CN104280551A - Duck tembusu virus E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit and preparation method thereof - Google Patents
Duck tembusu virus E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit adopting tembusu virus protein E as a coating antigen, and a preparation method and application thereof. The kit comprises a standard positive control duck serum, a standard negative control duck serum, a tembusu virus protein E (SEQ ID No.2) coated ELISA plate, a goat-anti-duck enzyme-marked secondary antibody, a TMB substrate color development solution and a stop solution. The prepared E-ELISA detection kit is used for antibody detection after duck tembusu virus infection.
Description
Technical field
The invention provides a kind of enzyme linked immunosorbent assay (E-ELISA) detection kit being envelope antigen with duck tembusu virus E protein, the present invention also provides the preparation method of described kit.
Background technology
Flavivirus (Flavivirus) is by arthropod-borne, belongs to flaviviridae family member.Its genome is made up of single-stranded positive RNA, is about 10.5kb, is made up of an open reading frame, coding 3 structural proteins and 7 non-structural proteins altogether, this virus does not have poly-A tail at 3 ' end, and virion is by a coat protein (C), a memebrane protein (E), a little non-glycosylated protein (M), 7 non-structural protein (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) composition.In most of flavivirus, E protein is positioned virion surface, virus with receptors bind, intrusion, cell fusion process in play an important role
[1-2].
Since in April, 2010, a kind of Novel infectious disease causing egg production sharply to decline raises district's large area outburst aquatic bird all over China, and the kind of infected duck comprises: Beijing duck, kind duck, sheldrake, goose etc.
[3-5].This disease cause up to 100% the incidence of disease and be less than 5% mortality ratio, laying rate was down to about 10% in 5 days.Find after cuing open inspection that sick duck has serious ovarian hemorrhage, oaritis, ovarian atrophy phenomenon.Because this disease causes sharply declining of reproductive capacity to bring serious economic loss to many plants.Through virus isolation and Identification, this viruses indentification is duck tembusu virus (in the present invention also referred to as " tembusu virus ").
This disease all has popular at some provinces, municipalities and autonomous regions of China in recent years, and feedwater poultry breeding industry causes economic loss heavy.Domestic and international is viral conventional separation andpreconcentration for the sick diagnostic method of tembusu virus.Virus isolation and Identification technology (diagnostic procedure needs 7-10 days time), time-consuming, effort, also needs the equipment such as professional and Electronic Speculum; Be difficult to promotion and application clinically.Be badly in need of setting up a kind of sensitivity, the sick antibody detection method of accurate, quick, easy duck tembusu virus for this reason.Enzyme linked immunosorbent assay (ELISA) is a kind of method being widely used in animal diseases diagnosis, is more suitable for detecting large batch of blood serum sample in the short time, has good specificity and higher sensitivity.
Along with our people's growth in the living standard, the fast development of aquatic bird aquaculture, the ratio of aquatic bird generation tembusu virus disease also rises thereupon.Therefore, Study and Development tembusu virus antibody detection kit is badly in need of, for the control of domestic tembusu virus provides technical support.In the report of external computer MSR Information system, proved that E protein is the main immunogenic albumen of tembusu virus, immunogenicity is good, and body can be induced to produce immunoprotective antibody.By the comparative analysis to domestic different separated strain sequence, design E protein PCR primer, after E protein Prokaryotic expression, purification, with the ELISA method of its a kind of quick detection tembusu virus antibody for envelope antigen establishes.
Summary of the invention
The invention provides a kind of enzyme linked immunosorbent assay (E-ELISA) detection kit being envelope antigen with duck tembusu virus E protein, described kit comprises the positive duck serum of standard control, the negative duck serum of standard control, duck tembusu virus E protein wrap the elisa plate of quilt, goat-anti duck ELIAS secondary antibody and other reagent, by positive for standard control duck serum, the negative duck serum of standard control, duck tembusu virus E protein wrap the elisa plate of quilt, goat-anti duck ELIAS secondary antibody and other reagent set dress, namely described kit is obtained, other compositions comprise sample diluting liquid, 10 × concentrated washing lotion, tmb substrate nitrite ion, stop buffer, described kit is characterised in that the envelope antigen of enzyme mark elisa plate is the tembusu virus E protein of the prokaryotic expression of purifying, wrap after by elisa plate after quantitatively and obtain, positive standard serum is for obtaining after duck tembusu virus immunity specific pathogen free (SPF) duck, and negative standards's serum is SPF duck serum.
E-ELISA detection kit prepared by the present invention is for the metainfective antibody test of duck tembusu virus.
As mentioned above, RNA is extracted from duck tembusu virus TA strain (being separated voluntarily), obtain the coding nucleotide sequence (SEQ ID No.1) of tembusu virus E protein through tembusu virus E protein gene-specific primer P1 and P2 (primer sequence is in table 1) amplification after reverse transcription, be cloned in suitable procaryotic cell expression carrier, in corresponding prokaryotic host cell, carry out expression and purification, obtain tembusu virus E protein (SEQ ID No.2).Bag is can be used for by elisa plate after quantitatively.
For ease of purifying, the N end band through the tembusu virus E protein of RT-PCR expression has His label, is convenient to purifying.
Art technology should be appreciated that arbitrarily, utilizes tembusu virus E protein bag can be carried out according to the conventional method of this area by elisa plate.Such as, the elisa plate of described E protein bag quilt can be prepared by following process:
Be 5 μ g/ml with 0.05mol/L carbonate buffer solution (pH value 9.6) by the dilution of the E recombinant protein of purifying; Joined by carbonate buffer solution containing recombinant protein in 96 hole ELISA Plate, 100 μ l/ holes, are placed in 4 DEG C (15 ~ 18h); Confide all the liquid in 96 orifice plates, add 10mmol/L PBST (pH value 7.4), 200 μ l/ holes, wash 3 times, each 5min; Confide all the liquid in 96 orifice plates, add confining liquid, be placed in 37 DEG C of 2.5h; Confide all the liquid in 96 orifice plates, add 10mmol/L PBST (pH value 7.4), 200 μ l/ holes, wash 3 times, each 5min.Thoroughly confide all the liquid in 96 orifice plates, 96 orifice plates being coated with His-E albumen are placed in 37 DEG C, dry 2h, are placed with in desiccant plastic bag.
Standard negative control serum is SPF duck serum, is taken a blood sample, conventionally extract serum by SPF duck.Standard positive control serum, for taking a blood sample with after duck tembusu virus (such as, duck tembusu virus TA strain) immune SPF duck, conventionally extracts serum.For for kit, conventionally can distinguish and extract the serum after SPF duck serum and duck tembusu virus immune duck in a large number, and as standard items deposit, again extract without the need to detecting at every turn.
Goat-anti duck ELIAS secondary antibody can be commercially available, such as, and the HRP goat-anti duck two anti-(article No.: 04-25-06) can bought from KPL company.
Other reagent in described kit comprise: sample diluting liquid, 10 × concentrated washing lotion, tmb substrate nitrite ion, stop buffer.Its composition and compound method as follows:
1) sample diluting liquid: each kit loading amount 1 bottle, 250ml/ bottle; The process for preparation of 10 × concentrated washing lotion (100mmol/L PBST, pH7.4) is as follows: KH
2pO
42.6g, Na
2hPO
4-12H
2o28.9g, NaCl87.1g, deionized water 800ml, adjust pH to 7.4, adds deionized water to 1L, adds 5ml Tween-20 in this solution.During use, 10 × concentrated washing lotion obtains sample diluting liquid according to 1: 9 (V/V) dilution.
2) preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of pH value 9.6 coating buffer
First liquid: Na
2cO
310.6g adds deionized water dissolving to 500ml
Second liquid: NaHCO
38.4g adds deionized water dissolving to 500ml
Get first liquid 16ml, second liquid 34ml, adds deionized water to 200ml, is 0.05mol/L pH value 9.6 carbonate buffer solution.
3) tmb substrate nitrite ion is purchased from Tian Gen company, each kit loading amount 1 bottle/box 50ml.
4) stop buffer: each kit loading amount 1 bottle of bottle, 250ml/ bottle; The preparation of stop buffer: 2mol/L sulfuric acid solution (2mol/L H
2sO
4), concentrated sulfuric acid solution concentration is 18mol/L, and the concentrated sulphuric acid and deionized water are prepared by 1: 9 can become 2mol/L sulfuric acid solution.
5) preparation of confining liquid: KH
2pO
40.26g, Na
2hPO
4-12H
2o2.89g, NaCl8.71g, skimmed milk power 50g, deionized water 800ml, adjust pH to 7.4, adds deionized water to 1L, adds 0.5ml Tween-20 in this solution.
The sample detected is duck serum.
The invention still further relates to the method for enzyme linked immunosorbent assay (E-ELISA) detection kit that preparation is envelope antigen with duck tembusu virus E protein, described method comprises the steps:
(1) prokaryotic expression purifying duck tembusu virus E protein, gets appropriate duck tembusu virus E protein bag by elisa plate;
(2) extract serum as standard control negative serum from SPF duck, extract serum as standard control positive serum from the SPF duck after duck tembusu virus (such as, duck tembusu virus TA strain) immunity;
(3) goat-anti duck ELIAS secondary antibody is commercially available, the HR goat-anti duck two anti-(article No.: 04-25-06) bought such as, but not limited to, KPL company;
(4) prepare sample diluting liquid, 10 × concentrated washing lotion, tmb substrate nitrite ion and stop buffer:
Sample diluting liquid: each kit loading amount 1 bottle, 250ml/ bottle; The process for preparation of 10 × concentrated washing lotion (100mmol/L PBST, pH7.4) is as follows: KH
2pO
42.6g, Na
2hPO
4-12H
2o28.9g, NaCl87.1g, deionized water 800ml, adjust pH to 7.4, adds deionized water to 1L, adds 5ml Tween-20 in this solution.During use, 10 × concentrated washing lotion obtains sample diluting liquid according to 1: 9 (V/V) dilution;
The preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of pH value 9.6 coating buffer
First liquid: Na
2cO
310.6g adds deionized water dissolving to 500ml
Second liquid: NaHCO
38.4g adds deionized water dissolving to 500ml
Get first liquid 16ml, second liquid 34ml, adds deionized water to 200ml, is 0.05mol/L pH value 9.6 carbonate buffer solution;
Tmb substrate nitrite ion purchased from Tian Gen company, each kit loading amount 1 bottle/box 50ml;
Stop buffer: each kit loading amount 1 bottle, 250ml/ bottle; The preparation of stop buffer: 2mol/L sulfuric acid solution (2mol/L H
2sO
4), concentrated sulfuric acid solution concentration is 18mol/L, and the concentrated sulphuric acid and deionized water are prepared by 1: 9 can become 2mol/L sulfuric acid solution;
The preparation of confining liquid: KH
2pO
40.26g, Na
2hPO
4-12H
2o2.89g, NaCl8.71g, skimmed milk power 50g, deionized water 800ml, adjust pH to 7.4, adds deionized water to 1L, adds 0.5ml Tween-20 in this solution;
(5) assemble: the above-mentioned elisa plate of duck tembusu virus E protein bag quilt and the composition of appropriate (2)-(4) are assembled into kit.
Therefore, the invention provides the following:
1. the sick E-ELISA detection kit of duck tembusu virus, described kit comprise standard positive control duck serum, standard negative control duck serum, duck tembusu virus E protein wrap the elisa plate of quilt, goat-anti duck ELIAS secondary antibody and other reagent.
2. the kit described in the 1st, wherein said standard positive control duck serum is the serum of the SPF duck of extracting personal duck tembusu virus immunity, and described standard negative control duck serum is extract the serum from SPF duck.
3. the kit described in the 1st, wherein said duck tembusu virus E protein wrap quilt elisa plate obtained by elisa plate by being used in duck tembusu virus E protein bag recombinant expressed in prokaryotic expression system.
4. the kit described in the 3rd, wherein said duck tembusu virus E protein is by be cloned in the nucleotide sequence of the coding tembusu virus E protein shown in SEQ ID No.1 in procaryotic cell expression carrier and to express obtained in prokaryotic.
5. the kit described in the 1st, wherein said goat-anti duck ELIAS secondary antibody is that the goat-anti duck two of HR mark resists.
6. the kit described in the 1st, other reagent wherein said comprise sample diluting liquid, 10 × concentrated washing lotion, tmb substrate nitrite ion, stop buffer.
7. the kit described in the 1st, the detection method of described kit comprises:
(1) reactions steps: duck blood serum sample to be measured, standard positive control duck serum and standard negative control duck serum are added respectively described duck tembusu virus E protein wrap in the independently hole of the elisa plate of quilt, 37 DEG C reaction 1h;
(2) with two anti-reactions steps: after being washed in every hole, add described goat-anti duck ELIAS secondary antibody respectively, at 37 DEG C of reaction 1h;
(3) development step: after the washing of every hole, add tmb substrate nitrite ion respectively and carry out chromogenic reaction;
(4) reading step: color development stopping is reacted, reading in microplate reader;
(5) determining step: when the ratio of the OD value of duck blood serum sample reacting hole to be measured and the OD value in standard negative control duck seroreaction hole is more than or equal to 2.1, represents that duck blood serum sample to be measured is for positive, illustrates that duck to be measured has infected duck tembusu virus; When above-mentioned ratio is less than 2.1, represent that duck blood serum sample to be measured is for negative, illustrates that duck to be measured does not have infected duck tembusu virus.
Beneficial effect of the present invention:
Compared with the existing method detecting duck tembusu virus wasted time and energy, the method utilizing kit of the present invention to detect duck tembusu virus has following advantage:
(1) save time, need only 2 hours, can testing result be known;
(2) without the need to isolated viral, duck serum to be checked is directly detected, simple to operate;
(3) multiple sample can be detected simultaneously, carry out high flux detection;
(4) kit of the present invention has the high specific for duck tembusu virus, and detect not by the impact of other virus infectionses, accuracy is high;
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1. SDS-PAGE and the Western blot of the E protein of purifying analyzes; Swimming band 1, Protein Marker; Swimming band 2, the E protein of purifying; Swimming band 3, the E protein of purifying.
Embodiment
Further describe the present invention referring to specific embodiment, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
Reagent used in following embodiment if no special instructions, is the reagent of the pure rank of commercially available analysis.
Embodiment 1. prokaryotic expression tembusu virus E protein
1) duck tembusu virus TA strain is extracted (by Zhang Yun and Liu Ming in Taian Shandong acquired original by TRIZOL instructions, now be stored in Harbin veterinary institute) RNA, increases through tembusu virus E protein gene-specific primer P1 and P2 (synthesis of TaKaRa company) after reverse transcription.1% agarose gel electrophoresis reclaims PCR primer, is connected obtains recombinant plasmid pET32-E with prokaryotic expression plasmid pET32a (purchased from Novagen company).
Table 1. increases the primer sequence of tembusu virus E protein gene
2) recombinant plasmid pET-E and pET32a carrier (blank) Transformed E .coli BL21 (DE3) competent cell respectively, the single colony inoculation of picking contains in the LB nutrient culture media of 100mg/L ampicillin in 2mL, 37 DEG C of shaken overnight, within 2nd day, be inoculated in containing in the antibiotic LB nutrient culture media of same concentrations with the ratio of 1: 100 (v/v), 37 DEG C of shaken cultivation reach 0.6 ~ 0.8 to A600, add IPTG to final concentration 1mmol/L to induce, collect and express bacterial sediment, carry out SDS-PAGE and Western Blot after ultrasonic treatment sex change and analyze.Result display molecular weight is that the destination protein of 60kDa exists (E protein of 54kDa adds the His label protein of 6kDa).
3) purge process of high-purity recombinant protein is as follows: by the induction bacterium liquid prepared in a large number after ultrasonic degradation, after 4 DEG C of centrifugal 10min of 10000g, collecting precipitation, after dissolving with the 20mmol/L PBS (pH value 7.4) containing 6mol/L urea and 0.5mol/L NaCl, use HisTrap with after 0.45 μm of frit
tMhP (GE Healthcare) carries out the purifying (operation by specification carries out) of albumen.Recombinant protein (His-E) after post purifying is analyzed through SDS-PAGE and Western Blot and is carried out purity analysis.Only near 60KDa, have single protein band, other position is with (see Fig. 1, swimming band 2 and 3) without obviously assorted.Purifying protein concentration is measured by the Biophotometer ultraviolet spectrophotometer of Eppendorf company.
The preparation of embodiment 2. standard female serum
Prepared by standard female serum: choose 60 age in days SPF ducks (purchased from Harbin veterinary institute Experimental Animal Center) sterile blood sampling, treat that blood clotting is placed on 37 DEG C of incubators and places 2h, be placed in 2 ~ 8 DEG C again and place 1h, then the serum of precipitation is placed in centrifugal bottle, centrifugal 15 ~ the 20min of 8000r/min, aseptic separation of serum, is placed in-20 DEG C of preservations.
Prepared by standard positive serum: through PBS damping fluid (pH value 7.4) 1: 100 (v/v) doubly dilution duck tembusu virus TA strain (1 × 10
5.3eID
50/ ml) be inoculated in 12 age in days duck embryos, gather in the crops duck embryo allantoic liquid dead in 85 ~ 96 hours.By duck embryo allantoic liquid and the equal-volume Seppic emulsifying agent of results (purchased from) Homogeneous phase mixing, 0.5ml/ inoculated with subcutaneous injections SPF test duck, inoculates 3 times, every minor tick 14 days, sterile blood sampling, as stated above separation of serum on the 7th after final vaccination, be placed in-20 DEG C of preservations.
Embodiment 3. tembusu virus E protein bag is by elisa plate
Be 5 μ g/ml with 0.05mol/L carbonate buffer solution (pH value 9.6) by the dilution of the His-E recombinant protein of purifying.Joined by carbonate buffer solution containing recombinant protein in 96 hole ELISA Plate, 100 μ l/ holes, are placed in 4 DEG C (15 ~ 18h).Confide all the liquid in 96 orifice plates, add 10mmol/L PBST (pH value 7.4), 200 μ l/ holes, wash 3 times, each 5min.Confide all the liquid in 96 orifice plates, add confining liquid, be placed in 37 DEG C of 2.5h.Confide all the liquid in 96 orifice plates, add 10mmol/L PBST (pH value 7.4), 200 μ l/ holes, wash 3 times, each 5min.Thoroughly confide all the liquid in 96 orifice plates, 96 orifice plates being coated with His-E albumen are placed in 37 DEG C, dry 2h, are placed with in desiccant plastic bag.
The assembling of embodiment 4. kit
1) each kit is selected can to detect the antibody of 500 samples time and medicine and reagent amount is assembled:
2) positive control: the standard positive control serum embodiment 2 be up to the standards prepared, by 2ml/ bottle quantitative separating, is placed in-20 DEG C of preservations;
3) negative control: the standard negative control serum embodiment 2 be up to the standards prepared, by 2ml/ bottle quantitative separating, is placed in-20 DEG C of preservations;
4) enzyme labelled antibody: the goat-anti duck two of the HRP mark that KPL company buys resists, each kit 50ml packing (1: 500 dilution), 4 DEG C of preservations.
5) other reagent: by liquor capacity quantitative separating, preserve under normal temperature.
Preparation and the concrete consumption of other reagent described are as follows:
A) sample diluting liquid: each kit loading amount 1 bottle/box 250ml; The process for preparation of 10 × concentrated washing lotion (100mmol/L PBST, pH7.4) is as follows: KH
2pO
42.6g, Na
2hPO
4-12H
2o28.9g, NaCl87.1g, deionized water 800ml, adjust pH to 7.4, add deionized water to 1L, add 5ml Tween-20 in this solution, mixing, then use 0.22 μm of filter degerming, aseptic subpackaged, every bottle is no less than 250ml.During use, 10 × concentrated washing lotion obtains sample diluting liquid according to 1: 9 (V/V) dilution.
B) preparation of coating buffer: 0.05mol/L carbonate buffer solution, the preparation of pH value 9.6 coating buffer
First liquid: Na
2cO
310.6g adds deionized water dissolving to 500ml
Second liquid: NaHCO
38.4g adds deionized water dissolving to 500ml
Get first liquid 16ml, second liquid 34ml, adds deionized water to 200ml, is 0.05mol/L pH value 9.6 carbonate buffer solution.
C) tmb substrate nitrite ion is purchased from Tian Gen company, each kit loading amount 1 bottle/box 50ml.
D) stop buffer: each kit loading amount 1 bottle, 50ml/ bottle; The preparation of stop buffer: 2mol/L sulfuric acid solution (2mol/L H
2sO
4), concentrated sulfuric acid solution concentration is 18mol/L, and the concentrated sulphuric acid and deionized water are prepared by 1: 9 can become 2mol/L sulfuric acid solution.
E) preparation of confining liquid: KH
2pO
40.26g, Na
2hPO
4-12H
2o2.89g, NaCl8.71g, skimmed milk power 50g, deionized water 800ml, adjust pH to 7.4, adds deionized water to 1L, adds 0.5ml Tween-20 in this solution.
6) according to the method for coating ELISA Plate 5 pieces of tembusu virus E protein bag quilt of embodiment 3.
Wherein, the preparation of ELIAS secondary antibody: 0.26gKH
2pO
4, 2.89gNa
2hPO
4-12H
2o, 8.71gNaCl, 800ml deionized water, the goat-anti duck antibody of 0.25ml horseradish peroxidase-labeled, adjust pH to 7.4, adds deionized water to 1L, adds 5ml Tween-20 in this solution.Degerming, aseptic subpackaged with 0.22 μm of filter, be no less than 50ml/ bottle.Preserve 2 ~ 8 DEG C of preservations, the term of validity 12 months.
The preparation of positive standard serum: dilute positive serum with containing 100mmol/L PBST (pH value 7.4), make its OD450 value between 1.0-1.2.Degerming, aseptic subpackaged with 0.22 μm of filter, be no less than 50ml/ bottle.2 ~ 8 DEG C of preservations, the term of validity 12 months.
Being formulated as follows of 100mmol/L PBST (pH value 7.4): 0.26gKH
2pO
4, 2.89gNa
2hPO
4-12H
2o, 8.71g NaCl, 800ml deionized water, 100 μ l thimerosals, adjust pH value to 7.4, add deionized water to 1L, add 5ml Tween-20 in this solution.
The preparation of standard negative control serum: dilute positive serum with containing 100mmol/L PBST (pH value 7.4), make its OD450 value between 1-1.2.0.26gKH
2pO
4, 2.89gNa
2hPO
4-12H
2o, 8.71g NaCl, 800ml deionized water, 15mg is phenol red, 100 μ l thimerosals, adjusts pH value to 7.4, adds deionized water to 1L, adds 5ml Tween-20 (add phenol red can sterilization, long shelf-life) in this solution herein.Degerming, aseptic subpackaged with 0.22 μm of filter, be no less than 50ml/ bottle.2 ~ 8 DEG C of preservations, the term of validity 12 months.
The detection code of embodiment 5. tembusu virus E-ELISA antibody assay kit
A. the dilution of sample: the blood serum sample taking from bird to be detected needs to dilute 100 times with sample diluting liquid: use multiple tracks sample injector and 96 orifice plates to dilute, recommendation following steps: the serum sample first drawing 10 μ l, join in the sample diluting liquid of 90 μ l, mixing, draw the liquid that 10 μ l dilute 10 times again to add in ELISA reaction plate hole, then add the sample diluting liquid of 90 μ l.
B. in three row's reacting holes, standard positive, negative control sera and the 100 times of tested blood serum samples diluted that volume is 100 μ l are added respectively, often kind of blood serum sample three repeating holes.
C. preservative film is covered, 37 DEG C of effect 1h.
D. striping, washes 3 times by the 1 × washing lotion of 200 μ l, and is blotted by the liquid in elisa plate on thieving paper.
E. in each reacting hole, add the enzyme labelled antibody (goat-anti duck ELIAS secondary antibody) of 100 μ l.
F. film is covered, 37 DEG C of effect 1h.
G. striping, washes 3 times by the 1 × washing lotion of 200 μ l, and is blotted by the liquid in elisa plate on thieving paper.
H. in each reacting hole, the tmb substrate nitrite ion of 100 μ l is added, jog plate 2s.
I. reaction plate is placed on dark and is in 37 DEG C of effect 15min.
J. in each reacting hole, add the stop buffer of 100 μ l.
K. gently wipe the foul of reaction plate bottom surface with soft thing, add in stop buffer 5 ~ 10min, elisa plate is put in microplate reader (Elx808, purchased from BioTek, USA), complete reading at 450 nm, and record result.
L. result judges the OD value in record microplate reader every hole of ELISA Plate under 450nm wavelength, first carries out the zeroing of blank well system, if after blank well zeroing, when a large amount of negative values appears in systems axiol-ogy, it is invalid that whole system measures; The OD value of each sample determination diplopore should be basically identical, if holes measured value difference is comparatively large, it is invalid that this ELISA Plate measures.If standard positive control value OD lower than or be near the mark negative control OD value time, it is also invalid to measure.When testing sample OD value is more than or equal to 2.1 with the ratio of standard negative control OD value detection, testing sample is positive, otherwise is negative.
Such as, gather clinical serum 10 parts, detect according to the operation steps of ELISA of the present invention from certain duck field, standard female serum OD value (repeating for three times) mean value is 0.172; Mean value is 0.415-0.873 to 10 parts of serum OD values to be checked (repeating for three times), and the ratio of standard female serum is: between 0.415/0.172=2.41 and 0.873/0.172=5.08, this value is greater than 2.1.Therefore detected 10 parts of duck serum are judged to be that tembusu virus infects the positive.
M. specific assay detects to goose parvovirus, avian influenza virus, Avian pneumo-encephalitis virus, reovirus, I type DHV, Muscovy duck parvovirus, duck plague virus positive serum the reaction that is all negative, when other serum virus 1: 100 times dilution, OD value < 0.31.This illustrates that kit of the present invention can specific detection duck tembusu virus.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, do not deviating from by under the condition of the spirit and scope of the present invention as defined in the claims, the change of various forms and details can be carried out wherein, the combination in any of various embodiment can be carried out.
List of references
[1]Lindenbach B D，et al.Flaviviridae：the viruses and their replication，In Knipe DM，Howley，PM(ed)，Fields virology，5th ed.Lippincott Williams&Wilkins，Philadelphia，PA，2007，1101-1152.
[2]Rice C M，Strauss J H.Production of flavivirus polypeptides by proteolytic processing.Semin.Virol，1990，1：357-367.
[3]Su J，et al.Duck egg-drop syndrome caused by BYD virus，a new Tembusu-related flavivirus.PLoS One，2011，6(3)：e18106.
[4]Liu，M.，Chen，S.Y.，Chen，Y.H.，Liu，C.G.，Shilong Chen，Xiuchen Yin，Gang Li，and Yun Zhang.Adapted Tembusu-Like Virus in Chickens and Geese in China.J ClinMicrobiol.2012，50(8)：2807-2809.
[5]Liu M，Liu，C.G.，Li G.，Li X.J.，Yin X.C.，Chen Y.H.，Zhang Y.，2012.Complete genomic sequence of duck flavivirus from China.J.Virol.86：3398-3399。
Claims (7)
1. the sick E-ELISA detection kit of duck tembusu virus, described kit comprise standard positive control duck serum, standard negative control duck serum, duck tembusu virus E protein wrap the elisa plate of quilt, goat-anti duck ELIAS secondary antibody and other reagent.
2. kit according to claim 1, wherein said standard positive control duck serum is the serum of the SPF duck of extracting personal duck tembusu virus immunity, and described standard negative control duck serum is extract the serum from SPF duck.
3. kit according to claim 1, wherein said duck tembusu virus E protein wrap quilt elisa plate obtained by elisa plate by being used in duck tembusu virus E protein bag recombinant expressed in prokaryotic expression system.
4. kit according to claim 3, wherein said duck tembusu virus E protein is by be cloned in the nucleotide sequence of the coding tembusu virus E protein shown in SEQ ID No.1 in procaryotic cell expression carrier and to express obtained in prokaryotic.
5. kit according to claim 1, wherein said goat-anti duck ELIAS secondary antibody is that the goat-anti duck two of HRP mark resists.
6. kit according to claim 1, other reagent wherein said comprise sample diluting liquid, 10 × concentrated washing lotion, tmb substrate nitrite ion, stop buffer.
7. kit according to claim 1, the detection method of described kit comprises:
(1) reactions steps: duck blood serum sample to be measured, standard positive control duck serum and standard negative control duck serum are added respectively described duck tembusu virus E protein wrap in the independently hole of the elisa plate of quilt, 37 DEG C reaction 1h;
(2) with two anti-reactions steps: after being washed in every hole, add described goat-anti duck ELIAS secondary antibody respectively, at 37 DEG C of reaction 1h;
(3) development step: after the washing of every hole, add tmb substrate nitrite ion respectively and carry out chromogenic reaction;
(4) reading step: color development stopping is reacted, reading in microplate reader;
(5) determining step: when the ratio of the OD value of duck blood serum sample reacting hole to be measured and the OD value in standard negative control duck seroreaction hole is more than or equal to 2.1, represents that duck blood serum sample to be measured is for positive, illustrates that duck to be measured has infected duck tembusu virus; When above-mentioned ratio is less than 2.1, represent that duck blood serum sample to be measured is for negative, illustrates that duck to be measured does not have infected duck tembusu virus.
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| CN201810399078.3A CN108627645A (en) | 2013-07-11 | 2013-07-11 | Duck tembusu virus disease E-ELISA detection kits and preparation method thereof |
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| CN105445458A (en) * | 2015-11-13 | 2016-03-30 | 山东省滨州畜牧兽医研究院 | ELISA (Enzyme-linked Immuno Sorbent Assay) detection kit for identifying duck Tembusu virus infected animals and inactivated vaccine animals |
| CN108676078A (en) * | 2018-05-23 | 2018-10-19 | 江苏省农业科学院 | Cause the application of the antigen of tembusu virus antibody-dependant humidification |
| CN109748971A (en) * | 2019-01-24 | 2019-05-14 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | A kind of ELISA antibody assay kit of duck tembusu virus and its application |
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| CN109580941A (en) * | 2018-11-19 | 2019-04-05 | 安徽农业大学 | A kind of indirect ELISA method detecting duck tembusu virus antibody |
| CN109557316A (en) * | 2018-12-03 | 2019-04-02 | 湖北省农业科学院畜牧兽医研究所 | A kind of detection method of duck Tan Busu Yolk antibody |
| CN114315991B (en) * | 2020-10-30 | 2024-06-11 | 广西壮族自治区动物疫病预防控制中心 | Duck flavivirus E protein and monoclonal antibody-based competition ELISA method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105445458A (en) * | 2015-11-13 | 2016-03-30 | 山东省滨州畜牧兽医研究院 | ELISA (Enzyme-linked Immuno Sorbent Assay) detection kit for identifying duck Tembusu virus infected animals and inactivated vaccine animals |
| CN108676078A (en) * | 2018-05-23 | 2018-10-19 | 江苏省农业科学院 | Cause the application of the antigen of tembusu virus antibody-dependant humidification |
| CN109748971A (en) * | 2019-01-24 | 2019-05-14 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | A kind of ELISA antibody assay kit of duck tembusu virus and its application |
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