CN116577515A - Method for early detection of cow fetal gender - Google Patents
Method for early detection of cow fetal gender Download PDFInfo
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- CN116577515A CN116577515A CN202310832488.3A CN202310832488A CN116577515A CN 116577515 A CN116577515 A CN 116577515A CN 202310832488 A CN202310832488 A CN 202310832488A CN 116577515 A CN116577515 A CN 116577515A
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
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- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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Abstract
The invention relates to a method for early detecting the sex of a cow fetus, which comprises the following steps: collecting cow blood and separating serum 28-35 days after cow breeding; inserting the cattle Mueller tube inhibition factor AMH test paper into serum for 5-10 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper develop or not after 15-18 minutes; the color development results were as follows: the test paper quality control line C does not develop color, the test line T develops color, and the test result is invalid; the quality control line C develops color, the detection line T does not develop color, and the cow fetus is a female calf; the quality control line C and the detection line T are both developed, which indicates that the cow fetus is a male calf. The sex of the cow and the fetus can be safely, conveniently, simply, quickly and accurately detected, the fetus is not damaged, no pollution is caused, the problem that the supply amount of the male cows and the market demand of the farm are disjointed is solved, and reliable technical support is provided for the market demand of the breeding cows and fattening bulls.
Description
Technical Field
The invention relates to a method for early detecting the sex of a cow fetus, belonging to the technical field of animal breeding.
Background
In the production process of male and female bison cows, two different industrial forms and market demands of breeding cows and fattening bulls are formed due to obvious differences in growth speed, application and the like of male and female cows. Under the general condition, after natural breeding of pregnant cows, the proportion of male calves to female calves is half, and the conception rate is lower than 50% in the large-scale production process due to the low conception rate of the conventional cow sex control frozen semen technology and frozen embryo transfer technology. The ultrasonic technology can only judge whether the cow is pregnant or not, but cannot identify the sex of the fetus in gestation period. Because the growth rate of the male calves is obviously superior to that of the female calves, the raising cost and income of the male calves are different, and the male calves need to be subjected to classified raising according to requirements, so that the raising cost is reduced as much as possible, and the market requirements are met. For example: when the cows need to be bred and expanded, only the fetuses of the cows are hoped to be all female calves, and even if the breeding cost is slightly high, the final cow income is not influenced; and the cows with male calves can lead the male calves of the cows to enter the production of fattening bull under the condition of the same feeding cost, so that the feeding economic benefit is ensured to the maximum extent. Thus, early fetal sex determination of cattle is of great significance in animal husbandry production.
Currently, the main methods for sex identification of cattle are: 1) The cytogenetic method has 100% accuracy, but the difficulty of obtaining high-quality metaphase chromosome disperse phase is great, the disperse phase obtaining rate is low, and the identification process is long. 2) Biochemistry not only reduces fetal viability due to potential cytotoxicity, but also makes it easy to misjudge a female fetus as male once the X-chromosome of some female fetuses has been inactivated in advance. The cytotoxicity analysis method has high accuracy and is easy to cause death of male fetuses. 3) Although the indirect immunofluorescence method does not damage the fetus and has a sex identification accuracy, it has a disadvantage that the assessment of fluorescence intensity has a certain subjectivity, which makes the sex identification result of the fetus unstable. 4) The blastula formation inhibition method is practical and simple, but is easy to misjudge a part of female fetuses with retarded development as male, and the fetuses just in the mulberry stage are difficult to obtain at the same time without in vitro insemination. 5) The male specific DNA probe method has high accuracy in identifying the sex of the fetus, but the method is limited in popularization and application because the fetus is easy to be damaged due to the large amount of fetal cells (15 or more) used for detection and the identification time is long, and each livestock needs to use the same male specific DNA probe. 6) The PCR amplified DNA fragment method has high discrimination rate, simple operation and high applicability, but the method amplifies large repeated fragments, and the discrimination accuracy rate still has instability in a certain proportion due to the high crossing rate between mammals. 7) The PCR amplification SRY sequence method has high sensitivity and correctness for identifying the sex of the fetus, simple and quick operation, but serious pollution, and needs strict operation program to control the pollution of DNA.
In addition, early fetal sex determination of cattle is limited to the blastula period of 7-8 days of breeding, and has different defects and defects, and no detection means for the fetal sex at 28-35 days after breeding exists, so that improvement of the prior art is needed.
Disclosure of Invention
The invention aims to provide a method for early detecting the sex of a cow fetus, which further improves the accuracy of early sex identification of the cow, ensures that the operation is simpler, more convenient and quicker, does not hurt the existing fetus, avoids pollution, ensures that the early sex identification technology enters a practical and productive stage, and provides reliable technical support for high-quality and high-volume production of animal husbandry.
The invention is completed by the following technical scheme: a method for early detection of cow fetal gender comprising the steps of:
1) Collecting 1-10 ml cow blood 28-35 days after cow breeding, standing in shade for 1-2 hr, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 5-10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15-18 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, so that the detection result is invalid and the detection needs to be repeated;
the test paper quality control line C develops color, and the detection line T does not develop color, so that the cow fetus is a cow calf;
the test paper quality control line C and the test line T are both developed, which shows that the cow fetus is a male calf, and the following comparison is carried out by using the developed color and an AMH colorimetric card:
31 When AMH is more than or equal to 0-2mIU/ml, indicating that the cow fetus is a male calf;
32 When AMH is more than or equal to 3-5mIU/ml, the cow fetuses are two male calves.
The step 2) of the test paper for detecting the AMH of the cattle Mueller pipe inhibition factor is prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of bovine serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg bovine Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: dissolving 100mg of bovine anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain an IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the cattle Mueller tube inhibition factor AMH test paper.
The invention has the following advantages and effects: by adopting the scheme, the sex of the cow fetus can be safely, conveniently, simply, quickly and accurately detected, the fetus is not damaged, the pollution is avoided, low-cost and high-quality guarantee is provided for subsequent feeding, and the development of animal husbandry is promoted. The detection result of the invention can meet the market demands of male calves and female calves, and can detect the number of male calves borne by the cows, so that pregnant cows are priced and sold or fed differently according to the gender of the fetus, thereby reducing the breeding cost and increasing the breeding income. Not only realizes the quick turnover and virtuous circle of invested funds, but also solves the problem of the dislocation of the actual supply quantity and market demand quantity of male cows in the existing production process, fundamentally meets the market demand of pregnant cows and quick fattening bulls, and provides reliable technical support for improving the supply demand of breeding cows and fattening bulls in the beef cattle industry in China.
Drawings
FIG. 1 is a schematic diagram of a structure of a test paper for detecting the AMH of the Muller pipe inhibition factor of cattle;
FIG. 2 is an AMH colorimetric card.
Detailed Description
The invention is further described below with reference to examples.
Example 1 bovine Muller tube inhibition factor AMH test paper was prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of bovine serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg bovine Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: dissolving 100mg of bovine anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain an IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the cattle Mueller tube inhibition factor AMH test paper.
Example 2a method for early detection of cow fetal gender comprising the steps of:
1) Collecting 2ml of cow blood 28 days after cow breeding, standing in shade for 12 hours, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 52 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, which indicates that the detection result is invalid and the detection needs to be repeated 2.
Example 3a method for early detection of cow fetal gender comprising the steps of:
1) Collecting 10 ml cow blood 30 days after cow breeding, standing in shade for 2 hr, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15 minutes:
3) The color development results were as follows:
and (3) developing color by a test paper quality control line C, wherein the detection line T does not develop color, and indicating that the cow fetus is a female calf.
Example 4a method for early detection of cow fetal gender comprising the steps of:
1) Collecting 5ml cow blood 33 days after cow breeding, standing in shade for 1.5 hr, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 8 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper develop or not after 16 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are both developed, and after the developed color is compared with an AMH colorimetric card:
AMH is 1mIU/ml, indicating that the cow fetus is a male calf.
Example 5a method for early detection of cow fetal gender comprising the steps of:
1) Collecting 8ml of cow blood 35 days after cow breeding, standing in shade for 2 hr, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 8 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper develop or not after 17 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are developed, and after the developed color is compared with an AMH colorimetric card:
AMH is 4mIU/ml, indicating that the cow fetuses are two male calves.
The effect of the invention is demonstrated by comparative experiments:
the method provided by the embodiment of the invention is adopted to detect 40 cows respectively, and the detection results are as follows:
the fetuses of 17 cows are female calves; the fetus with 19 cows is a male calf, wherein: the fetuses of 6 cows are 2 male calves, and the fetuses of 13 cows are 1 male calves; the sex of the fetuses is not detected in 4, and the ultrasonic detection result shows that the fetuses of the cows stop developing, and the fetuses apoptosis.
Claims (2)
1. A method for early detection of cow fetal gender comprising the steps of:
1) Collecting 1-10 ml cow blood 28-35 days after cow breeding, standing in shade for 1-2 hr, and separating serum;
2) Inserting the cattle Muller tube inhibition factor AMH test paper into the serum in the step 1) for 5-10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15-18 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, so that the detection result is invalid and the detection needs to be repeated;
the test paper quality control line C develops color, and the detection line T does not develop color, so that the cow fetus is a cow calf;
the test paper quality control line C and the test line T are both developed, which shows that the cow fetus is a male calf, and the following comparison is carried out by using the developed color and an AMH colorimetric card:
31 When AMH is more than or equal to 0-2mIU/ml, indicating that the cow fetus is a male calf;
32 When AMH is more than or equal to 3-5mIU/ml, the cow fetuses are two male calves.
2. The method for early detecting the sex of a cow fetus according to claim 1, wherein the step 2) the test paper for detecting the AMH of the cow's mullerian duct inhibition factor is prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of bovine serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg bovine Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: dissolving 100mg of bovine anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain an IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the cattle Mueller tube inhibition factor AMH test paper.
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