CN104804082B - A kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus and preparation method thereof - Google Patents
A kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus and preparation method thereof Download PDFInfo
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- CN104804082B CN104804082B CN201510152757.7A CN201510152757A CN104804082B CN 104804082 B CN104804082 B CN 104804082B CN 201510152757 A CN201510152757 A CN 201510152757A CN 104804082 B CN104804082 B CN 104804082B
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Abstract
The invention discloses a kind of Immunofluorescence test test strips of Quantitative detection Porcine epidemic diarrhea virus and preparation method thereof, test strips of the present invention contain sample pad, bonding pad, chromatographic film and water absorption pad, anti- PEDV single domain antibodies containing fluorescent microsphere mark on bonding pad, the single domain antibody have high specific and high sensitivity to antigen PEDV.The pollution of PEDV, available for field quick detection or test in laboratory is carried out, only needs 5 10min in PEDV viruses or feed plasma protein in immunology principle detection cultivation swine excrement of the present invention based on newfound specific single domain antibody and antigen；If it is used in conjunction with fluorescent quantitative detector, it can be achieved that quantitative detection；Simple to operate, professional training is not required in operating personnel, and specialized laboratories are not required, and overcomes the limitation of existing detection method, has good market prospects.
Description
Technical field
Immunofluorescence test paper strip and its preparation side the present invention relates to a kind of Quantitative detection Porcine epidemic diarrhea virus
Method.
Background technology
Pig epidemic diarrhea（porcine epidemic diarrhea, PED）It is by coronaviridae pig epidemic
A kind of acute infectious disease for encroaching on chitling road caused by diarrhea virus.It can cause the pig at each age that vomiting, diarrhea and dehydration occurs.
Each age pig is susceptible, especially the newborn piglet in 1 week old is endangered maximum.The incidence of newborn piglet is up to 60%-
100%, the death rate is up to 100%.Cause large quantities of death of piglet, slaughter rate of hogs declines, and causes market cut of pork deficiency, shadow
Pork price is rung to stablize.This disease is propagated widely in Europe and Asia, can all be caused every year to some countries and regions seriously
Economic loss, huge harm is brought to pig breeding industry.America ＆ Canada has been swept across respectively within 2013 and PEDV in 2014
Pig breeding industry.Canadian Food Inspection Agency（CFIA）Think that plasma protein powder is likely to be pig in March, 2014 issue bulletin
The arch-criminal of epidemic diarrhea.Because can add plasma protein powder in pig starter feed production, extensive for PEDV passes
Broadcast and provide potential chance.In order to reduce the risk that PEDV cause of diseases are carried in feed, we are badly in need of a kind of simple and quick side
Method is detected the PEDV cause of diseases in raw material, reduces the risk that feed carries cause of disease；Early diagnosis is made to disease at the same time, is carried
Before take prevention and control measure, and reduce the preferred approach for the loss that raiser brings by disease.
The method of currently used detection PEDV has Virus Isolation, RT-PCR, ELISA method, immunofluorescence technique（IF）
And colloidal gold.Wherein Virus Isolation, RT-PCR, ELISA method, immunofluorescence technique（IF）Must the method progress of Binding experiment room
Make a definite diagnosis, it is cumbersome time-consuming, it is necessary to which special experimental technique personnel and instrument and equipment, are only used for test in laboratory.Be not suitable for
In the use of Site Detection and basic unit veterinary staff.Further, since PEDV can cause persistent infection, this gives the clinic of PEDV
Detection brings huge difficulty.
On PEDV there is presently no similar product listing, in the market in quick diagnosis test strips China South Korea's import
PEDV colloidal gold colloidal gold detection test paper strips are being sold, but it can only carry out the qualitative of PEDV and cannot quantify, and cost is prohibitively expensive, commonly
Raiser makes can not afford；And its sensitivity is not very high.Therefore the quick determination method of PEDV is established, the infection for PEDV is early
Phase and field quick detection, are conducive to that the sow for infecting PEDV is immunized in time, prevent the diffusion of disease, be conducive to raise pigs
The sound development of industry；Quality control is carried out to the pollution sources of feedstuff plasma protein at the same time, reduces and is caused from feed approach
The risk of PEDV prevalences.The quick determination method of PEDV cause of diseases is developed with the nano antibody of anti-PEDV, is at home and abroad not yet appeared in the newspapers
Road.
The content of the invention
Above-mentioned in order to solve the problems, such as, the present invention is prepared for that a species specificity is high, high sensitivity receives by research
Meter Kang Ti（Anti- PEDV single domain antibodies）, and being capable of Quantitative detection Porcine epidemic diarrhea virus based on this Antibody preparation one kind
Immunofluorescence test test strips.
It is an object of the invention to provide a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus.
The technical solution used in the present invention is：
A kind of single domain antibody for being used to detect Porcine epidemic diarrhea virus, its amino acid sequence are such as SEQ ID NO：6 institutes
Show.
A kind of single domain antibody for being used to detect Porcine epidemic diarrhea virus, encodes the nucleosides of the single domain antibody amino acid sequence
Acid sequence such as SEQ ID NO：Shown in 3.
A kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus, comprising be attached on bottom plate and according to
Secondary closely coupled sample pad, bonding pad, chromatographic film and water absorption pad；Anti- PEDV containing fluorescent microsphere mark on the bonding pad
Single domain antibody；A detection line and a nature controlling line are provided with the chromatographic film, detection line is close close to bonding pad, nature controlling line
Water absorption pad, the polyclonal antibody of anti-PEDV is coated with detection line, be coated with nature controlling line can be marked with fluorescent microsphere it is anti-
The albumen that PEDV single domain antibodies combine；
The wherein described anti-PEDV single domain antibodies are single domain antibody described above.
Further, the albumen that the coated anti-PEDV single domain antibodies that can be marked with fluorescent microsphere are combined on above-mentioned nature controlling line
For rabbit-anti two-humped camel IgG.
Further, above-mentioned sample pad is glass fibre membrane.
Further, above-mentioned chromatographic film is nitrocellulose filter.
Further, above-mentioned water absorption pad is blotting paper.
Further, the distance between above-mentioned nature controlling line and detection line are 4.5 ~ 5.5mm.
Further, above-mentioned nature controlling line is coated with 0.5~1.5mg/mL rabbit-anti two-humped camels IgG, and dosage is 1 μ L/
cm。
Further, above-mentioned detection line is coated with the anti-PEDV polyclonal antibodies of 0.8~1.2 mg/mL, and dosage is 2 μ L/cm.
The beneficial effects of the invention are as follows：
1）The quick test paper technology of immunofluorescence prepared by the present invention is to apply nano-fluorescent grain（Fluorescent microsphere mark
PEDV single domain antibodies）Construct it is a kind of can qualitative simultaneous quantitative quick detection test paper.Operator need not train and professional knowledge,
It is easy to operate, if can obtain within 5 ~ 10 minutes as a result, being used in conjunction with fluorescent quantitation instrument, it can be achieved that quantitative detect, while the technology is clever
Sensitivity is substantially better than traditional colloidal gold test technology, has a vast market prospect.
2）Core of the antibody as immunity test strip detection technique, its specificity and affinity determine measurement result
Accuracy and sensitivity.Present invention employs the highly sensitive of specific preparation, the specific single domain heavy chains of PEDV（VHH）It is anti-
Body, is developed into the quick detection test paper of detection PEDV cause of diseases, is at home and abroad not reported at present.
Brief description of the drawings
Fig. 1 is the result figure that RT-PCR expands VHH genetic fragments in embodiment 1；
Fig. 2 is the result that SDS-PAGE detects PEDV single domain antibodies；
Fig. 3 is the structure diagram of PEDV immunofluorescence Rapid detection test strips, wherein 1 is plastic bottom board, 2 be sample
Pad, 3 be bonding pad, and 4 be nitrocellulose filter, and 5 be blotting paper, and 6 be detection line T, and 7 be nature controlling line C.
Embodiment
The preparation of 1 Porcine epidemic diarrhea virus single domain heavy chain antibody of embodiment
1）Animal immune
Subcutaneously subcutaneously carry out multiple spot with both hind leg in two-humped camel nape part and be inoculated with PEDV inactivated vaccines.First immunisation dosage
For 1mL/ heads, totally 2；Two exempt from after 7 days, 2 mL/ heads；It is spaced 14 days and is immunized once afterwards, immune 4 mL, are immunized 3 times altogether every time,
It is whole to be immunized 5 times altogether.7 days blood sampling 500 mL separation lymphocytes after last time is immune, extraction total serum IgE carries out real in next step
Test.
2）The structure in single domain antibody storehouse
Primer amplification VHH genetic fragments are designed, the best primer of effect, its sequence are filtered out by lot of experiments early period
Row are as follows：
VHH-F：5’- CCTTTCTATGCAGGCCCAGCCGGCCGCA-3’（SEQ ID NO：1）；
VHH-R：5’- GTTATTATTATTCAGATTATTATGCGGCC-3’ （SEQ ID NO：2）.
RT-PCR amplifications are carried out to the total serum IgE extracted using primer VHH-F and VHH-R, obtain size about 500bp's
Band（As shown in Figure 1）, by the purpose band and carrier pCANTAB-5E（Purchased from Guangzhou Chuan Wei Bioisystech Co., Ltd）Point
Do not pass throughSfiI andNotAfter I double digestions, reconnect and convert e. coli tg1, be coated with AmprLB tablets are screened, overnight
Lower all bacterium colonies are washed after culture uses the LB culture mediums containing 20% glycerine to preserve, and freezes spare in -70 DEG C, the single domain for obtaining anti-PEDV resists
Body gene pool.
Take above-mentioned single domain antibody gene pool and helper phage M13K07 carry out mixed culture 12 it is small when after, 4000rpm from
Heart 10min, takes supernatant in sterile centrifugation tube, and after ice bath 1h, 9000rpm centrifugation 15min, abandon supernatant, precipitate and suspended with PBS,
Then 12000g, which centrifuges 10min, ensures that bacterium is sufficiently separated, and collects supernatant, is bacteriophage single domain antibody storehouse.
3）The screening of specific single domain antibody
With PEDV antigen coat elisa plates, elutriation screening is carried out to above-mentioned bacteriophage single domain antibody storehouse, after 3 wheel screenings,
It is with PEDV specific reactions and higher with antigen binding sensitivity to screen 2 positive colonies, be respectively designated as Clone1 and
Clone2, carries out DNA sequencing analysis, sequencing result shows purpose base in Clone1, Clone2 respectively to the positive colony of acquisition
Because clip size is respectively 477bp（SEQ ID NO：3）, 474bp, through amino acid alignment, single domain resists in Clone1 and Clone2
The amino acid sequence of body is VHH heavy chain antibody sequences.
4）The induced expression of single domain antibody and verification
1. SDS-PAGE is tested
2 positive colonies obtained in above-described embodiment 1 are carried out using the primer containing EcoR I and BamH I sites
PCR amplification（Respectively using Clone1, Clone2 phage DNA as template）, wherein primer sequence is：
VHH-F1：5’- AGAATTCCCTTTCTATGCAGGCCCAGCCGGCCGCA-3’ （SEQ ID NO：4）,
VHH-R1：5’- ACGGATCCGTTATTATTATTCAGATTATTATGCGGCC-3’ （SEQ ID NO：5）；
It is connected after pcr amplified fragment gel is recycled through double digestion, endonuclease bamhi with expression vector pSMK, structure single domain resists
Body recombinant expression carrier, and convert e. coli bl21 and obtain recombinant bacterium BL21-1, BL21-2 respectively, through IPTG induced expressions,
Extract respectively, purify destination protein（As PEDV single domain antibodies）.Expression product is identified using SDS-PAGE, is as a result shown
Show, specific proteins band appears in 14.3KD or so, is consistent with expected size（As shown in Figure 2）.
2. antigen binding capacity is tested
The above-mentioned single domain antibody filtered out is detected with ELISA method（I.e. above-mentioned recombinant bacterium BL21-1 and BL21-2 expression
Destination protein）With the ability of antigen binding, the results show that recombinant bacterium BL21-1 expression destination protein（Mesh i.e. in Clone1 bodies
Gene code albumen）It is higher with the ability of PEDV antigen bindings, sensitivity higher, and the purpose of recombinant bacterium BL21-2 expression
The binding ability of albumen and PEDV antigens is relatively weak（As shown in table 1）.So purpose antibody of selection group bacterium BL21-1 expression
Albumen（The antibody that target gene encodes i.e. in Clone1 bodies）Carry out the development of follow-up test strips.
The binding ability of the single domain antibody expressed in 12 positive colonies of table and antigen PEDV detects
The preparation of 2 PEDV polyclonal antibodies of embodiment
Kunming mice is immunized using PEDV inactivated vaccines, only, totally 5, head carries out two for 7 days after exempting from and exempts from 0.5mL/, and 1mL/ is only；
Carried out being immunized 1 time every 14 days afterwards, 1mL/ only, is immunized 3 times altogether.Last 1 time it is immune after 7 days blood sampling detection serum effects
Valency, it is spare as PEDV polyclonal antibodies as a result to separate serum for positive collection whole blood.
The preparation of 3 PEDV immunofluorescence Rapid detection test strips of embodiment
1）The preparation of PEDV single domain antibodies
The recombinant bacterium BL21-1 obtained in above-mentioned experimental example 1 is subjected to IPTG induced expressions, extraction, purifying destination protein,
Up to PEDV single domain antibodies；
2）The preparation of PEDV single domain antibodies-fluorescent microsphere probe
Fluorescent microsphere carboxylated：By 100uL fluorescent microspheres（Purchased from Guangzhou Mei Jintai Bioisystech Co., Ltd）It is dissolved in 500
In μ L pure water, the NHS of the EDC and 6 mg of final concentration of 10 mg are then respectively adding, 27 DEG C of incubation 30min, 4 DEG C of reaction solution,
10000 rpm/min, centrifuge 1min, supernatant discarding, is repeated 3 times, and removes unnecessary EDC and NHS.Precipitation 500 μ L PBS weights
It is outstanding, ultrasonic 5min, you can.
The preparation of PEDV single domain antibodies-fluorescent microsphere probe：By the fluorescent microsphere solution of the above-mentioned carboxylated of 500 μ L with
100 μ L concentration are 0.2 ~ 1.0mg/mL steps 1）The PEDV single domain antibodies mixing of preparation, reacts at room temperature 3h；Then 5 μ L are added
10%BSA closes the remaining carboxyl of microballoon, is incubated 1.5h, 4 DEG C of 8000rpm/min centrifuge 5min, what supernatant discarding was not associated with
Antibody.Contain the pH7.2 PBS of 1%BSA, 2%Tween-20 and 25% trehalose with 200 μ L（Salt ionic concentration is 10mM）Weight
Outstanding, the PEDV single domain antibodies-fluorescent microsphere probe as prepared, 4 DEG C save backup.
3）The preparation of anti-PEDV single domain antibodies-fluorescent microsphere probe bonding pad
0.1% Tween-20 of polyester fiber film is handled into 10min, 60 DEG C of exhausting drying, by the anti-PEDV of above-mentioned preparation
Single domain antibody-fluorescent microsphere probe is sprayed on polyester fiber film combination pad by 1.5 μ L/cm, 37 DEG C of exhausting drying, drying at room temperature
Saved backup under environment.
4）The coating of nitrocellulose filter
Using automatic spray film instrument in nitrocellulose filter（NC films）Upper spray C, T line（As shown in Figure 3）.C lines are nature controlling line, bag
By rabbit-anti two-humped camel IgG, concentration is 0.5~1.5mg/mL, preferably 1 mg/mL, and discharge rate is 1 μ L/cm；T lines are detection line, bag
The PEDV polyclonal antibodies of the preparation of example 2 are carried out, concentration is 0.8~1.2 mg/mL, and discharge rate is 2 μ L/cm；C, between T lines
Distance is 5mm.The NC films sprayed are dried in 37 DEG C of incubators, the NC films after being coated with.
5）The preparation of sample pad
Glass fibre membrane is used and contains 2%（w/v）PVP, 3% Tween-20 and 2%（w/v）The 10mmol/L pH7.2 of PEG
PBS saturated process, 37 DEG C of exhausting drying, drying at room temperature condition save backup.
6）The assembling of PEDV immunofluorescence Rapid detection test strips
As shown in figure 3, by the above-mentioned sample pad 2 prepared, PEDV single domain antibodies-fluorescent microsphere probe bonding pad 3, it is coated with
Nitrocellulose filter 4, blotting paper 5 afterwards is pasted onto on plastic bottom board 1 successively by one end, sample pad 2 and 3 lap of bonding pad
Width be 2mm, bonding pad 3 and the width of 4 lap of nitrocellulose filter after coating be 2mm.
First, the nitrocellulose filter 4 after coating is pasted at the center of plastic bottom board 1；It is close in nitrocellulose filter 4
One end overlap joint of nature controlling line 7 pastes blotting paper 5；Bonding pad 3 is pasted in the other end overlap joint of nitrocellulose filter 4；Tying
The other end overlap joint for closing pad 3 pastes sample pad 2, obtains PEDV immunofluorescence Rapid detection test strips.
The judgement of above-mentioned ELISA test strip result：
1. if shows green fluorescence trace, expression detect detection line T and nature controlling line C at the same time under uv excitation light irradiation
As a result it is feminine gender, illustrates to be free of PEDV in detected sample, or its concentration is less than 104TCID50/mL；
2. detection line T does not show green fluorescence trace, only nature controlling line C under excitation light under uv excitation light irradiation
Shows green fluorescence trace；Or T lines are more shallow than C line color；Testing result is represented as the positive, illustrates to contain in tested sample
PEDV, and concentration is greater than or equal to 104TCID50/mL；
If PEDV Immunofluorescence tests test strips and fluorescent quantitative detector are used in conjunction, it can be achieved that quantitative detection.
4 PEDV Immunofluorescence test test strips specific tests of embodiment
The test strips prepared with embodiment 3 detect transmissible gastroenteritis of swine respectively（TGEV）, porcine rotavirus（Prov）'s
Cell culture fluid, swine fever virus（CSFV）, porcine pseudorabies virus（PRV）, pig blue-ear disease poison（PRRS）, foot and mouth disease virus（FMDV）
Positive and PEDV positives.The results show ELISA test strip PEDV positives are positive reaction, and other samples are equal
For feminine gender.Show that test strips specificity is good.
5 PEDV Immunofluorescence test paper strip sensitivity tests of embodiment
The cell culture fluid of PEDV is subjected to doubling dilution, sample concentration is respectively 107TCID50/mL、106TCID50/mL、
105TCID50/mL、104TCID50/mL、103TCID50/ mL, result of the test show, 107TCID50/mL、106TCID50/mL、
105TCID50/mL、104TCID50/ mL is the positive, and colloidal gold strip contrast common with import is found, the inspection of import test strips
Survey is limited to 105TCID50/ mL, fluorescent test paper strip of the invention are 10 times more sensitive than it.
6 PEDV Immunofluorescence test test strips clinical practices of embodiment
It is put into sample treatment liquid and stirs and evenly mixs after the excrement of pig is dipped with cotton swab, or takes a little plasma protein to add sample
Mixed in product treatment fluid, take supernatant 3-5 drops to be added dropwise in the sample of above-mentioned PEDV immunofluorescences Rapid detection test strip after 1-2 minutes
On pad, after sample-adding after 5-10min, the difference of the color of T lines and C lines is observed, whether to judge in sample to be tested containing PEDV diseases
Poison.If the concentration of PEDV is less than 10 in sample4 TCID50/ mL, then the colour developing of T lines is identical with C lines, is as a result feminine gender；If in sample
The concentration of PEDV is greater than or equal to 10 4 TCID50/ mL, then T lines are more shallow than C line color or can't see line completely, the more shallow then sample of color
The concentration of PEDV is higher in product, is as a result the positive., can if PEDV Immunofluorescence test paper strips are used in conjunction with fluorescent quantitative detector
Realize quantitative detection.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
<110>Guangdong Haida Husbandry and Veterinary Institute Co., Ltd.
<120>A kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus and preparation method thereof
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 28
<212> DNA
<213>Artificial sequence
<400> 1
cctttctatg caggcccagc cggccgca 28
<210> 2
<211> 29
<212> DNA
<213>Artificial sequence
<400> 2
gttattatta ttcagattat tatgcggcc 29
<210> 3
<211> 477
<212> DNA
<213>Artificial sequence
<400> 3
agaattccct ttctatgcag gcccagccgg ccgcatctgc aggagtctgg gggaggattg 60
gtgcaggctg ggggctctct gagactctcc tgtgcagcct ctggcgacac cgaaagtatc 120
gttaccatgg cctggttccg ccaggctcca gggaaggagc gtgagtttgt agcggctatt 180
acacggagtg gcgacagccg ggcgagtggt ggtatgacgg actatgcaga ctccgtggta 240
gcggctatta cacggagtgg cgacagccgg gcgagtggtg gtatgacgga ctatgcagac 300
tccgtgaagg gccgattcac catctccaga gacaacgcca agaacacggt gtatctgcaa 360
atgaacagcc tgaaacttga ggacacggcc gtttattact gtttagcaga caaaactacg 420
tattggaata ttccccggga tgagtatgac tactggggcc aggggaccca ggtcacc 477
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence
<400> 4
agaattccct ttctatgcag gcccagccgg ccgca 35
<210> 5
<211> 37
<212> DNA
<213>Artificial sequence
<400> 5
acggatccgt tattattatt cagattatta tgcggcc 37
<210> 6
<211> 159
<212> PRT
<213>Artificial sequence
<400> 6
Arg Ile Pro Phe Leu Cys Arg Pro Ser Arg Pro His Leu Gln Glu Ser
1 5 10 15
Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
20 25 30
Ala Ser Gly Asp Thr Glu Ser Ile Val Thr Met Ala Trp Phe Arg Gln
35 40 45
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly
50 55 60
Asp Ser Arg Ala Ser Gly Gly Met Thr Asp Tyr Ala Asp Ser Val Val
65 70 75 80
Ala Ala Ile Thr Arg Ser Gly Asp Ser Arg Ala Ser Gly Gly Met Thr
85 90 95
Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
100 105 110
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Leu Glu Asp
115 120 125
Thr Ala Val Tyr Tyr Cys Leu Ala Asp Lys Thr Thr Tyr Trp Asn Ile
130 135 140
Pro Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
145 150 155
Claims (10)
1. a kind of single domain antibody for being used to detect Porcine epidemic diarrhea virus, its amino acid sequence are such as SEQ ID NO：Shown in 6.
A kind of 2. single domain antibody for being used to detect Porcine epidemic diarrhea virus according to claim 1, it is characterised in that：Compile
The nucleotide sequence such as SEQ ID NO of the code single domain antibody amino acid sequence：Shown in 3.
3. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus, comprising being attached on bottom plate and successively
Closely coupled sample pad, bonding pad, chromatographic film and water absorption pad；It is characterized in that：Contain fluorescent microsphere mark on the bonding pad
The anti-PEDV single domain antibodies of note；Be provided with a detection line and a nature controlling line in the chromatographic film, detection line close to bonding pad,
Nature controlling line is coated with the polyclonal antibody of anti-PEDV on water absorption pad, detection line, and energy and fluorescent microsphere are coated with nature controlling line
The albumen that the anti-PEDV single domain antibodies of mark combine；
The wherein described anti-PEDV single domain antibodies are any single domain antibody of claim 1 or 2.
4. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The albumen that the coated anti-PEDV single domain antibodies that can be marked with fluorescent microsphere are combined on the nature controlling line is double for rabbit-anti
Peak camel IgG.
5. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The sample pad is glass fibre membrane.
6. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The chromatographic film is nitrocellulose filter.
7. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The water absorption pad is blotting paper.
8. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The distance between the nature controlling line and detection line are 4.5 ~ 5.5mm.
9. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The nature controlling line is coated with 0.5~1.5mg/mL rabbit-anti two-humped camels IgG, and dosage is 1 μ L/cm.
10. a kind of Immunofluorescence test paper strip of Quantitative detection Porcine epidemic diarrhea virus according to claim 3, its
It is characterized in that：The detection line is coated with the anti-PEDV polyclonal antibodies of 0.8~1.2 mg/mL, and dosage is 2 μ L/cm.
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| CN106596930A (en) * | 2015-10-15 | 2017-04-26 | 江苏维赛科技生物发展有限公司 | Immunofluorescence test strip for quantitatively detecting foot-mouth disease viruses |
| CN109517060B (en) * | 2015-12-25 | 2020-10-27 | 中国农业科学院兰州兽医研究所 | Porcine epidemic diarrhea virus M protein specific heavy chain antibody |
| CN108333369B (en) * | 2018-02-08 | 2019-09-24 | 中国科学院微生物研究所 | A kind of porcine reproductive and respiratory syndrome virus antibody detection method and its dedicated test card |
| CN109061159A (en) * | 2018-05-24 | 2018-12-21 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PEDV immune detection chromatograph test strip and its preparation method and application |
| CN108486069B (en) * | 2018-06-05 | 2021-10-26 | 中国农业科学院兰州兽医研究所 | Virus separation method for low-content sample of porcine epidemic diarrhea virus |
| CN108892723B (en) * | 2018-07-12 | 2021-09-21 | 内蒙古农业大学 | Single-domain heavy chain antibody for detecting porcine epidemic diarrhea virus, preparation method and application |
| CN109946458A (en) * | 2019-03-04 | 2019-06-28 | 武汉科前生物股份有限公司 | A kind of fluorescent test paper of quick detection Porcine epidemic diarrhea virus and preparation and application |
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| CN110187097B (en) * | 2019-05-28 | 2021-09-14 | 中国农业科学院哈尔滨兽医研究所 | Fluorescent quantitative detection test strip for porcine epidemic diarrhea virus and preparation method and application thereof |
| CN114146172B (en) * | 2021-12-07 | 2022-09-16 | 重庆市动物疫病预防控制中心 | Nano antibody vaccine capable of preventing porcine pseudorabies virus infection, preparation method and application |
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| CN103454419A (en) * | 2013-07-22 | 2013-12-18 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
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2015
- 2015-04-01 CN CN201510152757.7A patent/CN104804082B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101187665A (en) * | 2007-12-27 | 2008-05-28 | 北京博晖创新光电技术股份有限公司 | Enterovirus immunofluorescence chromatographic assay test paper and its preparation method |
| CN102766207A (en) * | 2012-07-18 | 2012-11-07 | 中国农业科学院兰州兽医研究所 | Bactrian camel heavy-chain (HC) variable-domain antibody resisting porcine circovirus 2 as well as preparation method and application thereof |
| CN103454419A (en) * | 2013-07-22 | 2013-12-18 | 东北农业大学 | Immune colloidal gold test strip for detecting porcine epidemic diarrhea virus as well as preparation method and application thereof |
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| Title |
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| Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA;Gerber Priscilla F et al;《Veterinary Journal》;20150401;33-36 * |
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