CN104698173B - A kind of duck embryo early sex authentication method and dedicated kit - Google Patents
A kind of duck embryo early sex authentication method and dedicated kit Download PDFInfo
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Abstract
The invention belongs to Fowl Sex detection technique field, specifically relate to method and dedicated kit that a kind of duck embryo early sex is identified.The present invention, by gathering hatching duck embryo allantoic liquid in early days, utilizes the monoclonal antibody for estradiol detection, and antigen antibody reaction, sets up duck embryo early sex authentication method.According to estradiol content in female embryo higher than 1ng/ml, in male embryo, estradiol content is less than 0.6ng/ml, it is determined that male and female sex.The present invention can identify male and female sex in early days in hatching and not affect the continuation growth of embryo, is of value to animal welfare and reduces vertical transmission of disease.
Description
Technical field
The invention belongs to Fowl Sex identification technology field, be specifically related to a kind of duck embryo early sex authentication method and special
Test kit.
Background technology
The sex appraisal method of duck generally outward appearance identification method, touch anus identification method, sound identification method and molecular methods.Outward
See identification method to distinguish according to the appearance traits feature of male and female duck, often have erroneous judgement；Although it is higher to touch anus identification method accuracy rate, have one
The risk of a little vertical transmission pathogenic bacteria cross infections, touches anus and requires higher to technology, and needing production experience to enrich could protect
Card accuracy rate；Sound identification method needs the syrinx of duck could distinguish after growing well, is difficult to distinguish going out shell to adolescence；Molecule reflects
Although method of determining is simply accurate, on usability chromosome, the clip size of (Z chromosome, W chromosome) special gene is different and judge
Period of embryo, sex, identifies that sex is to terminate fetal development as cost, it is impossible to after accomplishing to identify sex, embryo still can continue
Hatch shell.
Existing patent US6506570B1 utilizes radioimmunoassays to identify poultry gender, but, activity method is owing to depositing
In the harm polluted, it is rarely employed.And external test kit cost is the highest, general Study unit is difficult to bear high
Expensive cost.Domestic have Nanjing company to establish ELISA kit based on biotin double-antibody sandwich know-why, in inspection
After having surveyed the allantoic fluid sample that we collect, it is impossible to utilize testing result to distinguish male and female sex.And, set up the enzyme connection of little molecule
The general inapplicable biotin double-antibody sandwich know-why of immune reagent kit.Therefore, produce upper urgent need at present and develop a kind of method
Simply, reproducible, low cost, the sex appraisal method of convenient sampling and detection kit, can accurately quickly reflect in early days
Determine the embryo gender of duck, cost can be substantially reduced again.This method is easy to implement, and after sampling, embryo can continue to hatch shell,
Also replace out the separate artificial sex identification of male and female after shell, it is therefore prevented that cause vertical transmission of disease, no owing to turning over anus qualification
There is the animal welfare issues that unwanted drake or duck are killed and caused.
Although ELISA detection method and test kit are conventional technology, up to the present yet there are no poultry gender
Authentication method especially duck embryo sex appraisal method uses the report of indirect ELISA method,
Summary of the invention
It is an object of the invention to overcome the defect of prior art, utilize indirect ELISA method to set up a kind of duck embryo in early days
Sex appraisal method and dedicated kit.Present invention achieves and facilitate succinct acquisition test sample and set up a kind of duck embryo in early days
Sex appraisal method and dedicated kit, reduce the anus that turns over after going out shell because of tradition and identify that sex causes the continuation of vertical transmission disease
Popular drawback, identifies that to duck embryo early sex rate of accuracy reached is to 100%.
The present invention is achieved through the following technical solutions:
Applicant establishes the indirect ELISA method that a kind of duck embryo early sex is identified, its step includes preparing ELISA Plate
And sample treatment, specifically include the following step:
(1) taking out duck embryo from hatch machine, major part upwards, stands 3-5min, utilizes candler according to egg, finds extraction allantois
At the location of liquid, in the region of locating aperture extraction allantoic fluid, avoid, at vascularity, marking behind location, use 75% wine
Essence solution disinfection opening part, stretches into eggshell with disposable sterilized injector or pipettor again after piercing through eggshell with disposable aspiration needle and takes out
Take allantoic fluid, inject aseptic EP pipe, obtain allantoic fluid sample, be stored in-20 DEG C of refrigerators standby；
(2) preparation ELISA core reagent, described core reagent includes: the coated ELISA Plate of estradiol albumen is (purchased from Wuhan
Magnificent biological engineering company limited), estradiol monoclonal antibody (purchased from Wuhan Sino-American Biotechnology Company), standard substance,
Standard substance diluent, developer, stop buffer, cleaning mixture and ELIAS secondary antibody；Described standard substance are estradiol, described standard substance
Diluent is 1 × phosphate buffer containing 1% potassium chloride, and described developer is 3,3', 5,5'-tetramethyl benzidines,
Described stop buffer is the sulphuric acid of 2mol/l, and described cleaning mixture is 0.01mol/L PBST；Described ELIAS secondary antibody is goat
Anti-mouse IgG；
Prepare as follows:
1) being coated of ELISA ELISA Plate: using estradiol albumen as antigen, with the carbonate of the 0.05mol/L of pH 9.6
Buffer is diluted to be coated concentration 0.2ug/mL, is coated ELISA ELISA Plate, and (5g skimmed milk is dissolved in 100ml washing to add confining liquid
Liquid), the ELISA Plate that preparation is coated；
2) preparation of goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG-HRP enzyme labelled antibody, delay with 1 × phosphate
Rush liquid 1:600 by volume and be diluted to working concentration, then the membrane filtration by 0.22 μm is degerming, aseptic subpackaged rearmounted 2～8 DEG C of guarantors
Deposit standby；
3) preparation of developer: 3,3', 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and makes；
4) preparation of stop buffer: concentrated sulphuric acid is formulated as the hydrochloric acid of 2mol/L；
5) preparation of cleaning mixture:
A. storing solution A liquid and storing solution B liquid
The storing solution A liquid of 0.2mol/L: by the NaH of 6.2404g2PO4·2H2O is dissolved in 200ml ddH2In O；
The storing solution B liquid of 0.2mol/L: by the Na of 71.628g2HPO4·12H2O is dissolved in 1000ml ddH2O；
B.0.1mol/L PBST buffer, pH=7.2-7.4
By 0.2mol/L PBST buffer A liquid 95ml+, 0.2mol/L B liquid 405ml, ddH2O 500ml, NaCl 8.5g
With 0.05% tween 20 mixing；
C.0.01mol/L PBST buffer
Storing solution B liquid 202.5ml, ddH by the storing solution A liquid 47.5ml, 0.2mol/L of 0.2mol/L2O
NaOH 10ml and the tween 20 2.5ml mixing of 4747.5ml, NaCl 45g, 1N；
(3) determination of standard concentration gradient and detecting step:
1) determination of standard concentration gradient: standard substance 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. the core reagent of preparation and the sample to be tested of collection are moved to be balanced under 20-25 DEG C of room temperature at least 30min；
B. take one piece of ELISA Plate, every hole of ELISA Plate is added standard concentration gradient reagent successively, the most in order
In every hole, add duck embryo allantoic liquid 50 μ L to be checked, hereafter add estradiol monoclonal antibody 50 μ L, seal plate with plate, gently
Concussion mixing, reacts 30min in 37 DEG C；
C. take out the ELISA Plate of step b, dry the liquid in ELISA Plate hole, by the washing diluted of the amount in 250 μ L/ holes
Liquid washes plate 4 times, soaks 30 seconds every time, pats dry in absorbent paper；
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, react 30min in 37 DEG C；
E. taking out ELISA Plate, dry liquid in hole, the cleaning mixture diluted by the amount in 250 μ L/ holes washes plate 4 times, every time
Soak 30 seconds, absorbent paper pats dry；
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuges colour developing 15min；
G. in every hole of ELISA Plate, stop buffer 50 μ L is added, when the color in the aperture of ELISA Plate is transferred to yellow by blueness
Time be judged to terminate reaction；
H. (standard curve is public to use the microplate reader OD value in each hole of 450nm wavelength measurement after adding stop buffer in 5min immediately
Formula such as Fig. 3 and Fig. 4)；
I. the matching of standard curve formula: as it is shown on 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
Bring the OD value of sample into formula as shown in Figure 3 and Figure 4 and obtain the content of estradiol in sample；When the sample measured
When estradiol content in product is higher than 1ng/ml, it is determined that for female；When the estradiol content in the sample measured is less than 0.6ng/
Ml, it is determined that for male.
Based on said method, applicant has developed the ELISA kit that a kind of duck embryo early sex is identified, it includes solid phase
Carrier, ELISA Plate, developer, stop buffer and cleaning mixture；Described test kit also includes an anti-estradiol monoclonal antibody, enzyme mark
Two anti-goat anti-mouse IgG, standard substance, standard substance diluent and plate patch, described solid phase carrier is estradiol albumen, described
Standard substance are estradiol, and the concentration of described standard substance estradiol sets gradually as 0ng/ml, 0.05ng/ml, 0.15ng/ml,
0.45ng/ml, 1.0ng/ml, 2.0ng/ml gradient；Described standard substance diluent is 1 × phosphate containing 1% potassium chloride
Buffer, described developer is by 3,3', and 5,5'-tetramethyl benzidines are dissolved in dimethyl sulfoxide and obtain；Described stop buffer is
The sulphuric acid of 2mol/L.
The ELISA kit of the present invention can application in the indirect ELISA method that duck embryo early sex is identified, preferably
Jingjiang shelduck and change in vain duck embryonic early sex identify.
Compared with prior art the positive 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 reduce vertical pathophoresis.
(3) raw material sources of the present invention is stable, and method is simple, it is possible to grow early interim use at duck embryonic.
(4) the detection kit result that the present invention sets up is stable, reproducible, and duck embryo early sex is identified rate of accuracy reached
Relatively low to 100% and testing cost, it is suitable for grass-roots unit's popularization and application.
Accompanying drawing explanation
Fig. 1: be the indirect competitive ELISA method techniqueflow chart of the present invention.
The test kit assembling flow path of Fig. 2: the present invention.
Fig. 3: be the white standard curve and the standard curve formula of matching changing and making when duck allantoic fluid measures.
Fig. 4: be the standard curve and the standard curve formula of matching made during Jingjiang shelduck allantoic fluid mensuration.
Detailed description of the invention
Below in conjunction with example, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The collection of embodiment 1 early stage sample and the preparation of core reagent
1, the collection of duck embryo allantoic liquid
From hatch machine take out duck embryo (kind is Jingjiang shelduck or changes duck in vain, but is not limited to the two kind), major part to
On, stand 3-5min, utilize candler according to egg, find at the location of extraction allantoic fluid, in the district of locating aperture extraction allantoic fluid
Territory should be avoided at vascularity, marks, and with 75% alcohol disinfecting opening part, uses once after piercing through eggshell with disposable aspiration needle again
Property asepsis injector or pipettor stretch into eggshell extraction allantoic fluid, inject aseptic EP pipe, obtain allantoic fluid sample, be stored in-20
DEG C refrigerator is standby；
2, antigen protein (estradiol albumen) coated elisa plate is utilized
By the estradiol albumen (purchased from Wuhan Sino-American Biotechnology Company) of final concentration of 40 μ g/ml with being coated liquid
(25mmol/L carbonate buffer solution: Na2CO3 1.59g,NaHCO32.93g, uses ddH2O is settled to 1000ml, pH9.6) respectively
Carrying out volume ratio 1:200 dilution (final concentration is 0.2 μ g/ml), estradiol albumen presses every hole 100 μ l's with the volume ratio of 1:1
Amount is added in ELISA Plate (purchased from Wuhan Sino-American Biotechnology Company), puts and acts on 10～12h at 4 DEG C；Discard and be coated liquid, use
Cleaning mixture (see the cleaning mixture formula of test kit in " summary of the invention ") washs 2～3 times, each 2min, dries cleaning mixture, and every hole adds
Confining liquid (5g skimmed milk is dissolved in 100ml cleaning mixture) 100 μ l, puts incubation 100min at 37 DEG C；Pat dry, then put at 37 DEG C dry
30min, closes antigen coated microplate with tinfoil paper sealed membrane, puts and preserves at 4 DEG C.
3, the two anti-and preparation of core reagent
1) goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG (purchased from bio tech ltd of Google, Wuhan), uses
1 × phosphate buffer 1:100 by volume is diluted to working concentration, more degerming with 0.22 μm membrane filtration, aseptic subpackaged after
Put 2～8 DEG C to save backup；
2) preparation of developer: 3,3', 5,5'-tetramethyl benzidine (TMB) are dissolved in dimethyl sulfoxide (DMSO) and make；
3) preparation of stop buffer: concentrated sulphuric acid is formulated as the dilute sulfuric acid of 2mol/L content；
4) preparation of cleaning mixture:
A. storing solution A liquid and storing solution B liquid
0.2mol/L storing solution A liquid: by the NaH of 6.2404g2PO4·2H2O is dissolved in 200ml ddH2In O；
0.2mol/L storing solution B liquid: will, the Na of 71.628g2HPO412H2O is dissolved in 1000ml ddH2O；
B.0.1mol/L PBST buffer (pH=7.2-7.4):
Take storing solution A liquid 95ml, 0.2mol/L the B liquid 405ml, ddH of 0.2mol/L2O 500ml.NaCl 8.5g,
0.05% tween 20 mixes；
C.0.01mol/L PBST buffer
0.2mol/L storing solution A liquid 47.5ml, 0.2mol/L B liquid 202.5ml, ddH2O 4747.5ml, NaCl 45g,
The NaOH 10ml and tween 20 2.5ml of 1N mix；
5) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleaning mixture and obtains.
The determination of embodiment 2 estradiol standard substance diluted concentration and the making of standard curve
The present invention determines that through repeatedly groping condition determination the concentration of standard substance estradiol sets gradually as 0ng/ml,
0.05ng/ml, 0.15ng/ml, 0.45ng/ml, 1.0ng/ml, 2.0ng/ml.
Utilize CurveExpert software fit standard curve and obtain standard curve formula.
Detect according to following detecting step:
A. move to be balanced under 20-25 DEG C of room temperature by the reagent of preparation and the testing sample (i.e. duck embryo allantoic liquid) of collection
At least 30min；
B. take one piece of ELISA Plate, every hole of ELISA Plate is added standard concentration gradient successively, then presses in every hole
Order adds duck embryo allantoic liquid 50 μ L to be checked, is subsequently to added into estradiol monoclonal antibody (limited purchased from the magnificent biological engineering in Wuhan
Products) 50 μ L, seal plate with plate, shake mixing gently, react 30min in 37 DEG C；
C. taking out this ELISA Plate, dry the liquid in ELISA Plate hole, plate 4 washed by the cleaning mixture diluted by 250 μ L/ holes
Secondary, soak 30 seconds every time, absorbent 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
Reaction 30min；
E. taking out ELISA Plate, dry liquid in hole, the cleaning mixture diluted by 250 μ L/ holes is washed plate 4 times, is soaked every time
30 seconds, absorbent paper pats dry；
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuges colour developing 15min；
G. in every hole of ELISA Plate, stop buffer 50 μ L is added, when the color in the aperture of ELISA Plate is transferred to yellow by blueness
Time be judged to terminate reaction；
H. after adding stop buffer, in 5min, use the microplate reader OD value in each hole of 450nm wavelength measurement immediately.
I. the matching of standard curve formula: with CurveExpert software fit standard curve and obtain standard curve formula.
Such as Fig. 3, wherein: Y=1.6474x2-3.662x+0.6417；And such as Fig. 4, Y=1.3414x2-3.4603x+0.6809.
The application example 1 of embodiment 3 detection method
The conventional duck standard curve formula mensuration that changes in vain changing duck embryo allantoic liquid sample and foundation in vain is utilized to change duck embryo urine in vain
Estradiol content in capsule liquid, such that it is able to precise Identification changes duck male and female embryo gender in vain.Concrete grammar is as follows:
(1) take 14d embryo age changes duck (gather from duck field, Fujian province, but the invention is not restricted to this kind) embryo in vain
Allantoic fluid 100ul, totally 22 samples, judge according to the difference naked eyes of male and female gonad development profile and record sex, the most male
10, sample, 12, female sample, it 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 operating procedure).Criterion
Curve (see Fig. 3), Criterion curve computing formula Y=1.6474x2-3.662x+0.6417, all samples measures at ELISA
Not labelling male and female sex during estradiol content；
(3) according to the standard curve changing duck in vain described in Fig. 3 and computing formula Y=1.6474x2-3.662x+0.6417 counts
Calculate the content of estradiol in sample, and corresponding with the male and female sex of precedence record, finally according to estradiol content in male and female gonad
Distribution set up male and female sex determination's standard.And add up the accuracy of qualification result.Result such as table 1.
Table 1 14d embryo changes the testing result of estradiol content female, male in duck duck embryo age in vain
Calculate the estradiol content of all samples according to the standard curve described in Fig. 3, find estradiol in female gonads
Content is all higher than 1ng/ml, and in male gonad, estradiol content is entirely below 0.2ng/ml, the qualification rate of accuracy reached of the present invention
100%.
The application example 2 of embodiment 4 detection method
The Jingjiang shelduck standard curve formula utilizing conventional Jingjiang shelduck allantoic fluid sample and foundation measures Jingjiang shelduck
Estradiol content in allantoic fluid, such that it is able to precise Identification Jingjiang shelduck male and female embryo gender.
Concrete grammar is as follows:
(1) (sample picks up from Jingzhou City of Hubei Province, but the invention is not restricted to this to take the Jingjiang shelduck in 15d embryo age to 22d embryo age
Kind) embryo allantoic fluid 100ul, totally 119 samples, with the naked eye first determine whether and record all according to male and female Gonadal Morphology difference
Sex, 65, male sample, 54, female sample, it 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 operating procedure).With
CurveExpert software fit standard curve (Jingjiang shelduck content standard curve is shown in Fig. 4) also obtains standard curve formula, Y=
1.3414x2-3.4603x+0.6809, all samples is not labelling male and female sex when ELISA measures estradiol content；
(3) according to the standard curve shown in Fig. 4 and computing formula Y=1.3414x2-3.4603x+0.6809 calculates sample
Middle estradiol content, and corresponding with the male and female sex of precedence record, build finally according to the distribution of estradiol content in male and female gonad
Vertical male and female sex determination's standard.And add up the accuracy of qualification result.Result such as table 2 and table 3.
Estradiol content in table 2 15--22d embryo male Jingjiang shelduck in age allantoic fluid
Estradiol content in table 3 15--22d embryo female Jingjiang shelduck in age allantoic fluid
Calculate the estradiol content of all samples according to standard curve, find that in female gonads, estradiol content is the highest
In 1ng/ml, male gonad, estradiol content is entirely below 0.6ng/ml, identifies rate of accuracy reached 100%.Integrated embodiment 3 with
The testing result of embodiment 4, the test kit of the present invention according to estradiol content judge the standard of male and female sex as: female when measure
Glycol content is higher than 1ng/ml, it is determined that for female；When measuring estradiol content less than 0.6ng/ml, it is determined that for 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 simultaneously
(according to embodiment 2 operating procedure).Obtain the estradiol content result of 5 samples, calculate meansigma methods and standard deviation.CV (%)=
Standard deviation/meansigma methods × 100%.Result shows: the coefficient of variation of 5 Jingjiang shelduck samples is respectively 5.2%, 2.9%,
7.0%, 4.8%, 1.1%, the coefficient of variation is respectively less than 10%.By replica test of the present invention, show the ELISA side of the present invention
Method is reproducible.The results are shown in Table 4.
The replica test of table 4 duck embryo early sex authentication method
Embodiment 6
Utilize available reagent box detection duck embryo allantoic liquid content, the relatively Detection results of existing ELISA kit on market.
1, the collection of duck embryo allantoic liquid
Take out from hatch machine duck embryo (kind for changing duck in vain, source ibid, embryo age incubation period 14d-24d), major part upwards,
Stand 3-5min, utilize candler according to egg, find at location, vascularity should be avoided in the region of locating aperture extraction allantoic fluid
Place, marks, and with 75% alcohol disinfecting opening part, pierces through after eggshell again with disposable sterilized injector or shifting with disposable aspiration needle
Liquid device stretches into eggshell extraction allantoic fluid, injects aseptic EP pipe, obtains allantoic fluid sample, be stored in-20 DEG C of refrigerators standby；
2, the ELISA of the Nanjing company production based on biotin double-antibody sandwich know-why bought on market is utilized
The result that test kit detection, the component provided according to test kit and operating procedure acquisition are as shown in table 5:
1) fit standard curve equation: y=45.019-0.6336
2) utilize standard curve formula calculate estradiol content (seeing table 5):
The interpretation of result of estradiol content in table 5 existing ELISA kit detection duck embryo allantoic liquid
3) by obtain to change duck estradiol content value in vain corresponding with the sex of record, find variant male and female health check-up in period
The estradiol content gone out is not significantly different from, it is impossible to distinguish male and female sex by estradiol content.Comparing embodiment 6 and embodiment 4,
5, result shows, a kind of duck embryo early sex identifies that dedicated kit testing result discrimination is good, reproducible, reliable results.
Available reagent box cannot distinguish between estradiol content in male and female embryo's allantoic fluid.
Embodiment 7
Duck embryo early sex identifies the assembling of dedicated kit
Applicant assembles a kind of duck embryo early sex and identifies that dedicated kit (table 6) including:
6 one kinds of duck embryo early sexes of table identify that dedicated kit assembles
| Component | 96T | 48T |
| ELISA Plate | 96 holes | 48 holes |
| Standard substance | 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 |
| Cleaning mixture (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, press with 1 × phosphate buffer
Volume ratio 1:600 is diluted to working concentration, more degerming with 0.22 μm membrane filtration, and aseptic subpackaged rearmounted 2～8 DEG C save backup；
2) preparation of developer: 3,3', 5,5'-tetramethyl benzidine (TMB) are dissolved in dimethyl sulfoxide (DMSO) and make；
3) preparation of stop buffer: utilize concentrated sulphuric acid to be formulated as 2mol/L dilute sulfuric acid；
4) preparation of cleaning mixture:
A. storing solution A liquid and storing solution B liquid
0.2mol/L storing solution A liquid: by the NaH of 6.2404g2PO4·2H2O is dissolved in 200ml ddH2In O；
0.2mol/L storing solution B liquid: by the Na of 71.628g2HPO412H2O is dissolved in 1000ml ddH2O；
B.0.1mol/L PBST buffer (pH=7.2-7.4):
Take 0.2mol/L storing solution A liquid 95ml+0.2mol/L storing solution B liquid 405ml+ddH2O 500ml+NaCl 8.5g+
0.05% tween 20, mixing；
C.0.01mol/L PBST buffer:
Take 0.2mol/L storing solution A liquid 47.5ml+0.2mol/L storing solution B liquid 202.5ml+ddH2O 4747.5ml+
NaCl 45g+1N NaOH 10ml+ tween 20 2.5ml, mixing；
5) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleaning mixture and obtains.
3, test kit operating procedure:
A. the core reagent of preparation and the sample to be tested of collection are moved to be balanced under 20-25 DEG C of room temperature at least 30min；
B. take one piece of ELISA Plate, every hole of ELISA Plate is added standard concentration gradient successively and (sees the dense of embodiment 2
Degree gradient), then order adds duck embryo allantoic liquid 50 μ L to be checked in every hole of ELISA Plate, is subsequently to added into estradiol monoclonal anti
Body 50 μ L, seals plate with plate, shakes mixing gently, reacts 30min at 37 DEG C；
C. taking out ELISA Plate, dry the liquid in ELISA Plate hole, the cleaning mixture diluted by 250 μ L/ holes washes plate 4 times,
Soak 30 seconds every time, absorbent 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, in 37
30min is reacted at DEG C；
E. taking out ELISA Plate, dry liquid in hole, the cleaning mixture diluted by 250 μ L/ holes is washed plate 4 times, is soaked every time
30 seconds, absorbent paper pats dry；
F. in every hole of ELISA Plate, add chromophoric solution 100 μ L, shake gently, in 37 DEG C of lucifuges colour developing 15min；
G. in every hole of ELISA Plate, stop buffer 50 μ L is added, when the color in the aperture of ELISA Plate is transferred to yellow by blueness
Time be judged to terminate reaction；
H. after adding stop buffer, in 5min, use the microplate reader OD value in each hole of 450nm wavelength measurement immediately；
I. standard curve formula is utilized to be fitted (standard curve sees Fig. 3 and Fig. 4 respectively).
4, result judges
Bring the actual OD value of sample into standard curve formula and obtain the content of estradiol in sample；Female when measuring in sample
Glycol content is higher than 1ng/ml, it is determined that for female；It is less than 0.6ng/ml, it is determined that for male when measuring estradiol content in sample.
Embodiment in sum, is only presently preferred embodiments of the present invention, is not whole embodiments of the present invention, the most not
That the present invention is done any restriction, every according to the technology of the present invention essence, above example is done any simple modification, change and
Equivalent structure transformation, all still falls within the protection domain of technical solution of the present invention.
Leading reference:
[1] Gao Ji shakes. and the foundation of CD58 racing ELISA detecting method in lowlenthal serum, northwest agriculture and forestry science and technology is big
Learn journal (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 using estradiol-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 Steroid Biochemistry and Molecular
Biology.1991,38(3):339-343.
Claims (1)
1. the indirect ELISA method that duck embryo early sex is identified, its step includes preparing ELISA Plate and sample treatment, and it is special
Levy and be the following step:
(1) taking out duck embryo from hatch machine, major part upwards, stands 3-5min, utilizes candler according to egg, finds extraction allantoic fluid
At location, in the region of locating aperture extraction allantoic fluid, avoid, at vascularity, marking behind location, molten with 75% ethanol
Liquid disinfectant opening part, stretches into eggshell extraction urine with disposable sterilized injector or pipettor after piercing through eggshell with disposable aspiration needle again
Capsule liquid, injects aseptic EP pipe, obtains allantoic fluid sample, be stored in-20 DEG C of refrigerators standby；
(2) preparation ELISA core reagent, this core reagent includes: the coated ELISA Plate of estradiol albumen, estradiol monoclonal anti
Body, standard substance, standard substance diluent, developer, stop buffer, cleaning mixture and ELIAS secondary antibody；Described standard substance are estradiol, institute
The standard substance diluent stated is 1 × phosphate buffer containing 1% potassium chloride, and described developer is 3,3', 5,5'-tetramethyls
Base benzidine, described stop buffer is the dilute sulfuric acid of 2mol/l, and described cleaning mixture is 0.01mol/L PBST；Described enzyme mark
Two resist for goat anti-mouse IgG；
Prepare as follows:
1) being coated of ELISA ELISA Plate: using estradiol albumen as antigen, with the carbonate buffer of the 0.05mol/L of pH 9.6
Liquid is diluted to be coated concentration 0.2 μ g/mL, is coated ELISA ELISA Plate, adds confining liquid, the ELISA Plate that preparation is coated；
2) preparation of goat anti-mouse ELIAS secondary antibody: by goat anti-mouse IgG-HRP enzyme labelled antibody, with 1 × phosphate buffer
1:600 is diluted to working concentration by volume, then the membrane filtration by 0.22 μm is degerming, and aseptic subpackaged rearmounted 2～8 DEG C of preservations are standby
With；
3) preparation of developer: 3,3', 5,5'-tetramethyl benzidine is dissolved in dimethyl sulfoxide and makes；
4) preparation of stop buffer: concentrated sulphuric acid is formulated as the dilute sulfuric acid of 2mol/L；
5) preparation of cleaning mixture:
A. storing solution A liquid and storing solution B liquid
The storing solution A liquid of 0.2mol/L: by the NaH of 6.2404g2PO4·2H2O is dissolved in 200ml ddH2In O；
The storing solution B liquid of 0.2mol/L: by the Na of 71.628g2HPO4·12H2O is dissolved in 1000ml ddH2In O；
B.0.01mol/L PBST buffer
Storing solution B liquid 202.5ml, ddH by the storing solution A liquid 47.5ml, 0.2mol/L of 0.2mol/L2O 4747.5ml, NaCl
The NaOH 10ml and tween 20 2.5ml of 45g, 1N, mixing；
6) preparation of confining liquid: 5g skimmed milk is dissolved in 100ml cleaning mixture and obtains confining liquid；
(3) determination of standard concentration gradient and detecting step:
1) determination of standard concentration gradient: standard substance 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. move to be balanced at least under 20-25 DEG C of room temperature by the core reagent of preparation and the testing sample duck embryo allantoic liquid of collection
30min；
B. take one piece of ELISA Plate, every hole of ELISA Plate is added standard concentration gradient reagent successively, the most in order often
Hole adds duck embryo allantoic liquid 50 μ L to be checked, hereafter adds estradiol monoclonal antibody 50 μ L, seal plate with plate, shake gently
Mixing, reacts 30min in 37 DEG C；
C. taking out the ELISA Plate of step b, dry the liquid in ELISA Plate hole, the cleaning mixture diluted by the amount in 250 μ L/ holes is washed
Plate 4 times, soaks 30 seconds every time, pats dry in absorbent paper；
D. in the hole of ELISA Plate, add 100 μ L goat anti-mouse IgG-HRP, seal plate with plate, shake mixing gently, in 37 DEG C
Reaction 30min；
E. taking out ELISA Plate, dry liquid in hole, the cleaning mixture diluted by the amount in 250 μ L/ holes is washed plate 4 times, is soaked every time
30 seconds, absorbent paper pats dry；
F. in every hole of ELISA Plate, add developer 100 μ L, shake gently, in 37 DEG C of lucifuges colour developing 15min；
G. in every hole of ELISA Plate, add stop buffer 50 μ L, sentence when the color in the aperture of ELISA Plate is transferred to yellow by blueness
It is set to termination reaction；
H. the microplate reader OD value in each hole of 450nm wavelength measurement is used after adding stop buffer in 5min immediately；
I. the matching of standard curve formula: Y=1.6474X2-3.662X+0.6417 and Y=1.3414X2-3.4603X+
0.6809；
(4) result judges
Bring the OD value of testing sample into formula shown in i step and obtain the content of estradiol in sample；When in the sample measured
Estradiol content higher than 1ng/ml time, it is determined that for female；When the estradiol content in the sample measured is less than 0.6ng/ml, sentence
It is set to male.
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| CN105191831B (en) * | 2015-09-25 | 2017-12-12 | 中国农业大学 | A kind of embryo in egg sex appraisal method |
| DE102015226490A1 (en) * | 2015-12-22 | 2017-06-22 | Universität Leipzig | Procedure for in ovo gender determination of chicks |
| CN111387134B (en) * | 2020-03-06 | 2022-07-26 | 武汉市农业科学院 | A method for improving the reproductive performance of caged small and medium-sized meat ducks |
| CN117629987A (en) * | 2023-12-06 | 2024-03-01 | 华中农业大学 | Sex identification method for chick embryo eggs |
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 |
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