnoses test paper, it is characterised in that：The sample
Product pad is made up of mineral wool, nylon fiber or polyester fiber.
4. a kind of Porcine epidemic diarrhea virus as claimed in claim 1 quickly early diagnoses test paper, it is characterised in that：The knot
Pad is closed to be made up of mineral wool, nylon fiber or polyester fiber.
5. a kind of Porcine epidemic diarrhea virus as claimed in claim 1 quickly early diagnoses test paper, it is characterised in that：The fibre
Plain film layer is tieed up to be made up of nitrocellulose filter, pure cellulose film, carboxylated cellulose film or PVDF membrane.
6. a kind of Porcine epidemic diarrhea virus as claimed in claim 1 quickly early diagnoses test paper, it is characterised in that：Described
Diaphragm is laid with sample pad, pad and adsorptive pads.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720584578.5U CN206725585U (en) | 2017-05-24 | 2017-05-24 | A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720584578.5U CN206725585U (en) | 2017-05-24 | 2017-05-24 | A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper |
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| CN206725585U true CN206725585U (en) | 2017-12-08 |
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| CN201720584578.5U Expired - Fee Related CN206725585U (en) | 2017-05-24 | 2017-05-24 | A kind of Porcine epidemic diarrhea virus quickly early diagnoses test paper |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975544A (en) * | 2019-04-26 | 2019-07-05 | 安徽荣达生物技术有限公司 | A kind of PEDV IgY antibody colloidal gold test strip and its preparation method and application |
-
2017
- 2017-05-24 CN CN201720584578.5U patent/CN206725585U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109975544A (en) * | 2019-04-26 | 2019-07-05 | 安徽荣达生物技术有限公司 | A kind of PEDV IgY antibody colloidal gold test strip and its preparation method and application |
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| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171208 Termination date: 20180524 |