Fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit

Abstract

The invention discloses a fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit, and belongs to the technical field of animal epidemic disease detection. According to the kit, a fluorescence immunochromatography technology is utilized, a nitrocellulose membrane is coated with a specific antibody aiming at canine parvovirus and canine coronavirus, and a fluorescent marker is combined, so that the two viruses in a canine excrement sample are rapidly and accurately detected. The method is characterized in that an antibody combination and a reaction system are optimized, the detection sensitivity and specificity are remarkably improved, and cross reaction is effectively avoided. The kit is simple and convenient to operate, does not need professional equipment, can provide a detection result within 15-20 minutes, is suitable for on-site rapid screening in places such as pet hospitals, animal epidemic disease prevention and control institutions and farms, provides a powerful tool for early diagnosis and prevention and control of canine parvovirus and canine coronavirus, and has a good application prospect and popularization value.

Description

Technical Field

[0001] This application relates to the technical field of animal disease detection, and particularly relates to a fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit. Background Art

[0002] Canine parvovirus (CPV) belongs to the genus Parvovirus in the family Parvoviridae and is a single-stranded deoxyribonucleic acid virus. It is highly contagious to dogs and mainly causes diseases such as hemorrhagic enteritis and acute myocarditis in dogs. Canine coronavirus (CCV) belongs to the genus Coronavirus in the family Coronaviridae and mainly causes varying degrees of gastroenteritis in dogs, characterized by fatal watery diarrhea clinically. Sick dogs will suddenly fall ill, showing listlessness, loss of appetite, vomiting, and discharging foul-smelling, soft and mucus-containing feces. In severe cases, it will also lead to dehydration and weight loss.

[0003] Traditional clinical symptom observation is difficult to accurately distinguish between canine parvovirus and canine coronavirus infections because the symptoms of the two are somewhat similar and may be confused with other canine diseases, easily leading to misdiagnosis. Using the method of virus isolation and identification requires professional laboratory equipment and technical personnel, with complex operations and a long time-consuming process. It usually takes several days or even longer to obtain results, making it difficult to meet the needs of rapid diagnosis. Using serological tests such as neutralization tests and complement fixation tests, although they have high specificity, they also have problems such as cumbersome operations, long detection cycles, and high requirements for laboratory conditions, and are not convenient for widespread application in grass-roots units and pet hospitals. Using molecular biology detection such as polymerase chain reaction (PCR) technology, although it has high sensitivity and specificity, it requires professional molecular biology laboratory equipment and professional technical personnel for operation, with high detection costs, and strict requirements for sample collection, transportation, and storage conditions, restricting its rapid application in the clinical field.

[0004] Fluorescence immunochromatography is an immunological detection technology based on the specific binding of antigens and antibodies. Its basic principle is to use a fluorescent label to label an antibody or antigen. When the target antigen or antibody in the sample binds to the fluorescent-labeled specific antibody or antigen, under the action of chromatography, a detectable fluorescent signal is formed. This method usually consists of a sample pad, a conjugate pad, a reaction pad, an absorbent pad, etc. The sample reacts with the reagents on each pad during the chromatography process, and finally the result is judged by the presence or absence or intensity of the fluorescent signal.

[0005] Combined with the relationship between the intensity of the fluorescent signal and the concentration of the target substance, fluorescence immunochromatography can also achieve quantitative detection of virus antigens or antibodies in the sample, providing a more comprehensive reference basis for the diagnosis and treatment of diseases. The development and application prospects of fluorescence immunochromatography. Summary of the Invention

[0006] In view of this, the present invention provides a fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit, which utilizes fluorescent immunochromatography technology to coat specific antibodies against canine parvovirus and canine coronavirus on a nitrocellulose membrane, combined with fluorescent markers, to achieve rapid and accurate detection of the two viruses in canine fecal samples. Its innovation lies in the optimization of the antibody combination and reaction system, which significantly improves the detection sensitivity and specificity and effectively avoids cross-reactions. The kit is easy to operate, does not require professional equipment, and can issue test results within 15 to 20 minutes. It is suitable for on-site rapid screening in pet hospitals, animal disease prevention and control agencies, and farms, etc. It provides a powerful tool for the early diagnosis and prevention and control of canine parvovirus and canine coronavirus, and has good application prospects and promotion value.

[0007] One of the purposes of the present invention is to provide a canine parvovirus / canine coronavirus detection kit using fluorescent immunochromatography, comprising a VP2 antibody labeled with fluorescent microspheres, an M antibody labeled with fluorescent microspheres, a goat anti-mouse IgG antibody, a nitrocellulose membrane, a sample pad, a conjugate pad, an absorbent pad, and a PVC backing. The VP2 antibody labeled with fluorescent microspheres is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO: 1 and the VP2-VL shown in SEQ ID NO: 2 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys, and the M antibody labeled with fluorescent microspheres is an antibody obtained by coupling the M-VH shown in SEQ ID NO: 3 and the M-VL shown in SEQ ID NO: 4 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys.

[0008] One of the purposes of the present invention is to provide an assembly method for a fluorescent immunochromatography canine parvovirus test strip, comprising: using a film dispenser to spray 1 mg / mL of VP2-VH and VP2-VL buffer on the detection line (T1 line) of the NC membrane, 1 mg / mL of M-VH and M-VL buffer on the detection line (T2 line) of the NC membrane, and goat anti-mouse IgG on the quality control line (C line), with a spraying amount of 0.4 μL / cm. After spraying, the NC membrane is dried at 37°C for 2 hours. The distance between the T1 and T2 lines is 6 mm, and the distance between the T2 line and the C line is also 6 mm.

[0009] VP2 antibody labeled with fluorescent microspheres and M antibody labeled with fluorescent microspheres were mixed in equal amounts and sprayed on the conjugate pad using an ink dispenser. The spraying concentration was usually 1 mg / mL and the spraying volume was 0.4 μL / cm. The mixture was dried at 37°C for 2 hours. The sample pad and absorbent pad were respectively pasted on the PVC backing with nitrocellulose membrane and cut into 4 mm wide strips using a strip cutter to make test strips.

[0010] Among them, the fluorescent microsphere-labeled VP2 antibody is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO:1 and the VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys, and the fluorescent microsphere-labeled M antibody is an antibody obtained by coupling the M-VH shown in SEQ ID NO:3 and the M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys.

[0011] In a specific embodiment, the VP2-VH shown in SEQ ID NO:1 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:5 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VH into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the VP2-VH from the lysate.

[0012] In a specific embodiment, the VP2-VL shown in SEQ ID NO:2 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:6 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VL, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the VP2-VL from the lysate.

[0013] In a specific embodiment, the VP2-VH shown in SEQ ID NO:3 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:7 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the VP2-VH from the lysate.

[0014] In a specific embodiment, the M-VL shown in SEQ ID NO:4 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:8 into pET-28a(+) to obtain the expression plasmid pET-28a-M-VL, transforming pET-28a-M-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the M-VL from the lysate.

[0015] In a specific embodiment, the fluorescent microsphere-labeled VP2 antibody provided by the present invention is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain microspheres coupled with cyclopentapeptide; then, the VP2-VH activation solution is reacted with the microspheres coupled with the cyclopentapeptide, and then the VP2-VL activation solution is added to react.

[0016] In a specific embodiment, the fluorescent microsphere-labeled M antibody provided by the present invention is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain microspheres coupled with cyclopentapeptide; then, the activation solution of the M-VH is reacted with the microspheres coupled with the cyclopentapeptide, and then the activation solution of the M-VL is added to react.

[0017] One of the purposes of the present invention is to provide the use of antibodies against canine parvovirus and / or antibodies against canine coronavirus in the preparation of a canine parvovirus / canine coronavirus detection kit by fluorescent immunochromatography, wherein the antibodies against canine parvovirus include VP2-VH as shown in SEQ ID NO: 1 and VP2-VL as shown in SEQ ID NO: 2, and the antibodies against canine coronavirus include M-VH as shown in SEQ ID NO: 3 and M-VL as shown in SEQ ID NO: 4. Beneficial Effects

[0018] The antibodies against canine parvovirus and canine coronavirus provided by the present invention are designed for VP2 and M proteins, respectively, and unconventional VH peptides and VL peptides are obtained by recombinant expression. These variable regions are not only highly specific, but also can act as raw materials for rapid capture and identification of viruses to detect these two viruses.

[0019] The magnetic fluorescent microsphere-labeled antibody provided by the present invention can not only detect canine parvovirus / canine coronavirus simultaneously, but also eliminate interference and have higher sensitivity compared to the antibodies provided by the prior art and the prepared magnetic fluorescent microsphere-labeled antibody. In addition, the fluorescent signal of the magnetic fluorescent microsphere is stronger than that of the traditional colloidal gold or enzyme labeling, and the background noise is lower.

[0020] In addition, the present invention involves two detection lines (T1 and T2) through VP2-VH, VP2-VL, M-VH, M-VL, VP2 antibody labeled with fluorescent microspheres and M antibody labeled with fluorescent microspheres, which are used to detect canine parvovirus and canine coronavirus respectively, thus achieving simultaneous detection. Compared with traditional single virus detection test strips, this dual detection card can detect two viruses at the same time, reducing detection time and cost.

[0021] Furthermore, the test strip provided by the present invention adopts a highly specific antibody-antigen reaction, thereby reducing non-specific interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the SDS-PAGE picture of recombinantly expressed VP2-VH, VP2-VL, M-VH and M-VL.

[0023] Figure 2 Western Blot images of recombinantly expressed VP2-VH, VP2-VL, M-VH and M-VL.

[0024] Figure 3 Infrared images of alkynyl fluorescent microspheres (upper curve) and microspheres coupled with cyclopentapeptide (lower curve).

[0025] Figure 4 It is the SDS-PAGE picture of VP2 antibody and M antibody labeled with fluorescent microspheres.

[0026] Figure 5 The test strips provided for the experimental and control groups were used to detect the standard curves of VP2 and M proteins, respectively.

[0027] Figure 6 Actual photos of the test strips provided for the experimental group and the control group respectively containing recombinant porcine parvovirus VP2 protein, new coronavirus N protein, canine parvovirus VP2 protein and canine coronavirus M protein. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0029] Example 1: Screening of specific antibodies against canine parvovirus and canine coronavirus The present invention screens and prepares antibodies against canine parvovirus Capsid protein VP2 (GenBank: QDA34010.1) and antibodies against canine coronavirus Membrane protein M (GenBank: BAA02413.1).

[0030] By analyzing the VP2 antigen epitope and the M antigen epitope, the VH (heavy chain variable region) and VL (light chain variable region) that can specifically bind to the antigen are screened using a phage display library or a synthetic antibody library.

[0031] The VH and VL sequences obtained by screening for VP2 antigen are as follows: VP2-VH: QSELSSSGAEDASPGASDSMSCKASGDFDSRYTEMHWDKQRPGQGLEWIGFITPSRGYTDYDQRFDRDATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYEDYYSDEYWGQGTTLTVSS, SEQ ID NO:1 VP2-VL: ESDLTQSPEDLSLSPEEDATLSCDASQDVDEYDSWYQQKPGQAPRLLIYDESNDATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQESNDWPSLTFGAGTKLELK, SEQ ID NO:2 The VH and VL sequences obtained by screening for the M antigen are as follows: M-VH: SEQ ID NO:3 M-VL: DSDMTQSPEDLSLSPEEDATLSCDASQDVDEYDSWYQQKPGKAPKLLIYDESNDATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQESNDWPSLTFGAGTKLELK, SEQ ID NO:4 VH and VL are linked together through a linker peptide (such as (G4S)3, i.e. GGGGSGGGGSGGGGS) to form a single-chain antibody (scFv) of VH -Linker - VL. scFv can be expressed in E. coli, yeast or mammalian cells, or VH, VL and linker peptide chains can be obtained by recombinant expression protein, and then the corresponding single-chain antibody can be obtained by condensation reaction.

[0032] Example 2: Preparation of VP2-VH, VP2-VL, M-VH, M-VL 1. Target sequence The purpose of codon optimization is to improve the expression efficiency of the target protein in E. coli. The following is the nucleotide sequence after codon optimization (suitable for E. coli expression system): Target sequence of VP2-VH: cagtcggaactgtcgtcgagcggtgccgaagacgcgtcgccgggcgcgtcggacagcatgagctgcaaagcgagcggtgacttcgactcgcgtacggaacgcatgcacgactggcagcaacgtccgggccagggccagggcctggagtggatcggcgacatcaccccgtcgcgctacaccgactacgaccaacggttcgaccgagacgcgacgctgacgaccgacaagtcgtcgtcgaccgcttacatgcagctgtcgtcgctggactcggaagactcggcggtgtactgctgcgcgcgttactacgaggactactactcggacgagtactggggccaggggacgacgctggtgacgtcgtcg, SEQ IDNO:5 Target sequence of VP2-VL gaaagcgacctgacccagtcgccggaagacctgtcgctgtcgccggaagacgcgacgcgctgcgacgcgtcgcaggacgtcgacgagtacgactcgtggtaccagcagaaaccgggccaggccccgcgcctgctgatctacgacgagtccaacgacgcgacgggcatcccggcccgcttcagcggctcgggcagcgggaccgacttcacgctgacgatctcgtccctggaaccggacgacgcggtgtactgctgcagcaagagtccaacgactggccgtcgctgacgttcggcgcggggaccaagctggaactgaa, SEQ ID NO:6 Target sequence of M-VH gaaagcgacctggtggaatcgggcggtggtctggtgcagccgggcggttcgctgcgctcgagctgcgcggcgagcggtgacttcg acgaatcgtacgccatgtcttgggtgcggcaggccccgggcaaaggcctggagtgggtgtcggccatctcgggcagcggtggttcg acgtacgccgactcggtgaagggccgcttcacgatctcgcgcgacaactcgaagaacacgctgtacctgcagatgaactcgctgcgcgccgaggacaccgccgtgtactgctgcgcgcgcgactacggcgactactggggccaggggacgacgctggtgacggtgtcgtcg, SEQ ID NO:7 Target sequence of M-VL gactcggacatgacccagtcgccggaagacctgtcgctgtcgccggaagacgcgacgcgctgcgacgcgtcgcaggacgtcgacgagtacgactcgtggtaccagcagaaaccgggccaggccccgcgcctgctgatctacgacgagtccaacgacgc gacgggcatcccggcccgcttcagcggctcgggcagcgggaccgacttcacgctgacgatctcgtccctggaaccggacgacgcggtgtactgctgcagcaagagtccaacgactggccgtcgctgacgttcggcgcggggaccaagctggaactgaa, SEQ ID NO:8 Amplify target fragment PCR amplification was performed using the primers shown in Table 1, and the restriction enzyme effect was verified by agarose gel electrophoresis, and the target sequences of VP2-VH with restriction enzyme sites, VP2-VL with restriction enzyme sites, M-VH with restriction enzyme sites, and M-VL with restriction enzyme sites were recovered. The PCR amplification system was 2.0 μL 10×PCRBuffer, 1.6 μL (25 mM) MgCl 2, 0.4 μL dNTP Mix (10 mM each of four deoxynucleoside triphosphates), 0.4 μL upstream primer (10 μM), 0.4 μL downstream primer 2 (10 μM), 0.1 μL Taq DNA polymerase (5 U / μL), 50 ng template DNA, and ddH 2 O to 20 μL. The PCR amplification program was initial denaturation, 95°C, 5 minutes; denaturation, 95°C, 30 seconds, annealing, 55°C, 30 seconds, and extension at 72°C, 1 minute, which constituted one cycle. After 35 cycles, the final extension was at 72°C, 5 minutes, and the cells were stored at 4°C. The restriction sites in Table 1 are in capital letters.

[0033] Table 1 3. Enzyme digestion of pET-28a(+) pET-28a(+) (JHBIO, JH1207) was double-digested with NdeI and XhoI restriction endonucleases. The digestion reaction system was: 1 µg pET-28a(+), 1 µL (10 U / µL) NdeI, 1 µL (10 U / µL) XhoI, 5 µL 10× buffer, deionized water, made up to 50 µL, and digested at 37°C for 2h. After digestion, the digestion effect was verified by agarose gel electrophoresis, and the pET-28a(+) fragment was recovered.

[0034] 4. Recombinant expression steps The target sequences of VP2-VH with restriction site, VP2-VL with restriction site, M-VH with restriction site, and M-VL with restriction site were mixed with pET-28a(+) fragment at a molar ratio of 3:1, and ligated using T4 DNA ligase. The mixture was incubated at 16°C for 4 hours or overnight. The reaction system was 50 ng pET-28a(+) fragment, 150 ng insert, 1 µL (400 U / µL) T4 DNA ligase, and 2 µL 10× ligation buffer. Deionized water was added to make up to 20 µL.

[0035] Transform the ligation product into competent E. coli BL21 (DE3), screen positive clones on LB plates containing kanamycin (50 µg / mL), and verify positive clones by colony PCR or sequencing. Pick positive clones and inoculate them into LB liquid medium containing kanamycin (50 µg / mL), and culture them at 37°C with shaking overnight. Inoculate the overnight culture into fresh LB medium at a ratio of 1:100, and culture them at 37°C with shaking until OD600 reaches 0.6-0.8. Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.5 mM to induce protein expression, reduce the culture temperature to 16-25°C, and continue to culture with shaking for 12-16 hours to reduce inclusion body formation. Centrifuge the culture at 4°C and 5000 g for 10 minutes to collect the cells. Wash the cells with pre-cooled PBS buffer and collect the cells by centrifugation again.

[0036] Resuspend the cells in lysis buffer (e.g. 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). Use an ultrasonic disruptor to lyse the cells (operate on ice, power 200 W, work 2s, interval 5s, total time 10min). Centrifuge (4℃, 12000 g, 30 min), and collect the supernatant. Load the supernatant onto a pre-equilibrated Ni-NTA affinity chromatography column. Wash the column with wash buffer (e.g. 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-specifically bound proteins. Elute the target protein with elution buffer (e.g. 50 mM Tris-HCl, 300 mM NaCl, 250mM imidazole, pH 8.0). Dialyze the eluted protein into storage Tris-HCl buffer to remove imidazole. The protein is concentrated using an ultrafiltration centrifuge tube and freeze-dried to obtain the target protein.

[0037] The protein solution was prepared into 2 mg / mL using Tris-HCl buffer and then SDS-PAGE was performed. Figure 1 As shown, the control group is the expression result of E. coli transferred into pET-28a(+), and the other lanes are the results of VP2-VH, VP2-VL, M-VH and M-VL, respectively. It can be seen that protein bands of 16,500 Da, 14,300 Da, 15,400 Da and 14,300 Da appeared, respectively, which are consistent with the theoretical size.

[0038] The immunoaffinity of VP2-VH and VP2-VL with VP2 protein (CSB-YP355948JAK, CUSABIO), and the immunoaffinity of M-VH and M-VL with M protein (CSB-CF752619CHAH, CUSABIO) were verified by WB method. VP2-VH, VP2-VL, M-VH and M-VL were separated by 12% SDS-PAGE and transferred to PVDF membrane respectively; blocked with PBS buffer containing 3% BSA for 2h; then incubated PVDF membrane with VP2 protein or M protein at 4℃ overnight; washed the membrane 5 times with PBST, and incubated with HRP-labeled anti-His antibody at 37℃ for 1h; washed the membrane 5 times again, used ECL substrate colorimetric solution, and exposed to light for color in a dark box. Figure 2 As shown, VP2-VH and VP2-VL have immunoaffinity with VP2, and M-VH and M-VL have immunoaffinity with M, respectively.

[0039] Example 3: Preparation of fluorescent microsphere-labeled antibodies 1. Material preparation Azido peptide, Cyclo[RGDfK(Azido)]) also known as Cyclo[Arg-Gly-Asp-D-Phe-Lys(Azido)], cyclopentapeptide, Xi'an Qiyue Biotechnology Co., Ltd. Buffer: PBS, pH 7.4. Catalyst: CuSO 4 The catalyst system is composed of sodium ascorbate and other reagents: deionized water, ethanol, etc. for washing after the reaction.

[0040] Alkyne fluorescent microspheres (CNPCs microspheres) were prepared according to the method in Chapter 2 of “Zhang Peng, Preparation, Characterization and Application of Functionalized Polymer Porous Microspheres, Doctoral Dissertation of Fudan University, 2013”.

[0041] 2. Coupling steps Microsphere pretreatment: 5 mg of alkynyl fluorescent microspheres were suspended in 20 mL of PBS buffer and ultrasonicated for 15 min to ensure that the microspheres were evenly dispersed and to avoid agglomeration. A 5 mg / mL azido peptide solution was prepared in PBS.

[0042] 40 mL of azido peptide solution was mixed with 20 mL of alkynyl fluorescent microsphere solution, and copper sulfate with a final concentration of 0.5 mM and 5 mM ascorbic acid were added thereto. The mixture was stirred at room temperature in the dark for 4 h. After the solution was separated by magnetic adsorption, the microspheres were washed several times with PBS buffer to remove unreacted peptides, catalysts and other impurities. Then, deionized water or ethanol was used for the final washing to remove residual salts and other substances to obtain microspheres coupled with cyclopentapeptide. Figure 3 As shown, at 2100~2260 cm −1The alkynyl absorption peak basically disappeared, indicating that the azido peptide was successfully coupled to the alkynyl fluorescent microspheres.

[0043] 3. Condensation antibody The buffer solutions of VP2-VH, VP2-VL, M-VH and M-VL at 10 mg / mL and the buffer solution of microspheres coupled with cyclic pentapeptide at 50 mg / mL were prepared.

[0044] EDC and NHS were added to the buffers of VP2-VH, VP2-VL, M-VH and M-VL to make the final concentrations 0.2 M and 0.2 M, respectively, and the mixture was stirred gently at room temperature for 30 minutes. Two volumes of the buffer of the microspheres coupled with the cyclic pentapeptide were added to the activation solution of VP2-VH and reacted at room temperature for 4 hours. Then, an equal volume of the activation solution of VP2-VL was added to the activation solution of VP2-VL and reacted at room temperature for 4 hours. Unreacted proteins were removed by magnetic absorption, and the microspheres were washed several times with PBS buffer. The binding of the peptide to the protein was confirmed by SDS-PAGE to obtain the fluorescent microsphere-labeled VP2 antibody ( Figure 4 ).

[0045] After adding 2 volumes of the buffer solution of the microspheres coupled with the cyclic pentapeptide to the M-VH activation solution and reacting at room temperature for 4 hours, an equal volume of the M-VL activation solution was added to the M-VH activation solution and reacted at room temperature for 4 hours. Unreacted proteins were removed by magnetic adsorption, and the microspheres were washed several times with PBS buffer. The binding of the peptide and the protein was confirmed by SDS-PAGE to obtain the M antibody labeled with fluorescent microspheres ( Figure 4 ).

[0046] 4. Control group The magnetic fluorescent microspheres (Zhongke Keyou) were resuspended in pH 6.0 MES buffer, and EDC and 0.2 M sulfo-NHS were added at a final concentration of 0.2 M. The reaction was carried out at room temperature for 30 minutes, and the reaction was gently stirred during the reaction. After the reaction was completed, the microspheres were washed with MES buffer to remove the unreacted activator. A 10 mg / mL buffer of canine parvovirus antibody (MAB8293, R92F6, Merck) or canine coronavirus antibody (A041, Antibio) was prepared, and the buffer was reacted with 2 volumes of 50 mg / mL of the buffer for activating magnetic fluorescent microspheres at room temperature for 4 hours, and then the microspheres were washed with PBS or MES buffer to remove the unbound antibody. The coupled microspheres were resuspended in a blocking solution (such as 1% BSA) and incubated at room temperature for 1 hour. After the blocking was completed, the microspheres were washed with PBS to remove the unbound blocking agent. Resuspend the coupled and blocked magnetic fluorescent microspheres in an appropriate amount of storage solution (such as PBS containing 0.1% BSA) and store at 4°C to avoid repeated freezing and thawing.

[0047] Example 4. Preparation of fluorescent immunochromatographic strips The specific preparation steps of the fluorescent immunochromatographic test strip for simultaneously detecting canine parvovirus (CPV) and canine coronavirus (CCV) by fluorescent immunochromatography in this embodiment are as follows: 1. Material preparation Nitrocellulose membrane, sample pad, conjugate pad, absorbent pad, PVC backing, fluorescent microsphere-labeled VP2 antibody and fluorescent microsphere-labeled M antibody prepared in accordance with the above examples, goat anti-mouse IgG antibody, bovine serum albumin (BSA), Tris-HCl buffer, phosphate buffered saline (PBS), Tween-20 and blocking solution, etc.

[0048] 2. Assembly of test strips The buffer containing 1 mg / mL VP2-VH and 1 mg / mL VP2-VL was sprayed on the detection line (T1 line) of the NC membrane using a film dispenser, the buffer containing 1 mg / mL M-VH and M-VL was sprayed on the detection line (T2 line) of the NC membrane, and goat anti-mouse IgG was sprayed on the quality control line (C line). The spraying amount was 0.4 μL / cm. After the spraying was completed, the NC membrane was dried at 37°C for 2 hours. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6 mm.

[0049] Mix the VP2 antibody labeled with fluorescent microspheres and the M antibody labeled with fluorescent microspheres provided by the experimental group in equal amounts and spray them on the binding pad with an ink dispenser. The spraying concentration is usually 1 mg / mL and the spraying volume is 0.4 μL / cm. Dry at 37°C for 2 hours. Paste the sample pad and the absorbent pad on the PVC backing with nitrocellulose membrane respectively, cut them into 4 mm wide strips with a strip cutter, and make test strips. Dry and store them for later use. When using, treat the sample in a certain way and then drip it on the sample pad. A special sample addition hole can be made on the sample pad.

[0050] 3. Test strips for the control group A 1 mg / mL canine parvovirus antibody (MAB8293, R92F6, Merck) buffer solution was sprayed on the detection line (T1 line) of the NC membrane using a film dispenser, a 1 mg / mL canine coronavirus antibody (A041, Antibio) buffer solution was sprayed on the detection line (T2 line) of the NC membrane, and goat anti-mouse IgG was sprayed on the quality control line (C line). The spraying amount was 0.4 μL / cm. After the spraying was completed, the NC membrane was dried at 37°C for 2 h. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6 mm.

[0051] Mix the VP2 antibody labeled with fluorescent microspheres and the M antibody labeled with fluorescent microspheres provided by the control group in equal amounts and spray them on the binding pad with an ink dispenser. The spraying concentration is usually 1 mg / mL and the spraying volume is 0.4 μL / cm. Dry at 37°C for 2 hours. Paste the sample pad and the absorbent pad on the PVC backing with nitrocellulose membrane respectively, cut them into 4 mm wide strips with a strip cutter, and make test strips. Dry and store them for later use. When using, treat the sample to a certain extent before dripping it on the sample pad. A special sample addition hole can be made on the sample pad.

[0052] Example 5: Detection The following are the specific steps for detecting canine parvovirus by fluorescent immunochromatography: 1. Sample processing Fecal samples of dogs were collected, diluted with physiological saline, and centrifuged to obtain the supernatant as the test sample.

[0053] 2. Sample testing The sample to be tested is dripped onto the sample pad of the test paper. The sample is chromatographed on the test paper and combined with the magnetic fluorescent microspheres on the binding pad to form an antigen-antibody complex. The chromatography continues to the NC membrane, and the antigen-antibody complex combines with the sprayed detection antibody at the detection line 1 (T1) and the detection line 2 (T2) to form a sandwich complex. The quality control antibody at the quality control line (C line) is used to verify the effectiveness of the test paper.

[0054] Insert the test strip into the detection window of the fluorescence reader, and the fluorescence intensity of the test line 1 (T1), test line 2 (T2) and quality control line (C line) will be displayed on the display in the form of numerical values. According to the standard curve already entered in the instrument, the content of canine parvovirus and canine coronavirus in the sample is calculated to achieve quantitative detection.

[0055] 3. Interpretation of results If both the test line 1 (T1) and the quality control line (C line) show fluorescence or color reaction, it means that canine parvovirus is present in the sample. If both the test line 2 (T2) and the quality control line (C line) show fluorescence or color reaction, it means that canine coronavirus is present in the sample. If only the quality control line (C line) shows fluorescence or color reaction, and the test line 1 (T1) and the test line 2 (T2) have no reaction, it means that canine parvovirus and canine coronavirus are not present in the sample.

[0056] 4. Sensitivity test Canine parvovirus VP2 protein or canine coronavirus M protein was taken and diluted with the sample extract to obtain concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 ng / mL. After testing with the test strips prepared for the experimental group and the control group in Example 4, the fluorescence intensities of T1, T2, and C lines were detected by a fluorescence detector. The fluorescence intensity ratio of T1 / C or T2 / C was used as the ordinate and the mass concentrations of different standard products were used as the abscissa to draw a standard curve for the detection of the new coronavirus N-protein.

[0057] As a result, as the content of VP2 protein or M protein in the sample increases, the color of the detection line T gradually darkens. Using logEC50=logECF - (1 / HillSlope)*log(F / (100-F)); Y=Bottom + (Top-Bottom) / (1+10^((LogEC50-X)*HillSlope)), the dose-response curve is fitted, logEC50 is the logarithm of the half-maximum effective concentration, HillSlope is the Hill slope, Bottom and Top are the bottom and top of the curve respectively. Figure 5 It can be seen that the curves of the test strips in the experimental group for detecting VP2 protein and M protein have very good fitting effects, and the correlation coefficients are 0.9974 and 0.9961, respectively. The curves of the test strips in the control group for detecting VP2 protein and M protein have very good fitting effects, and the correlation coefficients are 0.9989 and 0.9989, respectively. However, the average T / C of the blank sample without antigen is 0.002, and the average T / C of the test strips in the control group for detecting VP2 protein and M protein is 10 -5 ng / mL and 10 -4 ng / mL samples, there was no significant difference in T / C value between them and the blank samples, indicating that the test strips provided by the control group could not detect 10 -5 ng / mL and 10 -4 ng / mL of VP2 protein or 10 -5 ng / mL and 10 -4 ng / mL of M protein was detected, and its sensitivity was not as good as that of the experimental group.

[0058] 5. Specificity test Recombinant porcine parvovirus VP2 protein (PhyCell®), COVID-19 N protein (Dongguan Feipeng Biotechnology Co., Ltd.), canine parvovirus VP2 protein and canine coronavirus M protein were prepared into 10 ng / mL sample solutions, and the test strips prepared in the experimental group and the control group in Example 4 were used for testing, and the T1, T2 and C lines were displayed by a fluorescence detector. Figure 6As shown, the test strips provided by the experimental group showed no color development for the recombinant porcine parvovirus VP2 protein and the new coronavirus N protein, but had obvious fluorescence display on the T1 line and T2 line for the canine parvovirus VP2 protein and the canine coronavirus M protein, respectively, indicating that the experimental group had obvious specificity for the canine parvovirus VP2 protein and the canine coronavirus M protein, respectively, and could exclude the interference of the recombinant porcine parvovirus VP2 protein and the new coronavirus N protein. In addition, although the test strips of the control group can show obvious fluorescence for the canine parvovirus VP2 protein and the canine coronavirus M protein, it also shows the T2 line for the detection of the canine parvovirus VP2 protein, indicating that its own specificity for the canine parvovirus VP2 protein and the canine coronavirus M protein is insufficient, and the interference of the two cannot be effectively excluded. In addition, the test strips of the control group also have fluorescent display for the recombinant porcine parvovirus VP2 protein, and its interference cannot be excluded.

[0059] 6. Actual sample test The test strips provided by the experimental group and the control group of the present invention were used to simultaneously collect canine fecal separation samples (detection of canine parvovirus) and throat swab samples (detection of canine coronavirus) for clinical comparison testing. In addition, the canine parvovirus (CPV) nucleic acid detection kit (PCR-fluorescent probe method, item number YB-11-1200, Yubo Bio) was used to detect the canine samples separated respectively, and the canine respiratory coronavirus RT-PCR kit (item number XY-P0055, XYBIO) was used to detect these throat swab samples.

[0060] For canine parvovirus, the sensitivity of the experimental group was 92.35%, 95%CI: 89.21%~98.92%; the specificity was 95.35%, 95%CI: 90.26%~98.16%. The sensitivity of the control group was 79.26%, 95%CI: 61.82%~85.36%; the specificity was 68.49%, 95%CI: 58.39%~79.26%. The sensitivity of the PCR-fluorescent probe method was 90.83%, 95%CI: 84.76%~93.29%; the specificity was 92.79%, 95%CI: 89.44%~96.09%.

[0061] For canine coronavirus, the sensitivity of the experimental group was 90.95%, 95%CI: 86.82%~94.33%; the specificity was 96.18%, 95%CI: 92.48%~99.08%. The sensitivity of the control group was 82.36%, 95%CI: 75.93%~89.08%; the specificity was 72.64%, 95%CI: 63.09%~81.82%. The sensitivity of the PCR-fluorescent probe method was 86.92%, 95%CI: 81.83%~92.39%; the specificity was 91.48%, 95%CI: 87.98%~95.42%.

[0062] The test results show that the fluorescent immunochromatography canine parvovirus / canine coronavirus test strip and detection kit provided by the present invention have good sensitivity and specificity, and a high consistency with clinical diagnosis, and can be used as an effective screening and diagnostic technology for canine parvovirus / canine coronavirus infection.

[0063] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit, characterized in that: Including fluorescent microsphere labeled VP2 antibody, fluorescent microsphere labeled M antibody, goat anti-mouse IgG antibody, nitrocellulose membrane, sample pad, conjugate pad, absorbent pad, PVC backing; The fluorescent microsphere-labeled VP2 antibody is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO:1 and the VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys, and the fluorescent microsphere-labeled M antibody is an antibody obtained by coupling the M-VH shown in SEQ ID NO:3 and the M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys.

2. A method for assembling a fluorescent immunochromatography canine parvovirus / canine coronavirus test strip, characterized in that: include: The buffer containing VP2-VH and VP2-VL was sprayed on the T1 detection line of the NC membrane, the buffer containing M-VH and M-VL was sprayed on the T2 detection line of the NC membrane, and the goat anti-mouse IgG was sprayed on the quality control line. After the spraying was completed, the NC membrane was dried at 37°C for 2h. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6mm; VP2 antibody labeled with fluorescent microspheres and M antibody labeled with fluorescent microspheres were mixed in equal amounts and sprayed on the conjugate pad using a film dispenser. After spraying, the NC membrane was dried at 37°C for 2 h. The sample pad and absorbent pad were respectively pasted on the PVC backing with nitrocellulose membrane and cut into 4 mm wide strips with a strip cutter to make test strips; Among them, the fluorescent microsphere-labeled VP2 antibody is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO:1 and the VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys, and the fluorescent microsphere-labeled M antibody is an antibody obtained by coupling the M-VH shown in SEQ ID NO:3 and the M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys.

3. The fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit according to claim 1, characterized in that: The VP2-VH shown in SEQ ID NO:1 is prepared by cloning the target sequence shown in SEQ ID NO:5 into pET-28a(+) to obtain an expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VH into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate; The VP2-VL shown in SEQ ID NO:2 is prepared by cloning the target sequence shown in SEQ ID NO:6 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VL, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the VP2-VL from the lysate.

4. The fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit according to claim 1, characterized in that: The VP2-VH shown in SEQ ID NO:3 is prepared by cloning the target sequence shown in SEQ ID NO:7 into pET-28a(+) to obtain an expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate; The M-VL shown in SEQ ID NO:4 is obtained by cloning the target sequence shown in SEQ ID NO:8 into pET-28a(+) to obtain an expression plasmid pET-28a-M-VL, transforming pET-28a-M-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacteria from the culture medium, lysing the bacteria, and purifying and collecting the M-VL from the lysate.

5. The assembly method according to claim 2, characterized in that: The fluorescent microsphere-labeled VP2 antibody is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain cyclopentapeptide-coupled microspheres; then reacting the VP2-VH activation solution with the cyclopentapeptide-coupled microspheres, and then adding the VP2-VL activation solution to react; The fluorescent microsphere-labeled M antibody is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain microspheres coupled with cyclopentapeptide; then reacting the M-VH activation solution with the microspheres coupled with cyclopentapeptide, and then adding the M-VL activation solution to react.

6. Use of antibodies against canine parvovirus and / or antibodies against canine coronavirus in the preparation of a canine parvovirus / canine coronavirus detection kit by fluorescent immunochromatography, wherein the antibodies against canine parvovirus include VP2-VH as shown in SEQ ID NO: 1 and VP2-VL as shown in SEQ ID NO: 2, and the antibodies against canine coronavirus include M-VH as shown in SEQ ID NO: 3 and M-VL as shown in SEQ ID NO: 4.

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