CN104698173A - Duck embryo early sex identification method and special kit - Google Patents
Duck embryo early sex identification method and special kit Download PDFInfo
- Publication number
- CN104698173A CN104698173A CN201510088392.6A CN201510088392A CN104698173A CN 104698173 A CN104698173 A CN 104698173A CN 201510088392 A CN201510088392 A CN 201510088392A CN 104698173 A CN104698173 A CN 104698173A
- Authority
- CN
- China
- Prior art keywords
- estradiol
- liquid
- elisa
- elisa plate
- plate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention belongs to the technical field of poultry sex detection, and in particular relates to a duck embryo early sex identification method and a special kit. The duck embryo early sex identification method is established by collecting a hatched early duck embryo allantoic fluid, making use of a monoclonal antibody for estradiol detection and carrying out antigen-antibody reaction. Male and female sexes are judged according to a characteristic that the content of estradiol in a female embryo is higher than 1ng/ml and the content of the estradiol in a male embryo is lower than 0.6ng/ml. The identification method disclosed by the invention can identify the female and male sexes in an early hatching period without an influence on the further development of the embryo; and the identification method is conducive to animal welfare and relieving the transmission of vertical diseases.
Description
Technical field
The invention belongs to Fowl Sex authenticate technology field, be specifically related to a kind of duck embryo early sex authentication method and dedicated kit.
Background technology
The sex appraisal method of duck is generally outward appearance identification method, touches anus identification method, sound identification method and molecular methods.Outward appearance identification method is distinguished according to the appearance traits feature of male and female duck, often has erroneous judgement; Although it is higher to touch anus identification method accuracy rate, there is the risk of some vertical transmission germ cross-infections, touch anus higher to technical requirement, need production experience to enrich guarantee accuracy rate; Sound identification method need the syrinx of duck grow good after could distinguish, going out shell to adolescency very difficult differentiation; Although molecular methods is simply accurate, different and the Sex estimation of the clip size of (Z chromosome, W chromosome) special gene on usability chromosome, identify that sex is to stop embryonic development for cost in embryonic period, embryonic phase, after can not accomplishing qualification sex, embryo still can continue to hatch shell.
Existing patent US6506570B1 utilizes radioimmunoassays to identify poultry gender, but activity method, owing to there is the harm of polluting, seldom uses now.And external kit cost is very high, general Study unit is difficult to bear expensive cost.Domestic have Nanjing company to establish ELISA kit based on biotin double-antibody sandwich know-why, after have detected the allantoic fluid sample that we collect, testing result cannot be utilized to distinguish male and female sex.And, set up the general inapplicable biotin double-antibody sandwich know-why of micromolecular enzyme linked immunological kit.Therefore, the upper urgent need of current production develops the sex appraisal method and detection kit that a kind of method is simple, reproducible, cost is low, conveniently sample, and accurately can identify the embryo gender of duck fast in early days, can greatly reduce costs again.This method is easy to implement, after sampling, embryo can continue to hatch shell, also alternative go out male and female are separated after shell artificial sex identification, prevent and cause vertical transmission of disease owing to turning over anus qualification, there is not the animal welfare issues that unwanted drake or duck are killed and caused.
Although ELISA detection method and kit are conventional technology, up to the present yet there are no the report that poultry gender authentication method especially adopts indirect ELISA method in duck embryo sex appraisal method,
Summary of the invention
The object of the invention is to the defect overcoming prior art, utilize indirect ELISA method to set up a kind of duck embryo early sex authentication method and dedicated kit.Present invention achieves and facilitate succinct acquisition test sample and set up a kind of duck embryo early sex authentication method and dedicated kit, reduce and cause the drawback that the continuation of vertical transmission disease is popular, to duck embryo early sex qualification rate of accuracy reached to 100% because tradition goes out the anus qualification sex of turning over after shell.
The present invention is achieved through the following technical solutions:
Applicant establishes the indirect ELISA method of a kind of duck embryo early sex qualification, and its step comprises preparation ELISA Plate and sample preparation, specifically comprises the following steps:
(1) from hatch machine, take out duck embryo, major part upwards, leaves standstill 3-5min, utilize candler according to egg, find the location place extracting allantoic fluid, extract the region of allantoic fluid at locating aperture, avoid vascular distribution place, mark behind location, with 75% alcoholic solution sterilization opening part, stretch into eggshell with disposable sterilized injector or pipettor again after piercing through eggshell with disposable aspiration needle and extract allantoic fluid, inject aseptic EP and manage, obtain allantoic fluid sample, be stored in-20 DEG C of refrigerators for subsequent use;
(2) ELISA core reagent is prepared, described core reagent comprises: the ELISA Plate (purchased from Wuhan Sino-American Biotechnology Company) of estradiol albumen bag quilt, estradiol monoclonal antibody (purchased from Wuhan Sino-American Biotechnology Company), standard items, standard items thinning agent, developer, stop buffer, cleansing solution and ELIAS secondary antibody; Described standard items are estradiol, and described standard items thinning agent is 1 × phosphate buffer containing 1% potassium chloride, and described developer is 3,3', 5,5'-tetramethyl benzidine, described stop buffer is the sulfuric acid of 2mol/l, and described cleansing solution is 0.01mol/L PBST; Described ELIAS secondary antibody is goat anti-mouse IgG;
Prepare as follows:
1) the bag quilt of ELISA ELISA Plate: using estradiol albumen as antigen, bag is diluted to by concentration 0.2ug/mL with the carbonate buffer solution of the 0.05mol/L of pH 9.6, bag is by ELISA ELISA Plate, add confining liquid (5g skimmed milk is dissolved in 100ml cleansing solution), preparation bag is by good ELISA Plate;
2) preparation of goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG-HRP enzyme labelled antibody, with 1 × phosphate buffer by volume 1:600 be diluted to working concentration, then use the membrane filtration of 0.22 μm degerming, aseptic subpackaged rearmounted 2 ~ 8 DEG C save backup;
3) preparation of developer: by 3,3', 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and makes;
4) preparation of stop buffer: the hydrochloric acid concentrated sulphuric acid being formulated as 2mol/L;
5) preparation of cleansing solution:
A. storing solution A liquid and storing solution B liquid
The storing solution A liquid of 0.2mol/L: by the NaH of 6.2404g
2pO
42H
2o is dissolved in 200ml ddH
2in O;
The storing solution B liquid of 0.2mol/L: by the Na of 71.628g
2hPO
412H
2o is dissolved in 1000ml ddH
2o;
B.0.1mol/L PBST damping fluid, pH=7.2-7.4
By 0.2mol/L PBST buffer A liquid 95ml+, 0.2mol/L B liquid 405ml, ddH
2o 500ml, NaCl 8.5g and the mixing of 0.05% Tween-20;
C.0.01mol/L PBST damping fluid
By the storing solution B liquid 202.5ml of the storing solution A liquid 47.5ml of 0.2mol/L, 0.2mol/L, ddH
2the NaOH 10ml of O 4747.5ml, NaCl 45g, 1N and Tween-20 2.5ml mixes;
(3) determination of standard concentration gradient and detecting step:
1) determination of standard concentration gradient: standard items gradient concentration is set and is followed successively by 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml and 2.0ng/ml;
2) detecting step:
A. carry out balancing at least 30min under the core reagent of preparation and the sample to be tested of collection being moved to 20-25 DEG C of room temperature;
B. get one piece of ELISA Plate, in every hole of ELISA Plate, add standard concentration gradient reagent successively, in every hole, then add duck embryo allantoic liquid 50 μ L to be checked in order, after this estradiol monoclonal antibody 50 μ L is added again, seal plate with plate, shake mixing gently, in 37 DEG C of reaction 30min;
C. take out the ELISA Plate of step b, dry the liquid in ELISA Plate hole, the cleansing solution diluted by the amount in 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
D. in the hole of ELISA Plate, add 100 μ L goat anti-mouse IgG (purchased from bio tech ltd of Google, Wuhan), seal plate with plate, shake mixing gently, in 37 DEG C of reaction 30min;
E. take out ELISA Plate, dry liquid in hole, the cleansing solution diluted by the amount in 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuge colour developing 15min;
G. in every hole of ELISA Plate, add stop buffer 50 μ L, when the color in the aperture of ELISA Plate transfers yellow to by blueness, be judged to be cessation reaction;
H. the OD value (typical curve formula as Fig. 3 and Fig. 4) of microplate reader in each hole of 450nm wavelength measurement is used immediately in 5min after adding stop buffer;
I. the matching of typical curve formula: as shown in Figure 3, Y=1.6474x2-3.662x+0.6417, and as shown in Figure 4, Y=1.3414x2-3.4603x+0.6809;
(4) result judges
The OD value formula brought into as shown in Figure 3 and Figure 4 of sample is obtained the content of estradiol in sample; When the estradiol content in the sample measured is higher than 1ng/ml, be judged to be female; When the estradiol content in the sample measured is lower than 0.6ng/ml, be judged to be male.
Based on said method, applicant has developed the ELISA kit of a kind of duck embryo early sex qualification, it comprises solid phase carrier, ELISA Plate, developer, stop buffer and cleansing solution; Described kit also comprises primary antibodie estradiol monoclonal antibody, ELIAS secondary antibody goat anti-mouse IgG, standard items, standard items thinning agent and plate paste, described solid phase carrier is estradiol albumen, described standard items are estradiol, and the concentration of described standard items estradiol sets gradually as 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml, 2.0ng/ml gradient; Described standard items thinning agent is 1 × phosphate buffer containing 1% potassium chloride, and described developer is by 3,3', and 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and obtains; Described stop buffer is the sulfuric acid of 2mol/L.
Application in the indirect ELISA method that ELISA kit of the present invention can be identified at duck embryo early sex, is preferably Jingjiang shelduck and changes the qualification of duck embryonic early sex in vain.
Compared with prior art good effect of the present invention is:
(1) sampling method of the present invention does not affect the continuation normal development of duck embryonic.
(2) detection method that the present invention sets up is beneficial to animal welfare and can reduces vertical transmission.
(3) raw material sources of the present invention is stablized, and method is simple, can grow early interim use at duck embryonic.
(4) the detection kit result set up of the present invention is stable, reproducible, and to duck embryo early sex qualification rate of accuracy reached to 100% and testing cost is lower, applicable grass-roots unit applies.
Accompanying drawing explanation
Fig. 1: be indirect competitive ELISA method techniqueflow chart of the present invention.
Fig. 2: kit assembling flow path of the present invention.
Fig. 3: be the white typical curve formula changing typical curve and the matching made when duck allantoic fluid measures.
Fig. 4: the typical curve made when being Jingjiang shelduck allantoic fluid mensuration and the typical curve formula of matching.
Embodiment
Below in conjunction with example, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 collection of sample in early stage and the preparation of core reagent
1, the collection of duck embryo allantoic liquid
From hatch machine, taking out duck embryo, (kind is Jingjiang shelduck or changes duck in vain, but be not limited to this two kinds), major part upwards, leave standstill 3-5min, utilize candler according to egg, find the location place extracting allantoic fluid, vascular distribution place should be avoided in the region extracting allantoic fluid at locating aperture, mark, with 75% alcohol disinfecting opening part, stretch into eggshell with disposable sterilized injector or pipettor again after piercing through eggshell with disposable aspiration needle and extract allantoic fluid, inject aseptic EP and manage, obtain allantoic fluid sample, be stored in-20 DEG C of refrigerators for subsequent use;
2, antigen protein (estradiol albumen) coated elisa plate is utilized
It is estradiol albumen (purchased from Wuhan Sino-American Biotechnology Company) coating buffer (the 25mmol/L carbonate buffer solution: Na of 40 μ g/ml by final concentration
2cO
31.59g, NaHCO
32.93g, uses ddH
2o is settled to 1000ml, pH9.6) volume ratio 1:200 dilution (final concentration is 0.2 μ g/ml) is carried out respectively, estradiol albumen is added in ELISA Plate (purchased from Wuhan Sino-American Biotechnology Company) with the volume ratio of 1:1 by the amount of every hole 100 μ l, acts on 10 ~ 12h at putting 4 DEG C; Discard coating buffer, wash 2 ~ 3 times, each 2min with cleansing solution (the cleansing solution formula see kit in " summary of the invention "), dry cleansing solution, every hole adds confining liquid (5g skimmed milk is dissolved in 100ml cleansing solution) 100 μ l, incubation 100min at putting 37 DEG C; Pat dry, drier 30min at putting 37 DEG C, with tinfoil paper sealed membrane, antigen coated microplate is closed, preserve at putting 4 DEG C.
3, the preparation of two anti-and core reagents
1) goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG (purchased from bio tech ltd of Google, Wuhan), with 1 × phosphate buffer by volume 1:100 be diluted to working concentration, use 0.22 μm of membrane filtration degerming again, aseptic subpackaged rearmounted 2 ~ 8 DEG C save backup;
2) preparation of developer: by 3,3', 5,5'-tetramethyl benzidine (TMB) is dissolved in dimethyl sulfoxide (DMSO) and makes;
3) preparation of stop buffer: the dilute sulfuric acid concentrated sulphuric acid being formulated as 2mol/L content;
4) preparation of cleansing solution:
A. storing solution A liquid and storing solution B liquid
0.2mol/L storing solution A liquid: by the NaH of 6.2404g
2pO
42H
2o is dissolved in 200ml ddH
2in O;
0.2mol/L storing solution B liquid: will, the Na of 71.628g
2hPO
412H2O is dissolved in 1000ml ddH
2o;
B.0.1mol/L PBST damping fluid (pH=7.2-7.4):
Get the storing solution A liquid 95ml of 0.2mol/L, 0.2mol/L B liquid 405ml, ddH
2o 500ml.NaCl 8.5g, 0.05% Tween-20 mixes;
C.0.01mol/L PBST damping fluid
0.2mol/L storing solution A liquid 47.5ml, 0.2mol/L B liquid 202.5ml, ddH
2naOH 10ml and the Tween-20 2.5ml of O 4747.5ml, NaCl 45g, 1N mix;
5) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleansing solution and obtains.
The determination of embodiment 2 estradiol standard items dilute concentration and the making of typical curve
The present invention sets gradually as 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml, 2.0ng/ml through the concentration of repeatedly groping condition determination confirmed standard product estradiol.
Utilize CurveExpert software fit standard curve and obtain typical curve formula.
Detect according to following detecting step:
A. carry out balancing at least 30min under the reagent of preparation and the testing sample (i.e. duck embryo allantoic liquid) of collection being moved to 20-25 DEG C of room temperature;
B. one piece of ELISA Plate is got, standard concentration gradient is added successively in every hole of ELISA Plate, then in every hole, be sequentially added into duck embryo allantoic liquid 50 μ L to be checked, after this estradiol monoclonal antibody (purchased from Wuhan Sino-American Biotechnology Company's product) 50 μ L is added, plate is sealed with plate, shake mixing gently, in 37 DEG C of reaction 30min;
C. take out this ELISA Plate, dry the liquid in ELISA Plate hole, the cleansing solution diluted by 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
D. in the hole of ELISA Plate, add 100 μ L goat anti-mouse IgG, seal plate with plate, shake mixing gently, in 37 DEG C of reaction 30min;
E. take out ELISA Plate, dry liquid in hole, the cleansing solution diluted by 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuge colour developing 15min;
G. in every hole of ELISA Plate, add stop buffer 50 μ L, when the color in the aperture of ELISA Plate transfers yellow to by blueness, be judged to be cessation reaction;
H. after adding stop buffer, the OD value of microplate reader in each hole of 450nm wavelength measurement is used immediately in 5min.
I. the matching of typical curve formula: obtain typical curve formula with CurveExpert software fit standard curve.As Fig. 3, wherein: Y=1.6474x2-3.662x+0.6417; And as Fig. 4, Y=1.3414x2-3.4603x+0.6809.
The application example 1 of embodiment 3 detection method
Utilize the conventional duck typical curve formula mensuration that changes in vain changing duck embryo allantoic liquid sample and foundation in vain to change estradiol content in duck embryo allantoic liquid in vain, thus duck male and female embryo gender can be changed in vain by precise Identification.Concrete grammar is as follows:
(1) duck that changes in vain of getting 14d embryo age (gathers from duck field, Fujian province, but the invention is not restricted to this kind) embryo's allantoic fluid 100ul, totally 22 samples, difference naked eyes according to male and female gonad development profile judge and record sex, wherein 10, male sample, 12, female sample, is put in-20 DEG C and saves backup;
(2) indirect competitive ELISA method [1] is utilized to measure estradiol content (according to embodiment 2 operation steps).Criterion curve (see Fig. 3), Criterion curve computing formula Y=1.6474x
2-3.662x+0.6417, all samples does not mark male and female sex when ELISA measures estradiol content;
(3) typical curve changing duck in vain according to Fig. 3 and computing formula Y=1.6474x
2the content of estradiol in-3.662x+0.6417 calculation sample, and corresponding with the male and female sex of precedence record, finally set up male and female sex determination standard according to the distribution of estradiol content in male and female sexual gland.And add up the accuracy of qualification result.Result is as table 1.
Table 1 14d embryo changes the testing result of female, male estradiol content in duck duck embryo age in vain
Typical curve according to Fig. 3 calculates the estradiol content of all samples, and find that in female gonads, estradiol content is all higher than 1ng/ml, in male gonad, estradiol content is all lower than 0.2ng/ml, qualification rate of accuracy reached 100% of the present invention.
The application example 2 of embodiment 4 detection method
The Jingjiang shelduck typical curve formula of conventional Jingjiang shelduck allantoic fluid sample and foundation is utilized to measure estradiol content in Jingjiang shelduck allantoic fluid, thus can precise Identification Jingjiang shelduck male and female embryo gender.
Concrete grammar is as follows:
(1) (sample picks up from Jingzhou City of Hubei Province to get the Jingjiang shelduck in 15d embryo age to 22d embryo age, but the invention is not restricted to this kind) embryo's allantoic fluid 100ul, totally 119 samples, all with the naked eye first judge according to male and female Gonadal Morphology difference and record sex, 65, male sample, 54, female sample, is put in-20 DEG C and saves backup;
(2) indirect competitive ELISA method is utilized to measure Jingjiang shelduck estradiol content (according to embodiment 2 operation steps).Obtain typical curve formula, Y=1.3414x with CurveExpert software fit standard curve (Jingjiang shelduck content standard curve is shown in Fig. 4)
2-3.4603x+0.6809, all samples does not mark male and female sex when ELISA measures estradiol content;
(3) typical curve according to Fig. 4 and computing formula Y=1.3414x
2estradiol content in-3.4603x+0.6809 calculation sample, and corresponding with the male and female sex of precedence record, finally set up male and female sex determination standard according to the distribution of estradiol content in male and female sexual gland.And add up the accuracy of qualification result.Result is as table 2 and table 3.
Estradiol content in table 2 15--22d embryo male Jingjiang shelduck allantoic fluid in age
Estradiol content in table 3 15--22d embryo female Jingjiang shelduck allantoic fluid in age
Calculate the estradiol content of all samples according to typical curve, find that in female gonads, estradiol content is all higher than 1ng/ml, in male gonad, estradiol content is all lower than 0.6ng/ml, qualification rate of accuracy reached 100%.The testing result of integrated embodiment 3 and embodiment 4, kit of the present invention according to estradiol content judges the standard of male and female sex as: when the estradiol content of mensuration is higher than 1ng/ml, be judged to be female; When mensuration estradiol content is lower than 0.6ng/ml, be judged to be male, this qualification result rate of accuracy reached 100%.
The replica test of embodiment 5 duck embryo early sex authentication method
The present embodiment ELISA Plate of the envelope antigen of 3 pieces of different batches detects 5 Jingjiang shelduck allantoic fluid samples (according to embodiment 2 operation steps) simultaneously.Obtain estradiol content result, calculating mean value and the standard deviation of 5 samples.CV (%)=standard deviation/mean value × 100%.Result shows: the coefficient of variation of 5 Jingjiang shelduck samples is respectively 5.2%, 2.9%, 7.0%, 4.8%, 1.1%, and the coefficient of variation is all less than 10%.By replica test of the present invention, show that ELISA method of the present invention is reproducible.The results are shown in Table 4.
The replica test of table 4 duck embryo early sex authentication method
Embodiment 6
Available reagent box on market is utilized to detect duck embryo allantoic liquid content, the Detection results of more existing ELISA kit.
1, the collection of duck embryo allantoic liquid
From hatch machine, taking out duck embryo, (kind is for change duck in vain, originate the same, incubation period 14d-24d embryo age), major part upwards, leave standstill 3-5min, utilize candler according to egg, searching location place, vascular distribution place should be avoided in the region extracting allantoic fluid at locating aperture, marks, with 75% alcohol disinfecting opening part, stretch into eggshell with disposable sterilized injector or pipettor again after piercing through eggshell with disposable aspiration needle and extract allantoic fluid, inject aseptic EP to manage, obtain allantoic fluid sample, be stored in-20 DEG C of refrigerators for subsequent use;
2, utilize Nanjing company that market is bought to detect based on the ELISA kit of the production of biotin double-antibody sandwich know-why, the component provided according to kit and operation steps obtain result as shown in table 5:
1) fit standard curve equation: y=45.019-0.6336
2) typical curve formulae discovery estradiol content (see table 5) is utilized:
The existing ELISA kit of table 5 detects the interpretation of result of estradiol content in duck embryo allantoic liquid
3) by obtain to change duck estradiol content value in vain corresponding with the sex of record, find that the individual estradiol content detected of variant male and female in period does not have significant difference, estradiol content difference male and female sex cannot be leaned on.Comparing embodiment 6 and embodiment 4,5, result shows, a kind of duck embryo early sex qualification dedicated kit testing result discrimination is good, reproducible, reliable results.Available reagent box can not distinguish estradiol content in male and female embryo allantoic fluid.
Embodiment 7
The assembling of duck embryo early sex qualification dedicated kit
Applicant assembles a kind of duck embryo early sex qualification dedicated kit (table 6) and comprising:
The assembling of a table 6 duck embryo early sex qualification dedicated kit
| Component | 96T | 48T |
| ELISA Plate | 96 holes | 48 holes |
| Standard items | 6x 1mL/ bottle | 6x 0.5mL/ bottle |
| Enzyme conjugates (ELIAS secondary antibody) | 1x 12mL/ bottle | 1x 6mL/ bottle |
| Antibody | 1x 7mL/ bottle | 1x 3.5mL/ bottle |
| Nitrite ion | 1x 12mL/ bottle | 1x 6mL/ bottle |
| Stop buffer | 1x 10mL/ bottle | 1x 5mL/ bottle |
| Sample dilution | 1x 50mL/ bottle | 1x 25mL/ bottle |
| Cleansing solution (10 ×) | 1x 50mL/ bottle | 1x 25mL/ bottle |
| Plate pastes | 4 | 4 |
2, the preparation of related reagent
1) goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG-HRP enzyme labelled antibody, with 1 × phosphate buffer by volume 1:600 be diluted to working concentration, then use 0.22 μm of membrane filtration degerming, aseptic subpackaged rearmounted 2 ~ 8 DEG C save backup;
2) preparation of developer: by 3,3', 5,5'-tetramethyl benzidine (TMB) is dissolved in dimethyl sulfoxide (DMSO) and makes;
3) preparation of stop buffer: utilize the concentrated sulphuric acid to be formulated as 2mol/L dilute sulfuric acid;
4) preparation of cleansing solution:
A. storing solution A liquid and storing solution B liquid
0.2mol/L storing solution A liquid: by the NaH of 6.2404g
2pO
42H
2o is dissolved in 200ml ddH
2in O;
0.2mol/L storing solution B liquid: by the Na of 71.628g
2hPO
412H2O is dissolved in 1000ml ddH
2o;
B.0.1mol/L PBST damping fluid (pH=7.2-7.4):
Get 0.2mol/L storing solution A liquid 95ml+0.2mol/L storing solution B liquid 405ml+ddH
2o 500ml+NaCl 8.5g+0.05% Tween-20, mixing;
C.0.01mol/L PBST damping fluid:
Get 0.2mol/L storing solution A liquid 47.5ml+0.2mol/L storing solution B liquid 202.5ml+ddH
2o 4747.5ml+NaCl 45g+1NNaOH 10ml+ Tween-20 2.5ml, mixing;
5) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleansing solution and obtains.
3, kit operation steps:
A. carry out balancing at least 30min under the core reagent of preparation and the sample to be tested of collection being moved to 20-25 DEG C of room temperature;
B. one piece of ELISA Plate is got, standard concentration gradient (concentration gradient see embodiment 2) is added successively in every hole of ELISA Plate, then order adds duck embryo allantoic liquid 50 μ L to be checked in every hole of ELISA Plate, after this estradiol monoclonal antibody 50 μ L is added, plate is sealed with plate, shake mixing gently, at 37 DEG C, react 30min;
C. take out ELISA Plate, dry the liquid in ELISA Plate hole, the cleansing solution diluted by 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
D. in every hole of ELISA Plate, add 100 μ L goat anti-mouse IgG, seal plate with plate, shake mixing gently, at 37 DEG C, react 30min;
E. take out ELISA Plate, dry liquid in hole, the cleansing solution diluted by 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
F. in every hole of ELISA Plate, add chromophoric solution 100 μ L, shake gently, in 37 DEG C of lucifuge colour developing 15min;
G. in every hole of ELISA Plate, add stop buffer 50 μ L, when the color in the aperture of ELISA Plate transfers yellow to by blueness, be judged to be cessation reaction;
H. after adding stop buffer, the OD value of microplate reader in each hole of 450nm wavelength measurement is used immediately in 5min;
I. typical curve formula is utilized to carry out matching (typical curve is respectively see Fig. 3 and Fig. 4).
4, result judges
Bring the actual OD value of sample into content that typical curve formula obtains estradiol in sample; When estradiol content in working sample is higher than 1ng/ml, be judged to be female; When estradiol content in working sample is lower than 0.6ng/ml, be judged to be male.
Embodiment in sum; it is only preferred embodiment of the present invention; it is not whole embodiment of the present invention; any restriction neither be done to the present invention; everyly according to the technology of the present invention essence, any simple modification, change and equivalent structure transformation are done to above embodiment, all still belong in the protection domain of technical solution of the present invention.
Leading reference:
[1] Gao Ji shakes. the foundation of CD58 racing ELISA detecting method in lowlenthal serum, Journal of Northwest Sci Tech University of Agriculture and Forestry (natural science edition .2009,37 (8): 25-30.
[2]Mikhail G,,et al..Radioimmunoassay of plasma estrone and estradiol.Steroids.1970,15(3):333–352.
[3]Dhar TK,Samanta AK,Ali E.Homogeneous enzyme immunoassay of estradiol usingestradiol-3-0-carboxymethyl ether as hapten.Steroids.1988,51(5-6):519-526
[4]Jabri JE.Enzyme immunoassay for plasma estradiol using a monoclonal antibody.The Journal of SteroidBiochemistry and Molecular Biology.1991,38(3):339-343.
Claims (3)
1. an indirect ELISA method for duck embryo early sex qualification, its step comprises preparation ELISA Plate and sample preparation, it is characterized in that the following step:
(1) from hatch machine, take out duck embryo, major part upwards, leaves standstill 3-5min, utilize candler according to egg, find the location place extracting allantoic fluid, extract the region of allantoic fluid at locating aperture, avoid vascular distribution place, mark behind location, with 75% alcoholic solution sterilization opening part, stretch into eggshell with disposable sterilized injector or pipettor again after piercing through eggshell with disposable aspiration needle and extract allantoic fluid, inject aseptic EP and manage, obtain allantoic fluid sample, be stored in-20 DEG C of refrigerators for subsequent use;
(2) prepare ELISA core reagent, this core reagent comprises: the ELISA Plate of estradiol albumen bag quilt, estradiol monoclonal antibody, standard items, standard items thinning agent, developer, stop buffer, cleansing solution and ELIAS secondary antibody; Described standard items are estradiol, and described standard items thinning agent is 1 × phosphate buffer containing 1% potassium chloride, and described developer is 3,3', 5,5'-tetramethyl benzidine, described stop buffer is the dilute sulfuric acid of 2mol/l, and described cleansing solution is 0.01mol/L PBST; Described ELIAS secondary antibody is goat anti-mouse IgG;
Prepare as follows:
1) the bag quilt of ELISA ELISA Plate: using estradiol albumen as antigen, is diluted to bag by concentration 0.2ug/mL with the carbonate buffer solution of the 0.05mol/L of pH 9.6, wraps by ELISA ELISA Plate, add confining liquid, and preparation bag is by good ELISA Plate;
2) preparation of goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG-HRP enzyme labelled antibody, with 1 × phosphate buffer by volume 1:600 be diluted to working concentration, then use the membrane filtration of 0.22 μm degerming, aseptic subpackaged rearmounted 2 ~ 8 DEG C save backup;
3) preparation of developer: by 3,3', 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and makes;
4) preparation of stop buffer: the dilute sulfuric acid concentrated sulphuric acid being formulated as 2mol/L;
5) preparation of cleansing solution:
A. storing solution A liquid and storing solution B liquid
The storing solution A liquid of 0.2mol/L: by the NaH of 6.2404g
2pO
42H
2o is dissolved in 200ml ddH
2in O;
The storing solution B liquid of 0.2mol/L: by the Na of 71.628g
2hPO
412H
2o is dissolved in 1000ml ddH
2o;
B.0.1mol/L PBST damping fluid, pH=7.2-7.4;
By 0.2mol/L PBST buffer A liquid 95ml+, 0.2mol/L B liquid 405ml, ddH
2o 500ml, NaCl 8.5g and 0.05% Tween-20, mixing;
C.0.01mol/L PBST damping fluid
By the storing solution B liquid 202.5ml of the storing solution A liquid 47.5ml of 0.2mol/L, 0.2mol/L, ddH
2o 4747.5ml, the NaOH 10ml of NaCl45g, 1N and Tween-20 2.5ml, mixing;
6) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleansing solution and obtains confining liquid;
(3) determination of standard concentration gradient and detecting step:
1) determination of standard concentration gradient: standard items gradient concentration is set and is followed successively by 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml and 2.0ng/ml;
2) detecting step:
A. carry out balancing at least 30min under the testing sample duck embryo allantoic liquid of the core reagent of preparation and collection being moved to 20-25 DEG C of room temperature;
B. get one piece of ELISA Plate, in every hole of ELISA Plate, add standard concentration gradient reagent successively, in every hole, then add duck embryo allantoic liquid 50 μ L to be checked in order, after this estradiol monoclonal antibody 50 μ L is added again, seal plate with plate, shake mixing gently, in 37 DEG C of reaction 30min;
C. take out the ELISA Plate of step b, dry the liquid in ELISA Plate hole, the cleansing solution diluted by the amount in 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
D. in the hole of ELISA Plate, add 100 μ L goat anti-mouse IgG, seal plate with plate, shake mixing gently, in 37 DEG C of reaction 30min;
E. take out ELISA Plate, dry liquid in hole, the cleansing solution diluted by the amount in 250 μ L/ holes washes plate 4 times, and each immersion 30 seconds, thieving paper pats dry;
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuge colour developing 15min;
G. in every hole of ELISA Plate, add stop buffer 50 μ L, when the color in the aperture of ELISA Plate transfers yellow to by blueness, be judged to be cessation reaction;
H. the OD value of microplate reader in each hole of 450nm wavelength measurement is used immediately in 5min after adding stop buffer;
I. the matching of typical curve formula: as shown in Figure 3, Y=1.6474x2-3.662x+0.6417, and as shown in Figure 4, Y=1.3414x2-3.4603x+0.6809;
(4) result judges
The OD value formula brought into as shown in Figure 3 and Figure 4 of testing sample is obtained the content of estradiol in sample; When the estradiol content in the sample measured is higher than 1ng/ml, be judged to be female; When the estradiol content in the sample measured is lower than 0.6ng/ml, be judged to be male.
2. an ELISA kit for duck embryo early sex qualification, comprises solid phase carrier, ELISA Plate, developer, stop buffer and cleansing solution; It is characterized in that: described kit also comprises primary antibodie estradiol monoclonal antibody, ELIAS secondary antibody goat anti-mouse IgG, standard items, standard items thinning agent and plate paste, described solid phase carrier is estradiol albumen, described standard items are estradiol, and the concentration of described standard items estradiol sets gradually as 0ng/ml, 0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml, 2.0ng/ml gradient; Described standard items thinning agent is 1 × phosphate buffer containing 1% potassium chloride, and described developer is by 3,3', and 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and obtains; Described stop buffer is the sulfuric acid of 2mol/L.
3. ELISA kit according to claim 2 Jingjiang shelduck and change in vain duck embryonic early sex qualification in application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510088392.6A CN104698173B (en) | 2015-02-26 | 2015-02-26 | A kind of duck embryo early sex authentication method and dedicated kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510088392.6A CN104698173B (en) | 2015-02-26 | 2015-02-26 | A kind of duck embryo early sex authentication method and dedicated kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104698173A true CN104698173A (en) | 2015-06-10 |
| CN104698173B CN104698173B (en) | 2016-08-17 |
Family
ID=53345512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510088392.6A Active CN104698173B (en) | 2015-02-26 | 2015-02-26 | A kind of duck embryo early sex authentication method and dedicated kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104698173B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105191831A (en) * | 2015-09-25 | 2015-12-30 | 中国农业大学 | Method for embryonated egg sex identification |
| CN108603874A (en) * | 2015-12-22 | 2018-09-28 | 奥凡多公司 | Method for sex identification in ovum gallinaceum |
| CN111387134A (en) * | 2020-03-06 | 2020-07-10 | 武汉市农业科学院 | Method for improving reproductive performance of small and medium-sized meat ducks cultured in cages |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995023237A1 (en) * | 1994-02-28 | 1995-08-31 | Agritech Technologies, Ltd. | Methods of determining the gender of poultry |
| CN1232545A (en) * | 1996-10-04 | 1999-10-20 | 恩布里克斯公司 | Method of sorting birds in ovo |
| CN1766626A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting estradiol and detection method thereof |
| CN202372504U (en) * | 2011-11-29 | 2012-08-08 | 武汉优尔生科技股份有限公司 | Estradiol enzyme linked immunosorbent assay kit |
-
2015
- 2015-02-26 CN CN201510088392.6A patent/CN104698173B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995023237A1 (en) * | 1994-02-28 | 1995-08-31 | Agritech Technologies, Ltd. | Methods of determining the gender of poultry |
| CN1232545A (en) * | 1996-10-04 | 1999-10-20 | 恩布里克斯公司 | Method of sorting birds in ovo |
| CN1766626A (en) * | 2005-11-03 | 2006-05-03 | 北京望尔生物技术有限公司 | ELISA kit for detecting estradiol and detection method thereof |
| CN202372504U (en) * | 2011-11-29 | 2012-08-08 | 武汉优尔生科技股份有限公司 | Estradiol enzyme linked immunosorbent assay kit |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105191831A (en) * | 2015-09-25 | 2015-12-30 | 中国农业大学 | Method for embryonated egg sex identification |
| CN105191831B (en) * | 2015-09-25 | 2017-12-12 | 中国农业大学 | A kind of embryo in egg sex appraisal method |
| CN108603874A (en) * | 2015-12-22 | 2018-09-28 | 奥凡多公司 | Method for sex identification in ovum gallinaceum |
| CN111387134A (en) * | 2020-03-06 | 2020-07-10 | 武汉市农业科学院 | Method for improving reproductive performance of small and medium-sized meat ducks cultured in cages |
| CN111387134B (en) * | 2020-03-06 | 2022-07-26 | 武汉市农业科学院 | Method for improving reproductive performance of small and medium-sized meat ducks cultured in cages |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104698173B (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103575913B (en) | The urinalysis test card of microdose urine protein/UCr | |
| CN107422129A (en) | A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit | |
| CN104897895B (en) | The magnetic immunochemiluminescence detection kit of a kind of gentamicin and application thereof | |
| CN101377500A (en) | Free prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN106290889A (en) | The detection method of AFB1 | |
| CN104698173A (en) | Duck embryo early sex identification method and special kit | |
| CN103091494B (en) | Aflatoxin M 1chemical luminescence ELISA detection kit and using method | |
| CN102998466A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit | |
| CN103901034A (en) | Detection reagent strip for detecting microalbumin in urine and preparation method of detection reagent strip | |
| CN104076154A (en) | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof | |
| CN104558084B (en) | The extracting method of steroid hormone in woods musk deer ight soil | |
| CN101315379B (en) | Reagent kit for detecting Ractopamine and application thereof | |
| CN101368953A (en) | Chemical luminescence ELISA detection reagent kit of ciprofloxacin | |
| CN101377498A (en) | Prostate gland specificity antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof | |
| CN112649610A (en) | Detection kit and detection method for methamphetamine drugs in hair | |
| CN103102319B (en) | Melamine hapten and its preparation method and application | |
| CN101545910A (en) | Ferritin chemoluminescence immunoassay quantitative measuring kit and preparation method thereof | |
| CN109254145A (en) | For improving the dilution of matrix effect between fresh serum and third party's Quality Control | |
| CN106771150A (en) | A kind of ELISA detection kit and detection method for detecting dimethomorph residual | |
| CN103645320B (en) | A kind of AMOZ chemiluminescent enzyme-linked immunosorbent immunity quick detection kit | |
| CN101949923A (en) | Enzyme-linked immunization kit of domoic acid and detection method thereof | |
| CN104897651A (en) | Chemiluminescent kit for aflatoxin M1 and application thereof | |
| CN105092563B (en) | Enzyme linked immunological kit and preparation and the method that pepsin detection is carried out using it | |
| CN107703294A (en) | A kind of ELISA method for being used to detect cLHRH antibody titers in chicken serum | |
| CN103163291A (en) | Kit and operation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: 430208 Hubei Province, Wuhan city Jiangxia District Jin Kou Jie Chang Shan Cun Jin Road No. 2 paper Patentee after: ANIMAL HUSBANDRY AND VETERINARY SCIENCE INSTITUTE, WUHAN ACADEMY OF AGRICULTURAL SCIENCE & TECHNOLOGY Address before: 430208 Hubei Province, Wuhan city Jiangxia District Road No. 2 jin Kou Jie paper Changshan Patentee before: WUHAN Research Institute OF ANIMAL AND VETERINARY SCIENCE |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170328 Address after: 430075 Hubei Province, East Lake hi tech Zone, No. two high tech Road, No. 388, building C22, biomedical park, building 5, building Patentee after: WUHAN SERVICEBIO TECHNOLOGY CO.,LTD. Address before: 430208 Hubei Province, Wuhan city Jiangxia District Jin Kou Jie Chang Shan Cun Jin Road No. 2 paper Patentee before: ANIMAL HUSBANDRY AND VETERINARY SCIENCE INSTITUTE, WUHAN ACADEMY OF AGRICULTURAL SCIENCE & TECHNOLOGY |