A nanobody against foot-and-mouth disease of sheep and its application in semen preservation and detection

Abstract

The present invention provides a nano-antibody for foot-and-mouth disease in sheep, which can widely identify the foot-and-mouth disease virus that has been prevalent in Asia in recent years, has no cross-reaction with other viruses, and has high specificity. It can be used to develop a kit for detecting the presence or level of foot-and-mouth disease virus in samples, especially in the semen of rams, enabling rapid sample detection and promising application prospects. Furthermore, the present application provides a method for detecting foot-and-mouth disease virus specifically in sheep semen. This method can be used to detect not only raw semen but also frozen semen, thus completing an upstream and downstream process chain and filling a gap in semen testing in my country's livestock industry.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a sheep foot-and-mouth disease nano-antibody and its application in semen preservation and detection. Background Art

[0002] Foot-and-mouth disease in sheep and goats develops from the foot-and-mouth disease virus, which belongs to the genus Orthomavirus in the family Picornaviridae. The virus is polymorphic and variable, divided into seven distinct serotypes (O, A, C, Asian I, South African I, II, and III) and 65 subtypes based on antigenic differences. There is no cross-immunity between subtypes. The foot-and-mouth disease virus is highly adaptable to the environment, resistant to low temperatures and susceptible to desiccation. It is insensitive to phenols, alcohol, and chloroform, but highly sensitive to sunlight, high temperatures, and acid and alkali conditions.

[0003] Foot-and-mouth disease (FMD) in sheep is characterized by an outbreak in late autumn, intensification in winter, abating in spring, and essentially subsiding in summer. The disease lasts two to three weeks, with an 80% incidence rate in adult sheep and a low mortality rate. The incidence in lambs can reach 90%, with a mortality rate of approximately 40%. Lambs often die from myocarditis caused by the aggressive form of FMD. Affected sheep experience fever and loss of appetite. Blisters and erosions develop on the inner lips, gums, tongue, and cheeks. The skin between the toes and around the hoof crowns becomes red, hot, swollen, and painful, followed by blisters and ulcers. These symptoms can lead to pain, lameness, and even kneeling or lying on the ground. In my country, FMD is the most common infectious disease in the livestock industry and carries significant consequences. The mortality rate can reach 20% or even 50%, necessitating culling. Consequently, FMD symptoms inflict significant economic losses on breeders, shortening the lifespan of cashmere goats and affecting the health of their offspring.

[0004] The virus can be transmitted through air, dust, secretions and excretions of infected animals, such as blisters, saliva, milk, feces, urine, and semen, as well as contaminated feed, bedding, and clothing of people who come into contact with infected sheep. Furthermore, dust containing the virus can be spread by wind up to 50 to 100 kilometers away, so foot-and-mouth disease often spreads over long distances. The infection rate is nearly 100%. Generally speaking, the mortality rate for adult sheep with foot-and-mouth disease is between 5% and 20%, and for lambs, it is between 50% and 80%. These high mortality rates make prevention and subsequent treatment crucial.

[0005] Artificial insemination is an essential technology in modern sheep production, and semen cryopreservation is a major innovation in artificial insemination technology. It solves the problem of long-term semen storage, freeing it from time, location, or the lifespan of the breeding stock. This allows for the full utilization of the genetic performance of high-quality breeds at their peak, maximizing and improving the utilization rate of high-quality breeding rams, significantly enhancing genetic progress, and accelerating the development and improvement of breeds. Furthermore, short-term progeny testing of high-quality breeding rams is crucial for preserving and restoring the superior characteristics of a particular breed or individual ram, and is of great significance and application value in pedigree renewal, introduction of new breeds, reducing production costs, and protecting breed resources. However, foot-and-mouth disease virus (FMD) can be transmitted through semen. In 2014, semen samples from 62 rams of five different breeds at 49 breeding farms in five regions of Shandong Province were tested, and the positive rate for FMD virus was 14.46%. Uncontaminated semen is a prerequisite for safe production. Furthermore, if semen or semen bottles are contaminated with FMDV during semen collection or transportation, it can be devastating to sheep production and reproduction. However, in practice, especially in rural farms, it is difficult to avoid FMDV contamination using detection methods such as RT-PCR / PCR and immunofluorescence. There is a lack of simple methods for FMDV detection, and there is a lack of standardized testing methods specifically for frozen sheep semen. Summary of the Invention

[0006] In order to solve the above technical problems, the purpose of the present invention is:

[0007] Provided is a sheep foot-and-mouth disease nanoantibody FMDV-Nano, the amino acid sequence of which is shown in SEQ ID NO.2. The nanoantibody can rapidly detect foot-and-mouth disease virus in sheep semen samples, thereby achieving rapid detection and identification of frozen sheep semen.

[0008] Furthermore, the rapid test is to use the above nanoantibodies to prepare colloidal gold test strips according to conventional means to detect foot-and-mouth disease virus in sheep semen;

[0009] Further,

[0010] The rapid detection test strip for foot-and-mouth disease virus provided by the present invention comprises a base plate, a water-absorbing pad, an NC membrane, a gold pad, and a sample chromatography pad, wherein the water-absorbing pad, the NC membrane, the gold pad, and the sample chromatography pad are sequentially attached from top to bottom on the base plate, wherein the nano antibody FMDV-Nano labeled with colloidal gold is provided on the gold pad;

[0011] The NC membrane is provided with a detection area and a quality control area separated from each other. The detection area is sprayed with FMDV coated antigen, and the quality control area is sprayed with antibodies that specifically bind to the colloidal gold-labeled nano antibody FMDV-Nano.

[0012] Furthermore, the present invention provides a method for preparing a rapid detection test strip for foot-and-mouth disease virus.

[0013] 1) Purify the nanoantibody FMDV-Nano and dialyze for later use;

[0014] 2) Colloidal gold-labeled nanoantibody FMDV-Nano;

[0015] 3) BSA blocking reaction;

[0016] 4) After centrifugation, wash the precipitate with PBS containing 1% BSA;

[0017] 5) Spray the gold label pad after re-dissolving;

[0018] 6) Coating with NC membrane;

[0019] 7) Dry at room temperature and low humidity; paste absorbent paper, NC membrane, gold pad, and sample chromatography pad from top to bottom on the bottom plate, and cut them for use;

[0020] 8) Application testing of colloidal gold test strips revealed a detection limit of <10 ng / mL, which meets the requirements for rapid FMDV detection.

[0021] Furthermore, the present invention provides a ram semen detection diluent with readily available raw materials and convenient preparation.

[0022] The ram semen detection diluent comprises: 25-35 g of Tris, 13-17 g of anhydrous citric acid, 9-13 g of anhydrous glucose, 0.05-0.2 g of cysteine, 0.01-0.05 g of glutathione, 0.6 g of penicillin, and 1 g of streptomycin, dissolved in 1000 ml of pure water; wherein, by mass, the ratio of Tris to anhydrous citric acid is 1.9-2:1.

[0023] Furthermore, the present invention provides a rapid detection method for frozen sheep semen, the method comprising:

[0024] 1) Keep the room temperature above 25°C. Place 0.25ml of frozen sheep semen in a 37°C water bath and gently shake for 40 seconds. Remove the semen and quickly dry the tube. Cut the semen tube and allow it to flow into a preheated centrifuge tube. Mix gently and observe after 10-15 minutes.

[0025] 2) Use ram semen test diluent and incubated thawed semen to perform isothermal dilution, diluent: frozen semen = 9:1

[0026] 3) Conduct testing using the aforementioned rapid test strips for foot-and-mouth disease virus and establish a testing catalog.

[0027] Furthermore, the present invention provides a method for rapid detection of sheep semen, the method comprising:

[0028] 1) Collect ram semen using conventional methods;

[0029] 2) Preheat the ram semen test diluent to 30-35°C, depending on the semen temperature, and perform isothermal dilution at a ratio of diluent to original semen of approximately 99:1.

[0030] 3) Conduct testing using the aforementioned rapid test strips for foot-and-mouth disease virus and establish a testing catalog.

[0031] Furthermore, the present invention provides an application of the above-mentioned rapid test strip for foot-and-mouth disease virus, which is for non-disease diagnosis purposes. In addition to the above-mentioned semen quality control, it also includes but is not limited to: food testing, water sample testing, import and export product safety monitoring, environmental sample testing and other aspects.

[0032] Beneficial effects

[0033] The sheep foot-and-mouth disease nanoantibodies provided by the present invention can widely identify the sheep foot-and-mouth disease virus that has been prevalent in Asia in recent years, have no cross-reactions with other viruses, and have high specificity. They can be used to develop test kits for the presence or level of foot-and-mouth disease virus in samples, especially in the semen of rams, to achieve rapid detection of samples and have good application prospects. At the same time, this application provides a method for detecting foot-and-mouth disease virus specifically for sheep semen. It can be used not only for raw semen, but also for frozen semen, realizing an upstream and downstream process chain, filling the gap in semen testing in my country's animal husbandry industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the predicted 3-D structure of the antigen epitope fusion polypeptide F12;

[0035] Figure 2A 2B is a schematic diagram of the enzyme cleavage of the fusion antigen, and 2B is the SDS-PAGE verification of the antigen epitope fusion polypeptide after purification by nickel column chromatography, wherein lane M is the protein marker; lanes a / b / c / d are four repeats.

[0036] Figure 3 The cross-sectional structure of the test strip, where 1 is the PVC base, 2 is the sample chromatography pad, 3 is the gold pad, 4 is the NC membrane, and 5 is the absorbent pad paper;

[0037] Figure 4 shows a normal picture (A) and an abnormal picture (B) of ram sperm. DETAILED DESCRIPTION

[0038] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, laboratory procedures in cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0039] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0040] Example 1

[0041] Firstly, homology analysis was performed on the VP1 major antigenic epitopes of the more popular FMDV O types (O / ZK / 93, O / Mya / 98, O / HK / 93, O / BY / CHA / 2010) and AsiaI types (Asia1 / JL / 05, IND 63 / 72, YNBS / 58), and the VP2 antigenic epitopes of O types of foot-and-mouth disease virus (O / ZK / 93, O / Mya / 98, O / HK / 93, O / BY / CHA / 2010). Bioanalysis was used to screen candidate antigenic fragments, and some amino acid residues were replaced or optimized based on bioinformatics analysis. Finally, the amino acid sequence of the epitope fusion peptide F12 was determined to be KGYDQELATSPE FGSGESVKRLDPSDTRDEHYPSDVRQSVRSKLAPTTYGEESTRRESSRRGDLAALARRVNNRLDTTQDRRKQKIIAPITRL (SEQ ID NO. 1).

[0042] An epitope fusion polypeptide expression cassette was constructed using overlap PCR. This cassette was then ligated and cloned into PET-28a (purchased from Invitrogen, Catalog No. A11499) using double enzyme digestion. Single clones were selected to verify insertion orientation. Plasmids with the correct insertion orientation were sent to Invitrogen for sequencing. The plasmids with the correct insertion orientation were designated PET-28a-F12. The recombinant positive plasmid PET-28a-F12 was expressed, verified by SDS-PAGE, and purified by nickel column chromatography to obtain a soluble fusion protein (see Figure 2).

[0043] Example 2 Immuno-panning process of natural single-domain antibodies against antigen epitope fusion polypeptides

[0044] (1) Amplify the established natural single-domain antibody phage library: add 100 μL glycerol bacterial library to 2×YT medium, add 20 MOI helper phage when OD600=0.5, let it stand for 30 minutes, centrifuge and resuspend the precipitate with 2×YT medium, culture for another 1 hour, add antibiotics and culture for 16 hours before centrifugation. The supernatant is precipitated with pre-cooled PEG-NaCl (1 / 4 volume) and resuspended in 1 mL PBS to obtain the amplified single-domain antibody library;

[0045] (2) Immunotube panning: The purified antigen epitope fusion peptide F12 with biotin label was coated on the immunotube at 50 μg / tube overnight. After removing the coating solution, the tube was washed 3 times, blocked with 2 mL BSA (1%) for 2 h, washed 3 times with PBST, and 100 μL of the single domain antibody library amplified in the above step (1) was added as the primary antibody. The tube was incubated at 37°C for 2 h, washed 3 times with PBST, and eluted with Glycine-HCl (pH 2.2). The eluate was adjusted to pH 7.4 with Tris-HCl to obtain the first round of panning natural single domain antibody library.

[0046] (3) The first round natural single domain antibody library obtained in step (2) is amplified according to step (1) to obtain the first round natural single domain antibody resuspended library, and then the immunotube panning step (2) is repeated, except that the primary antibody is added to 100µL of the amplified first round natural single domain antibody resuspended library, and finally the second round natural single domain antibody library is obtained; (4) The second round natural single domain antibody library obtained in step (3) is amplified according to step (1) to obtain the second round natural single domain antibody resuspended library, and then the immunotube panning step (2) is repeated, except that the primary antibody is added to 100µL of the amplified second round natural single domain antibody resuspended library, and finally the third round natural single domain antibody library is obtained.

[0047] Example 3 ELISA identification of single clones

[0048] (1) Amplification of the selected single positive clones by shaking for antibody expression: The natural single domain antibody library from the third round of panning was inoculated into 2×YT medium, and the OD 600nm =0.5, add 20 MOI helper phage, let it stand for 30 minutes, resuspend the precipitate with 2×YT medium after centrifugation, and culture it for another 1 hour, then spread it on a 2×YT plate containing antibiotics and culture it overnight. The next day, pick 40 single colonies and inoculate them into 2×YT medium. When OD600=0.5, add 20 MOI helper phage, let it stand for 30 minutes, resuspend the precipitate with 2×YT medium after centrifugation, and culture it again, and add IPTG to induce expression for 8 hours.

[0049] (2) ELISA identification: 100µL / well of the purified antigen epitope fusion peptide F12 with a biotin tag was coated on the ELISA plate overnight. After removing the coating solution, the plate was washed three times. The plate was blocked with 200µL / well BSA (3%) for 2h and washed three times with PBST. 100µL / well of the single-domain antibody library amplified in step (1) was added as the primary antibody (the library construction vector contained M13). The plate was incubated at 37℃ for 2h and washed three times with PBST. M13-HRP was added as the secondary antibody. After termination, the OD450nm value was detected. The result was interpreted as positive if the OD450nm value was three times higher than that of the control group.

[0050] Example 4 Sequencing and Identification of Positive Clones

[0051] Plasmids were extracted from the corresponding bacterial cultures of the clones that tested positive by ELISA in Example 3 and sequenced using universal primers for the plasmid vector. The gene sequences of the individual clones were analyzed using the sequence alignment software MEGA 6.0. Strains with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while those with different sequences were considered different clones. This yielded a specific sheep foot-and-mouth disease nanoantibody, FMDV-Nano, with the sequence HVQLVESGGGSVQAGRPASLGASFNYNNWSKGRFTISQDKAKDTALYYCAAGGNTRGKEAPGVSFTNGLNVERANKLKPEDTAISQDKAKNTLYYCAYYCAAGGWGQGTQVTVSSS (SEQ ID NO. 2).

[0052] Example 5 Detection of Antibody Type Specificity

[0053] Using an indirect ELISA, the coating agents were 100 ng / well of the following: VP1 protein of O / ZK / 93, VP1 protein of Asia1 / JL / 05 strain, VP2 protein of O / ZK / 93, hemagglutinin protein H protein of Peste des Petits Ruminants virus (PPRV), ORFV059 protein of Oral Fever Virus (ORFV), P32 protein of Goat Pox Virus (GTPV), ompA protein of Chlamydophila abortus (Ca), and LPS protein of Brucella ovis. The nanoantibody was tested (Table 1). The results showed that the antibody reacted positively to the aforementioned FMD viruses, but was negative for other viruses and bacteria commonly found in sheep, confirming that the nanoantibody is a FMD-specific antibody.

[0054] Table 1 Antibody type specificity test results

[0055] Detection object result Detection object result Detection object result O / ZK / 93 +++ peste des petits ruminants virus - Chlamydia abortus - Asia1 / JL / 05 strain +++ Sheep scab virus - Brucella capitis - O / ZK / 93 +++ Goatpox virus - -

[0056] Example 6 Neutralizing Activity and Stability Evaluation of Sheep Foot-and-Mouth Disease Specific Antibody FMDV-Nano

[0057] The micro-cell neutralization test was used in the laboratory for verification. The sheep foot-and-mouth disease specific antibody FMDV-Nano was diluted with physiological saline at a ratio of 1:2 to 1:128. One immune serum was used as a positive control and was also diluted at a ratio of 1:2 to 1:128. Each dilution was repeated 5 times. 100 TCID 50 The virus solution was added to each well. A serial dilution well for the negative serum control was also set up, and an equal amount of dilution solution was added. All culture plates were placed in a 37°C cell culture incubator for 2 hours. After neutralization, a Vero cell suspension was added to each well and the cells were incubated at 37°C for 5 days. A normal cell control was also set up. After 5 days, each well was observed and counted for pathological changes, indicating that 50% of the cells were protected from 100 TCID. 50 The highest dilution of virus-infected serum was used as the antibody titer for that serum, and the entire test was repeated three times. The final titer of the sheep foot-and-mouth disease-specific antibody FMDV-Nano was 1:128, and the neutralization titer of the positive control serum was 1:64 (≥1:4 was considered positive). Neutralization activity assays demonstrated that the sheep foot-and-mouth disease-specific antibody FMDV-Nano had a high neutralization titer, with a coefficient of variation of <3% across three replicates, demonstrating the good stability of this single-domain antibody.

[0058] Example 7 Construction of a sheep frozen semen detection system

[0059] The rapid detection test strips for foot-and-mouth disease virus were prepared using conventional methods in the art ( Figure 3 ),

[0060] 1) Purify the nanoantibody FMDV-Nano and dialyze for later use;

[0061] 2) Colloidal gold-labeled nanoantibody FMDV-Nano;

[0062] 3) BSA blocking reaction;

[0063] 4) After centrifugation, wash the precipitate with PBS containing 1% BSA;

[0064] 5) Spray the gold label pad after re-dissolving;

[0065] 6) Coating with NC membrane;

[0066] 7) Dry at room temperature and low humidity; paste absorbent paper, NC membrane, gold pad, and sample chromatography pad from top to bottom on the bottom plate, and cut them for use;

[0067] 8) Application testing of colloidal gold test strips revealed a detection limit of <10 ng / mL, which meets the requirements for rapid FMDV detection.

[0068] Prepare the diluent for cryopreservation of sheep semen according to the following components:

[0069] Weigh 25g of Tris, 13g of anhydrous citric acid, 9g of anhydrous glucose, 0.05g of cysteine, 0.01g of glutathione, 0.6g of penicillin, and 1g of streptomycin, and dissolve them in 1000ml of pure water. After complete dissolution, centrifuge at 4000 rpm for 45 minutes and collect the supernatant. The mass ratio of Tris to anhydrous citric acid is 2:1. Meanwhile, adjust the ratio of the above ingredients within a certain range as a reference example.

[0070] They are:

[0071] Reference 1: Dissolve 32.3g of Tris, 17g of anhydrous citric acid, 13g of anhydrous glucose, 0.2g of cysteine, 0.05g of glutathione, 0.6g of penicillin, and 1g of streptomycin in 1000ml of pure water. Centrifuge at 4000 rpm for 60min after full dissolution, and collect the supernatant. The mass ratio of Tris to anhydrous citric acid is 1.9:1.

[0072] Reference 2: Dissolve 30g Tris, 15g anhydrous citric acid, 10g anhydrous glucose, 0.1g cysteine, 0.05g glutathione, 0.6g penicillin, and 1g streptomycin in 1000ml of pure water. Centrifuge at 4000 rpm for 60min after full dissolution, and collect the supernatant. The ratio of Tris to anhydrous citric acid is 2:1 by mass.

[0073] Reference 3: Dissolve 28.5g Tris, 15g anhydrous citric acid, 9g anhydrous glucose, 0.1g cysteine, 0.01g glutathione, 0.6g penicillin, and 1g streptomycin in 1000ml of pure water. Centrifuge at 4000 rpm for 60min after complete dissolution, and collect the supernatant. The mass ratio of Tris to anhydrous citric acid is 1.9:1.

[0074] Establishment of a freezing system for breeding sheep semen

[0075] Semen from adult Hu sheep rams was collected by pseudo-vaginal method, and semen motility was measured. If the semen met the requirements (motility greater than 80%), it was cryopreserved. The preservation method was as follows: semen from the same Hu sheep was added to the above-mentioned Hu sheep semen freezing solution to make the sperm density 2×10 8 After mixing evenly, add into the cryopreservation tube, place the cryopreservation tube at 4℃ for 2h, then place it 5cm away from the liquid nitrogen surface for 10min, and finally put it into liquid nitrogen for storage.

[0076] Keep the room temperature above 25°C. Place the frozen 0.25ml of goat semen in a 37°C water bath and gently shake for 40 seconds. Remove the semen and quickly wipe off the water on the tube. Cut the capillary tube and allow the semen to flow into the preheated centrifuge tube. Mix gently and observe after 10-15 minutes.

[0077] The thawed semen after incubation was diluted isothermally with ram semen test diluent, with the diluent:frozen semen ratio of 9:1.

[0078] Use the above-mentioned rapid test strips for foot-and-mouth disease virus to conduct testing and establish a testing catalog.

[0079] The corresponding indexes of the thawed sheep semen obtained by the above different schemes were tested (the frozen storage time was 10 days).

[0080] Viability test: Automated testing was performed using the Tianyuan Orui CASA system. Giemsa staining was used to determine deformity and acrosome integrity, while PI staining was used to determine plasma membrane integrity. The test results are shown in the following table:

[0081] vitality Deformity rate Plasma membrane integrity Acrosome integrity rate Reference 1 66.1±3.6 9.5±2.6 80.2±2.1 76.4±4.7 Reference 2 63.2±4.8 12.3±3.9 75.9±3.8 72.3±2.6 Reference 3 72.0±3.3 8.1±4.2 81.0±3.3 84.1±3.9 Example 7 68.9±2.5 7.3±3.2 85.8±4.3 83.2±3.4

[0082] The results showed that the effectiveness of the aforementioned ram semen test diluents was relatively stable within a certain range. Rapid testing for foot-and-mouth disease virus (FMDV) was also performed on the four samples, using positive serum as a control. The results showed that none of the samples contained the corresponding FMDV, while the positive control showed two bands. This demonstrates that the aforementioned detection method and kit can be used for large-scale sample screening and on-farm use, achieving excellent results while saving time, effort, and breeding costs.

[0083] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A sheep foot-and-mouth disease nano antibody FMDV-Nano, the amino acid sequence of the sheep foot-and-mouth disease nano antibody is: HVQLVESGGGSVQAGRPASLGASFNYNNWSKGRFTISQDKAKDTALYYCAAGGNTRGKEAPGVSFTNGLNVERANKLKPEDTAISQDKAKNTLYYCAYYCAAGGWGQGTQVTVSSS.

2. A nucleic acid fragment comprising a nucleotide sequence encoding the sheep foot-and-mouth disease nanobody FMDV-Nano according to claim 1.

3. A vector comprising the nucleic acid fragment of claim 2. A host cell comprising the nucleic acid fragment of claim 2 or the vector of claim 3.

5. Use of the sheep foot-and-mouth disease nanoantibody FMDV-Nano according to claim 1, the nucleic acid fragment according to claim 2, the vector according to claim 3, and the host cell according to claim 4 in preparing a detection reagent for detecting sheep foot-and-mouth disease.

6. A rapid test strip for foot-and-mouth disease virus, characterized in that The rapid test strip comprises a base plate, absorbent pad paper, NC membrane, gold pad, and sample chromatography pad, and the absorbent pad paper, NC membrane, gold pad, and sample chromatography pad are sequentially pasted on the base plate from top to bottom, wherein the gold pad is provided with the nano antibody FMDV-Nano as claimed in claim 1 labeled with colloidal gold.

7. Use of the rapid test strip according to claim 6 in preparing a kit for detecting sheep foot-and-mouth disease.

8. A method for rapid detection of frozen sheep semen for non-diagnostic purposes using the rapid test strip according to claim 6, the method comprising the following steps: 1) Keep the room temperature above 25°C. Place 0.25ml of frozen sheep semen in a 37°C water bath and gently shake for 40 seconds. Remove the semen and quickly dry the tube. Cut the semen tube and allow it to flow into a preheated centrifuge tube. Mix gently and observe after 10-15 minutes. 2) Isothermally dilute the thawed semen after incubation with ram semen test diluent at a ratio of 9:1; 3) Conduct testing using rapid test strips and establish a testing catalog; The specific components of the ram semen test diluent are: Tris 25-35g, anhydrous citric acid 13-17g, anhydrous glucose 9-13g, cysteine 0.05-0.2g, glutathione 0.01-0.05g, penicillin 0.6g, and streptomycin 1g, dissolved in 1000ml of pure water, where the mass ratio of Tris to anhydrous citric acid is 1.9-2:1.

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