A colloidal gold test strip for detecting group A rotavirus antibodies, its preparation method and application

Abstract

This invention discloses a method for preparing and applying a colloidal gold test strip for detecting group A rotavirus antibodies. The gold-labeled protein used in this test strip is a prokaryotically expressed and soluble recombinant RVA-VP6-3P protein. This protein has abundant group-specific antigenic epitopes, ensuring the specificity and sensitivity of the gold-labeled protein, reducing the economic cost of obtaining and purifying the target protein, and laying the foundation for the establishment and widespread application of antibody detection methods. The test strip is assembled by labeling colloidal gold with RVA-VP6-3P, coating the detection line with SPA, and coating the control line with the monoclonal antibody 1M1F9, which specifically recognizes RVA-VP6-3P. The test strip shows no cross-reactivity with positive serum antibodies against other common swine viruses and features convenient operation, low time consumption, high sensitivity, and strong specificity. Detection of group A rotavirus antibodies can be completed in 5-15 minutes, facilitating immediate clinical use.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a method for preparing and applying a colloidal gold test strip for detecting group A rotavirus antibodies. Background Technology

[0002] Rotavirus, a double-stranded RNA virus belonging to the genus Rotavirus in the family Reoviridae, can cause acute diarrhea in human newborns and young animals in various species, including cattle, sheep, and pigs. It is an important pathogen causing gastrointestinal infectious diseases. Adult animals and adults are mostly asymptomatic carriers, while young animals and children infected may experience diarrhea, vomiting, and other clinical manifestations, which can lead to dehydration and death in severe cases. Rotavirus infection poses a significant threat to the health of humans and various animals, and has become a major public health issue globally. Currently, common methods for detecting rotavirus antibodies include enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT), while antigen detection methods include PCR, quantitative real-time PCR, and virus isolation. These methods largely rely on specialized operators and laboratory equipment, and are time-consuming, making them unsuitable for rapid screening in farms and other on-site settings. Therefore, there is an urgent need to establish a rapid, simple, and accurate method for detecting rotavirus.

[0003] Rotaviruses have different serogroups and serotypes. Based on the antigenic characteristics of VP6, they can be divided into seven serogroups (AG). Group A rotaviruses are the main pathogens causing gastrointestinal diseases in humans and animals. Rotaviruses that can infect pigs mainly include four serogroups (A, B, C, and E), with group A rotaviruses being the most common, accounting for over 90% of cases of rotavirus-induced diarrhea in piglets around weaning. The VP6 protein of group A rotaviruses is highly conserved among different strains and is an important target protein for establishing detection methods for group A rotaviruses. Currently, some laboratories have attempted to express RVA-VP6 protein through prokaryotic systems, but all have expressed it in inclusion body form, which cannot ensure the activity of the target protein. Furthermore, the use of denaturing agents limits the application scenarios of this protein. Although some laboratories have successfully expressed soluble RVA-VP6 using eukaryotic expression systems, this method has low yield and high expression and purification costs, limiting its large-scale application. Therefore, developing a rapid and economical RVA detection technology based on soluble and efficient antigen expression is of great significance.

[0004] Colloidal gold immunochromatography is a rapid on-site serological testing method. Its ease of operation, rapid reaction, and lack of specialized equipment make it ideal for on-site screening. The basic principle of this technology is to use colloidal gold-labeled antigens or antibodies as tracers. The antigens and specific antibodies react on a nitrocellulose membrane, accumulating at specific test lines to form a visible color reaction. Based on this technology platform, developing a test strip capable of efficiently detecting group A rotavirus antibodies holds promise for overcoming the shortcomings of existing testing systems.

[0005] In summary, serotype A rotavirus has shown a high prevalence in my country's aquaculture industry in recent years, causing considerable economic losses. Although some laboratories have established corresponding detection methods, these methods still have significant room for improvement and optimization. Therefore, this invention provides a method for preparing and applying a colloidal gold test strip for detecting serotype A rotavirus antibodies. Summary of the Invention

[0006] To address the current limitations and significant room for improvement in rapid detection technologies for group A rotavirus antibodies, this invention provides a method for preparing and applying a colloidal gold test strip for detecting group A rotavirus antibodies. This test strip offers a rapid, effective, and relatively economical method for detecting group A rotavirus antibodies.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention claims protection for a colloidal gold test strip for detecting group A rotavirus antibodies. The test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad, which are sequentially stacked and pasted on a PVC base plate. The gold-labeled pad is coated with colloidal gold-labeled RVA-VP6-3P recombinant protein. The nitrocellulose membrane is provided with a detection line (T) and a control line (C). The detection line is coated with Staphylococcus aureus protein A (SPA), and the control line is coated with a monoclonal antibody 1M1F9 capable of specifically recognizing the RVA-VP6-3P recombinant protein.

[0009] Furthermore, the RVA-VP6-3P recombinant protein is a soluble protein expressed via a prokaryotic expression system, and its amino acid sequence contains multiple tandem repeats of at least one conserved B-cell epitope of the group A rotavirus VP6 protein. Even further, the conserved B-cell epitope is tandemly repeated three times; the amino acid sequence of the RVA-VP6-3P recombinant protein is shown in SEQ ID NO:2.

[0010] Furthermore, the amino acid sequence of the heavy chain of the monoclonal antibody 1M1F9 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain is shown in SEQ ID NO:5. The test strip of this invention is used to detect antibodies induced by infection with different strains of group A rotavirus.

[0011] In the specific implementation scheme, the RVA-VP6-3P recombinant protein is composed of tandem epitopes specific to group A rotavirus. To increase the abundance of epitopes, three copies of the screened epitope sequences are tandemly combined, further enhancing the epitope abundance and reactivity of the target protein, laying the foundation for the specificity and sensitivity of the detection method. The control line of the test strip of this invention uses the monoclonal antibody 1M1F9, which can recognize the RVA-VP6-3P recombinant protein, while the detection line uses SPA protein. SPA protein can bind to IgG from various mammals, and the test strip of this invention can detect the presence of group A rotavirus antibodies in the serum of various animals.

[0012] Secondly, this invention claims protection for a method for preparing the above-mentioned colloidal gold test strip for detecting group A rotavirus antibodies, the method comprising the following steps:

[0013] (1) Preparation of gold labeling pads: The purified RVA-VP6-3P recombinant protein was mixed with colloidal gold solution for labeling. The labeled complex was then sprayed onto the pretreated gold labeling pads and dried for later use.

[0014] (2) Preparation of coating membrane: Staphylococcus aureus A protein solution and monoclonal antibody 1M1F9 solution that can specifically recognize the RVA-VP6-3P recombinant protein were sprayed onto nitrocellulose membrane to form detection line and control line, respectively, and then dried for later use;

[0015] (3) Assemble the test strip: Paste the pretreated sample pad, gold label pad, coated nitrocellulose membrane and absorbent pad in sequence onto the PVC base plate and cut it into shape.

[0016] Furthermore, prior to step (1), the process includes expressing and purifying the RVA-VP6-3P recombinant protein, wherein a prokaryotic expression vector containing a Strep-II tag is used and purification is performed using Strep-Tactin affinity chromatography.

[0017] Furthermore, the blocking solution used for the gold-labeled pad in pretreatment step (1) comprises Tris-HCl, PVP-10 and Tween 20; the blocking solution used for the sample pad in pretreatment step (3) comprises Tris-HCl, casein and Tween 20.

[0018] In a specific implementation scheme, the specific steps for expressing the RVA-VP6-3P recombinant protein in step (1) are as follows:

[0019] (11) Screening conserved B-cell epitopes of VP6 protein and optimizing them in tandem, and commissioning Shanghai Sangon Biotech to synthesize the tandem sequence.

[0020] (12) Construct a prokaryotic recombinant expression vector and express, purify and identify the recombinant protein.

[0021] In a specific implementation plan, the sample pad and gold label pad need to be pretreated by soaking them in a sealing solution for 10 minutes and drying them in an oven at 37°C for 30-150 minutes to obtain the pretreated sample pad and gold label pad.

[0022] Preferably, the gold-labeled pad sealing solution is composed of the following raw materials: 10mM Tris-HCl, 0.3% (v / v) Tween 20, and 1% (m / v, g / 100ml) PVP-10.

[0023] Preferably, the sample pad sealing solution is composed of the following ingredients: 10mM Tris-HCl, 0.3% (v / v) Tween 20, and 1% (m / v, g / 100ml) casein.

[0024] Thirdly, the present invention seeks protection for the use of the recombinant RVA-VP6-3P protein with the amino acid sequence shown in SEQ ID NO:2 in the preparation of antibody products for detecting group A rotavirus.

[0025] Fourthly, the present invention claims protection for the use of the colloidal gold test strip as described above in the detection of group A rotavirus antibodies in samples for non-diagnostic purposes.

[0026] Fifthly, the present invention claims protection for a reagent kit comprising the colloidal gold test strip as described above.

[0027] The test strip of the present invention can be used to detect or assist in the detection of group A rotavirus antibodies in serum, and to detect or assist in the detection of whether a sample or animal to be tested is infected with group A rotavirus.

[0028] In the specific technical solution, the judgment criteria for the colloidal gold test strip for detecting group A rotavirus antibodies are as follows: no band on the control line indicates the test strip is invalid; bands on both the control line and the test line indicate a positive result; and bands on the control line but no band on the test line indicate a negative result.

[0029] In a specific implementation plan, the product may be a reagent kit.

[0030] In specific implementation schemes, the animals mentioned include pigs, cattle, horses, cats, dogs, etc.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The gold-labeled protein used in the test strip of this invention is a prokaryotically expressed and soluble recombinant RVA-VP6-3P protein. This protein has abundant group-specific antigenic epitopes, ensuring the specificity and sensitivity of the detection method.

[0033] 2. The test strip of the present invention has good specificity and shows no cross-reactivity with positive sera of porcine circovirus type 2, classical swine fever virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine transmissible gastroenteritis virus, and foot-and-mouth disease virus.

[0034] 3. The test strip of the present invention still shows a clear T-line band even when the positive sample is diluted 1:12800 times, indicating very high sensitivity.

[0035] 4. The test strip of the present invention is simple and quick to operate. No other instruments or reagents are required during the detection process. Simply add the serum to be tested to the sample well, and the test result can be determined in about 5-15 minutes.

[0036] 5. The rapid test strips of this invention are easy to carry and store, can meet the needs of different farmers, and have broad market prospects and significant economic and social benefits.

[0037] 6. The establishment of the detection method in this invention provides reliable technical support for the monitoring of group A rotavirus antibodies. Attached Figure Description

[0038] Figure 1 SDS-PAGE image (A) and WB image (B) of the purified RVA-VP6-3P recombinant protein.

[0039] Figure 2 This is a schematic diagram of the assembly of colloidal gold test strips.

[0040] Figure 3 This is a diagram illustrating the specificity of colloidal gold test strips.

[0041] Figure 4 This is a graph showing the sensitivity test of colloidal gold test strips. Detailed Implementation

[0042] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0043] Example 1: Screening, optimization, and synthesis of tandem epitope sequences

[0044] 1. Experimental Methods

[0045] The VP6 protein sequence of group A rotavirus was analyzed using epitope screening and analysis software systems such as BepiPred-2.0 and ABCpred to predict and analyze candidate B-cell epitopes of this protein. Further validation and selection of B-cell epitope sequences were performed, and conserved epitope sequences in the VP6 rotavirus protein were chosen to enhance the broad-spectrum reactivity of the antigen protein. Finally, the selected conserved B-cell epitopes were tandemly optimized to three copies for use as the detection antigen. The above sequences were codon-optimized using a prokaryotic expression system and synthesized by Sangon Biotech (Shanghai) Co., Ltd., ligated into the pCold-TF-StrepII vector, and transformed into BL21(DE3) competent cells. The amino acid sequence information is shown in SEQ ID NO:2, and the nucleotide sequence of the encoded gene after codon optimization is shown in SEQ ID NO:1.

[0046] Example 2 Preparation of soluble RVA-VP6-3P recombinant protein

[0047] 1. Experimental Methods

[0048] The bacterial strain was activated overnight at 37°C, then inoculated at a 1:100 (v:v) ratio to further propagate until OD600 = 0.6. IPTG was then added to a final concentration of 0.1 mM, and the mixture was transferred to a 15°C shaker for 24 hours of further incubation. After incubation, the cells were centrifuged at 6000 rpm for 10 min at 4°C. The supernatant was collected and sonicated. The recombinant protein was purified using IBA Strep-Tactin® XT4flow®. Mouse group A rotavirus-positive serum was used as the primary antibody, and the recombinant protein was identified by Western blot using goat anti-mouse secondary antibody containing HRP.

[0049] The specific steps for purifying the recombinant protein using IBA Strep-Tactin®XT 4flow® in the above-described method for preparing soluble RVA-VP6-3P recombinant protein are as follows:

[0050] ① Add 5 volumes of PBS to the affinity chromatography column to replace its storage buffer.

[0051] ② After centrifuging the collected supernatant at 8000 rpm for 20 min at 4℃, add the supernatant to the affinity chromatography column and incubate overnight on a rotating shaker.

[0052] ③ After binding, the supernatant is released and washed with 10 times the volume of washing buffer in the affinity chromatography column. The washing buffer formula is: 100mM Tris, 150mM NaCl, 1mM EDTA.

[0053] ④ Add 2-4 mL of biotin-containing eluent for elution. The eluent formulation is: 100 mM Tris, 150 mM NaCl, 1 mM EDTA, and 50 mM biotin. Collect the eluent and prepare a protein sample for Western blot identification.

[0054] ⑤ The harvested purified protein sample was added to a 30 kDa Millipore ultrafiltration tube for ultrafiltration concentration. The concentration conditions were: centrifugation at 5200 rpm for 30 min. When the volume reached approximately 500 μL, 10 mL of PBS solution was added to replace the storage medium. The PBS was replaced twice in total, and the final volume was concentrated to 200-300 μL. After determining the protein concentration using the Bradford method, the purified protein was aliquoted and stored at -80℃ for later use.

[0055] The specific steps for Western blot analysis of the recombinant protein in the above-mentioned method for preparing soluble RVA-VP6-3P recombinant protein are as follows:

[0056] ① Prepare filter paper and NC membrane and immerse them in transfer solution.

[0057] ② Take out the runnated protein gel, cut off the top layer of stacking gel, and place it in the following order: three layers of filter paper, protein gel, NC membrane, three layers of filter paper. After removing air bubbles, place it in a semi-dry machine and run it at a constant current of 250 mA for 25 min.

[0058] ③ Remove the transferred membrane, add 10 mL of 5% skim milk powder in PBS and block at room temperature for 2 hours. Discard the blocking solution and wash with PBST 5 times, 5 minutes each time.

[0059] ④ Add RVA-positive mouse serum and incubate overnight at 4°C. After incubation, remove the membrane and wash it 5 times with PBST for 10 min each time.

[0060] ⑤ Add goat anti-mouse secondary antibody containing HRP (1:10000 dilution), incubate at room temperature (25±5℃) for 60 min, and then wash with PBST 5 times for 10 min each time.

[0061] ⑥ Scan the membrane: Take 500 μL each of the luminescent solution A and B, mix them well, and add them to the NC membrane in the dark. After they are fully combined, put the membrane into the membrane scanning instrument for scanning and take a picture for storage.

[0062] This invention enables the prokaryotic soluble expression of the recombinant RVA-VP6-3P protein, ensuring the specificity and reactivity of the target protein (see...). Figure 1 This method helps improve the specificity and sensitivity of detection, while also having low expression costs, making it easy to promote and apply.

[0063] Example 3: Preparation of monoclonal antibodies for quality control lines

[0064] (1) Animal immunization: The RAV-VP6 protein expressed by inclusion bodies was purified and set aside. 100 μg of VP6 protein was mixed with ISA15 adjuvant and subcutaneously injected into 7-8 week old BALB / c mice. A total of 4 immunizations were performed, with an interval of 2 weeks between each immunization. After the 4th immunization, the mice were injected with 50 μg of VP6 protein for a shock immunization. The mice were dissected 3 days later, and the spleen was taken under sterile conditions for use.

[0065] (2) Monoclonal antibody screening: The monoclonal antibodies used were screened using single B cell screening technology, including B cell enrichment screening, BGE high-throughput expression, and positive clone screening. The supernatants of different B cells prepared in the preliminary stage were harvested for later use. Highly reactive antibodies were screened using ELISA plates coated with RVA-VP6-3P protein. After reading the values ​​with an ELISA reader, the monoclonal antibody with the best reactivity was selected and named 1M1F9. The heavy chain gene sequence (as shown in SEQ ID NO:3) and light chain gene sequence (as shown in SEQ ID NO:5) of this B cell were obtained by gene sequencing. The amino acid sequence of the protein encoded by the heavy chain gene sequence is shown in SEQ ID NO:4, and the amino acid sequence of the protein encoded by the light chain gene sequence is shown in SEQ ID NO:6.

[0066] (3) Monoclonal antibody preparation: The heavy and light chain gene sequences obtained from sequencing were synthesized by Nanjing Detai Biotechnology Co., Ltd. and inserted into the EcoRI and BamHI restriction sites of the pcDNA3.4 vector to construct a plasmid for expressing the heavy and light chains of monoclonal antibodies. The plasmid was used to transiently transfect CHO cells to express monoclonal antibodies. The culture supernatant was then purified by Protein A agarose beads to obtain antibodies for later use. The final antibody concentration was 4 mg / mL, which was used for the quality control line of the colloidal gold test strip described in this invention.

[0067] Example 4

[0068] 1. Preparation of gold-labeled proteins

[0069] A colloidal gold solution with a particle size of 30 nm and a concentration of 1% (m / v, g / 100 ml) was prepared using the sodium citrate reduction method.

[0070] (1) Colloidal gold solutions with pH gradients were prepared using 0.2M K2CO3 at pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. 125 µL of each solution was added to the microwells. Sufficiently high concentrations of the target protein (40 µg / ml) were added to the colloidal gold solutions at different pH gradients. After mixing thoroughly, the solutions were reacted at room temperature (25±5℃) for 15 min. 125 µL of 10% NaCl solution (m / v, g / 100 ml) was added to each well and allowed to stand at room temperature for 5 min. The color change of the solution was observed. The group with no color change or the darkest color was considered to be the optimal pH for labeling stable colloidal gold. The optimal labeling pH was confirmed to be 6.0.

[0071] (2) Under optimal pH conditions, the optimal labeled protein concentration was determined. 125 μL of colloidal gold solution was added to each well, and the final protein concentration was adjusted to 5, 7, 9, 11, 13, and 15 μg / mL. After mixing, the mixture was reacted at room temperature (25±5℃) for 15 min. 125 µL of 10% NaCl solution (m / v, g / 100 ml) was added to each well and the mixture was allowed to stand at room temperature for 5 min. The color change of the solution was observed. The minimum protein concentration with no color change was considered to be the optimal protein concentration for stabilizing colloidal gold. The optimal labeling concentration was confirmed to be 11 ug / ml.

[0072] (3) Add recombinant RVA-VP6-3P recombinant protein with an optimal labeling concentration of 11 μg / mL and label it in a silanized glass beaker. Stir during the process to ensure thorough labeling. After labeling for 30 min, add BSA at a final concentration of 1% (m / v, g / 100ml) per milliliter of colloidal gold to block unbound colloidal gold.

[0073] (4) Centrifuge the labeled complex at 2000 rpm at 4℃ for 5 min to remove unlabeled colloidal gold particles, collect the supernatant, centrifuge at 4℃ and 12000 rpm for 30 min, and carefully remove the supernatant; wash twice with gold-labeled protein washing buffer (10mM Tris-HCl, 1% (m / v) BSA, 0.3% (v / v) Tween 20), and add resuspension buffer (10mM Tris-HCl, 1% (m / v) BSA, 0.3% (v / v) Tween 20, 30% (m / v) sucrose), and resuspend in 1 / 5 of the original liquid volume. After resuspension, store at 4℃ in the dark for later use.

[0074] 2. Preparation of gold-labeled pads

[0075] Cut the gold-labeled pad (Shanghai Jieyi Biotechnology Co., Ltd.) into strips 30 cm long and 0.6 cm wide. Immerse the gold-labeled pad in a gold-labeled pad blocking solution (10 mM Tris-HCl, 1% PVP-10 (m / v, g / 100 ml), 0.3% Tween 20) for 10 min, remove and dry at 37℃ for 2 h. Spray the gold-labeled protein onto the gold-labeled pad at a rate of 8 μL / cm using a spraying instrument (XYZ large platform 3D stylus spraying gold sprayer HM3260). Place the gold-labeled pad in a vacuum drying oven and dry at 37℃ for 1 h.

[0076] 3. Assembly of test strips

[0077] The colloidal gold test strip consists of a PVC base plate, a sample pad, a gold-labeled pad, an NC membrane, and an absorbent pad. SPA protein (Beijing Solarbio Science & Technology Co., Ltd.) was diluted to a final concentration of 0.4 mg / mL with 0.01 M Tris (pH=6.0), and the monoclonal antibody 1M1F9 prepared in Example 3 was diluted to 0.4 mg / mL. These were sprayed onto the nitrocellulose membrane at a rate of 1 μL / cm to form the detection line (T line) and control line (C line). The sample pad was cut into strips 30 cm long and 1.8 cm wide, soaked in sample pad blocking solution (10 mM Tris-HCl, 1% casein (m / v), 0.3% (v / v) Tween 20) for 10 min, and then dried at 37°C for 2 h. The absorbent pad was also cut into strips 30 cm long and 1.8 cm wide. The NC membrane, gold-labeled pad, sample pad, and absorbent pad were then arranged according to... Figure 2 As shown, the strips are attached sequentially to the PVC base plate and then cut into 0.3cm wide colloidal gold test strips, which are then inserted into the cartridge.

[0078] Example 5

[0079] The results judgment criteria and principles of the rapid test strips of this invention:

[0080] In this invention, after 100 μL of serum diluted 1:100 with sample diluent is added to the sample well of the rapid test strip, the serum to be tested, through a siphon effect, carries the antibodies and colloidal gold-labeled proteins in the serum to diffuse together towards the nitrocellulose membrane, and finally diffuses into the absorbent pad at the handle end. During the diffusion process, the antibodies in the serum to be tested can bind to the gold-labeled protein and bind to SPA on the T line of the nitrocellulose membrane, forming a brown-red mark. The gold-labeled protein binds to the RVA-VP6-3P recombinant protein monoclonal antibody on the C line, forming a brown-red mark. If there are no group A rotavirus antibodies in the serum, the test strip will only show one C line mark; if there are group A rotavirus-related antibodies in the serum, both the C line and the T line of the test strip will show brown-red marks; if there are no marks on the test strip, it indicates that the test strip is invalid or the operation is incorrect. Figure 2 ).

[0081] Example 6

[0082] Specificity testing of Group A rotavirus antibody test strips

[0083] The test strip was used to detect positive sera for porcine circovirus type 2, classical swine fever virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine transmissible gastroenteritis virus, and porcine foot-and-mouth disease virus to evaluate its specificity. Results are as follows: Figure 4 As shown, when the serum was diluted 1:100, the C line of the test strip showed a band, proving the test strip's effectiveness; the T line of group A rotavirus-positive serum showed a band, while the T lines of other sera showed no bands, indicating no non-specific cases and demonstrating good specificity of the test strip. The results are as follows. Figure 3 As shown.

[0084] Example 7

[0085] Sensitivity test of Group A rotavirus antibody test strips

[0086] The sensitivity of the test strip developed in this invention to detect positive serum for group A rotavirus was tested. The positive serum was diluted 50 times, then repeatedly diluted 2 times, and finally diluted to 25,600 times before detection. The results are as follows. Figure 4 As shown, when the serum was diluted to 1:12800, the colloidal gold test strip still showed a clear T-line band, but when diluted to 1:25600, the T-line band became very faint and almost invisible to the naked eye. Figure 4 Therefore, the detection limit of this test strip is 1:12800, indicating high sensitivity.

[0087] Example 8

[0088] Stability testing of Group A rotavirus antibody test strips

[0089] The assembled group A rotavirus colloidal gold antibody test strip was added to a silica desiccant and then vacuum-sealed in a storage bag, stored in a light-protected incubator at 37°C or at room temperature. The test strip was tested monthly with serially diluted positive serum to evaluate its detection performance. Results showed that the test strip could be stored at 37°C for one month and at room temperature for six months without any change in sensitivity or specificity, indicating that the test strip prepared in this invention has good stability.

[0090] Example 9

[0091] Serum sample detection using Group A rotavirus antibody test strips

[0092] Sixteen collected porcine serum samples (8 positive and 8 negative) were diluted 1:100 and added to the rotavirus A antibody test strip established in this invention. Results were evaluated within 15 minutes. A positive result was defined as having bands in both the T and C lines, while a negative result was defined as having a band only in the C line. All serum samples were tested in duplicate. Furthermore, the results were compared with those obtained using the ELISA method to determine the concordance rate (Table 1).

[0093] Table 1. Concordance rates between rotavirus group A colloidal gold test strips and ELISA assays.

[0094] Group A rotavirus antibody test strip ELISA Compliance rate Positive 8 8 100% Negative 8 8 100% Positive rate 50% 50%

[0095] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0096] sequence list

[0097] SEQ ID NO:1 (RVA-VP6-3P recombinant protein encoding gene):

[0098]

[0099] SEQ ID NO: 2 (RVA-VP6-3P recombinant protein):

[0100] MNHKVHHHHHHMQVSVETTQGLGRRVTITIAADSIETAVKSELVNVAKKVRIDGFRKGKVPMNIVAQRYGASVRQDVLGDLMSRNFIDAIIKEKINPAGAPTYVPGEYKLGEDFTYSVEFEVYPEVELQGLEAIEVEKPIVEVTDADVDGMLDTLRKQQATWKEKDGAVEAEDRVTIDFTGSVDGEEFEGGKASDFVLAMGQGRMIPGFEDGIKGHKAGEEFTIDVTFPEEYHAENLKGKAAKFAINLKKVEERELPELTAEFIKRFGVEDGSVEGLRAEVRKNMERELKSAIRNRVKSQAIEGLVKANDIDVPAALIDSEIDVLRRQAAQRFGGNEKQALELPRELFEEQAKRRVVVGLLLGEVIRTNELKADEERVKGLIEEMASAYEDPKEVIEFYSKNKELMDNMRNVALEEQAVEAVLAKAKVTEKETTFNELMNQQASAGLEVLFQGPSAGLVPRGSGGIEGRHMELGTLESLSKTLKDARDKIVECMDEMARESQRNGIADYIENWNLQNRPAVANLFPQFPPGMNWTELITTGGIGNLPIRNWNFDFGLLGTTLLNLDAPNIFPYSASFTLNDNLQRVFTVASIRSMLVKGPGPGKLSLSKTLKDARDKIVECMDEMARESQRNGIADYIENWNLQNRPAVANLFPQFPPGMNWTELITTGGIGNLPIRNWNFDFGLLGTTLLNLDAPNIFPYSASFTLNDNLQRVFTVASIRSMLVKGPGPGLESLSKTLKDARDKIVECMDEMARESQRNGIADYIENWNLQNRPAVANLFPQFPPGMNWTELITTGGIGNLPIRNWNFDFGLLGTTLLNLDAPNIFPYSASFTLNDNLQRVFTVASIRSMLVKEFHHHHHHGGGSWSHPQFEKGGGSWSHPQFEKSR

[0101] SEQ ID NO:3 (encoding the heavy chain nucleotide sequence of monoclonal antibody 1M1F9):

[0102]

[0103] SEQ ID NO:4 (Monoclonal antibody 1M1F9 heavy chain amino acid sequence):

[0104] MGWSCIILFLVATATGVHSEVQLLQSGPELVKPGASMKISCKASGYSFTGYNINWVKESHGKNLEWIGLINPYNGGSSYNQKFKGKATLTVNRSSSTAFMELRSLTSEDSAVYYC VRWGGQFFDSWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0105] SEQ ID NO:5 (encoding the light chain nucleotide sequence of monoclonal antibody 1M1F9):

[0106] ATGGGCTGGAGCTGCATCATCCTGTTCCTCGTGGCTACAGCTACCGGAGTGCACAGCGACATCCAGATGACTCAGTCTCCAGCCTTTCTATCTGCATCTGTGGGAGAAACTGTCATCATGACATGTCAAGCAAGTGAGAATGTTAAAAGTAATTTAGCATGGTATCAAAAGAGACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACAAACTTAGCAGATGGTGTGCCATCTAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGATTATCAACGACCTGCTGCCTGAAGATTTTGGGACTTATTATTGTCAACATTTATATGGTATTCCGTACACATTCGGAGGGGGGACCGAGCTGGAAATAGAAAGGGCCGACGCAGCTCCTACCGTGTCTATCTTCCCCCCTAGCAGCGAGCAGCTGACATCAGGAGGAGCTAGCGTGGTCTGCTTCCTGAACAACTTCTACCCCAAGGACATCAACGTCAAGTGGAAGATCGACGGCAGCGAGAGGCAGAACGGCGTGCTGAACTCTTGGACCGACCAGGATAGCAAGGACAGCACCTACAGCATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCGGCACAACAGCTACACCTGCGAGGCTACACACAAGACCAGCACCAGCCCCATCGTGAAGAGCTTCAACCGGAACGAGTGC

[0107] SEQ ID NO:6 (Amino acid sequence of the light chain of monoclonal antibody 1M1F9):

[0108] MGWSCIILFLVATATGVHSDIQMTQSPAFLSASVGETVIMTCQASENVKSNLAWYQKRQGKSPQLLVYAATNLADGVPSRFSGSGSGTQFSLIINDLLPEDFGTYYCQHLYGIPYTFGGGTELEIERADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

Claims

1. A colloidal gold test strip for detecting group A rotavirus antibody, the test strip comprising a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially laminated and pasted on a PVC base plate, characterized in that, The gold label pad is coated with the RVA-VP6-3P recombinant protein labeled with colloidal gold; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with Staphylococcus aureus A protein, and the quality control line is coated with monoclonal antibody 1M1F9 capable of specifically recognizing the RVA-VP6-3P recombinant protein; The amino acid sequence of the RVA-VP6-3P recombinant protein is shown as SEQ ID NO: 2; The amino acid sequence of the heavy chain of the monoclonal antibody 1M1F9 is shown as SEQ ID NO: 3, and the amino acid sequence of the light chain is shown as SEQ ID NO:

5.

2. The preparation method of the colloidal gold test strip for detecting group A rotavirus antibodies as described in claim 1, characterized in that, The method comprises the following steps: (1) Preparation of gold label pad: mix the purified RVA-VP6-3P recombinant protein with colloidal gold solution for labeling, spray the labeled compound on the pretreated gold label pad, and dry for standby; (2) Preparation of coated membrane: Staphylococcus aureus A protein solution and monoclonal antibody 1M1F9 solution capable of specifically recognizing the RVA-VP6-3P recombinant protein are sprayed on the nitrocellulose membrane respectively to form the detection line and the quality control line, and dried for standby; (3) Assemble the test strip: paste the pretreated sample pad, gold label pad, coated nitrocellulose membrane and water absorption pad on the PVC bottom plate in order, and cut into shape.

3. The method of claim 2, wherein, Before step (1), the step of expressing and purifying the RVA-VP6-3P recombinant protein is further included, wherein a prokaryotic expression vector containing Strep-II tag is used, and Strep-Tactin affinity chromatography is used for purification.

4. The method of claim 2, wherein, The blocking solution for pretreating the gold label pad in step (1) comprises Tris-HCl, PVP-10 and Tween 20; the blocking solution for pretreating the sample pad in step (3) comprises Tris-HCl, casein and Tween 20.

5. The method of claim 4, wherein, The blocking solution of the gold label pad comprises 10mM Tris-HCl, 0.3% Tween 20 and 1% PVP-10; the blocking solution of the sample pad comprises 10mM Tris-HCl, 0.3% Tween 20 and 1% casein.

6. The use of the RVA-VP6-3P recombinant protein with the amino acid sequence shown as SEQ ID NO: 2 in the preparation of a product for detecting group A rotavirus antibodies.

7. The use of the colloidal gold test strip according to claim 1 in detecting group A rotavirus antibodies in a sample for non-diagnostic purposes.

8. A kit characterized in that, The kit comprises the colloidal gold test strip according to claim 1. The kit comprises the colloidal gold test strip according to claim 1.

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