Latex duplex immunochromatography test strip for rapidly identifying and detecting TGEV and PEDV and preparation method of latex duplex immunochromatography test strip

Abstract

The invention belongs to the technical field of biology, and particularly relates to a latex duplex immunochromatography test strip for rapidly identifying a porcine transmissible gastroenteritis virus (TGEV) and a porcine epidemic diarrhea virus (PEDV) and a preparation method of the latex duplex immunochromatography test strip. Soluble TGEV N protein and PEDV N protein with immunogenicity and reactivity are respectively obtained by utilizing a prokaryotic expression system, the soluble TGEV N protein and the soluble PEDV N protein are used as immunogenicity to immunize mice, and specific monoclonal antibodies of the TGEV N protein and the PEDV N protein are respectively obtained by utilizing cell fusion and subcloning screening technologies. The antibody variable region gene sequence is obtained by using a nested PCR amplification technology, and can be subsequently obtained by using a gene engineering or protein engineering method. Then, the latex duplex immunochromatography test strip for detecting TGEV and PEDV, which is convenient, rapid and high in sensitivity, and the preparation method are researched and developed on the basis of specific monoclonal antibodies of TGEV N protein and PEDV N protein.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a latex double immunochromatographic test strip for rapidly distinguishing porcine transmissible gastroenteritis virus (TGEV) from porcine epidemic diarrhea virus (PEDV) and a preparation method thereof. Background Art

[0002] Clinically, diarrheal viruses often exist in the form of mixed infections of two or more viruses, which potentially increases the difficulty of prevention and control. In the absence of an effective vaccine, it is crucial to establish an antigen diagnosis method that can quickly and conveniently identify and detect multiple viruses simultaneously to prevent and control the spread of the disease.

[0003] Porcine transmissible gastroenteritis coronavirus (TGEV) and porcine epidemic diarrhea virus (PEDV) are two enteric coronaviruses that cause diarrhea in piglets. Their clinical symptoms and epidemiology are very similar, with watery diarrhea, vomiting, dehydration, and especially high mortality in newborn piglets. Commonly used etiological detection methods for both pathogens include virus isolation and identification, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and real-time quantitative PCR. However, these methods take a long time to detect, are complex to operate, require specific or expensive large instruments and professional technicians, and are difficult to meet the requirements of rapid, cheap, and efficient monitoring in grassroots farms.

[0004] Lateral immunochromatography technology is a technology that couples different signal markers on the surface of antigens or antibodies to achieve macroscopic visualization or microscopic antigen-antibody reaction by signal amplification. This technology is easy to operate and can be completed without the need for professional personnel. At the same time, it does not require the use of specific instruments, and the test results can be obtained with the naked eye. It is also time-saving and can be completed within 15 minutes. It greatly shortens the detection time. Its commonly used signal markers are mainly colloidal gold particles and latex microspheres. Although there are a variety of markers on the market, colloidal gold particles and latex microspheres are still the ones that can be used for large-scale production.

[0005] TGEV N protein is an important nucleocapsid structural protein that can stimulate the body to produce a large number of antibodies in the early stages of viral infection. Similar to TGEV N protein, PEDV N protein, as the main nucleocapsid protein, is abundantly expressed and has good gene sequence conservation, and can stimulate the body's specific antibodies in the early stages of viral infection. Therefore, both N proteins have important application value in immunological diagnosis and new vaccine development. Summary of the invention

[0006] The present invention uses a prokaryotic expression system to obtain immunogenic and reactive soluble TGEV N protein and PEDV N protein, respectively, and uses them as immunogens to immunize mice, and uses cell fusion and subclone screening techniques to obtain specific TGEV N protein and PEDV N protein monoclonal antibodies, respectively. The antibody variable region gene sequence is obtained by nested PCR amplification technology, and can be subsequently obtained by genetic engineering or protein engineering methods. Then, based on the TGEV N protein and PEDV N protein specific monoclonal antibodies, a convenient, rapid and highly sensitive latex double immunochromatographic test strip and preparation method for detecting TGEV and PEDV are developed.

[0007] The present invention specifically includes the following contents:

[0008] The present invention provides a latex double immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus. The test strip comprises a PVC bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad; a quality control line C, a detection line T1 and a detection line T2 are provided on the nitrocellulose membrane. The conjugation pad is sprayed with latex microspheres labeled with TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 as capture antibodies, the detection line T1 is coated with PEDV N protein monoclonal antibody 1E1 as a detection antibody, the detection line T2 is coated with TGEV N protein monoclonal antibody 2A9 as a detection antibody, and the quality control line C is coated with goat anti-mouse IgG.

[0009] The heavy chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of GFNIKDTY, a CDR2 with an amino acid sequence of IDPADGYT, and a CDR3 with an amino acid sequence of ARPGTLDY. The heavy chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of GYTFTDFN, a CDR2 with an amino acid sequence of INPNNGRS, and a CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0010] The light chain variable region of the monoclonal antibody 1B7 of the present invention includes a CDR1 with an amino acid sequence of KSVSTSGYSY, a CDR2 with an amino acid sequence of LVS, and a CDR3 with an amino acid sequence of QHIRELTR. The light chain variable region of the monoclonal antibody 2A9 of the present invention includes a CDR1 with an amino acid sequence of KSLLHSN, a CDR2 with an amino acid sequence of QMS, and a CDR3 with an amino acid sequence of AQNLEFPWT.

[0011] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1B7 of the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0012] The DNA sequence encoding the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO:5, and the DNA sequence encoding the light chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO:6; the DNA sequence encoding the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO:7, and the DNA sequence encoding the light chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO:8.

[0013] The heavy chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of IgG1 or IgG2b type. The light chain constant region of the monoclonal antibody against the N protein of porcine transmissible gastroenteritis coronavirus of the present invention is of Kappa type.

[0014] The heavy chain variable region of the monoclonal antibody 1F10 of the present invention includes a CDR1 with an amino acid sequence of GYTFTTYY, a CDR2 with an amino acid sequence of IYPGNINT, and a CDR3 with an amino acid sequence of ARISSALPY. The heavy chain variable region of the monoclonal antibody 1E1 of the present invention includes a CDR1 with an amino acid sequence of GYTFTAYV, a CDR2 with an amino acid sequence of INPYNDRT, and a CDR3 with an amino acid sequence of ARDDDDYEEGFAY.

[0015] The light chain variable region of the monoclonal antibody 1F10 of the present invention includes a CDR1 with an amino acid sequence of RSLLNTSSQKSY, a CDR2 with an amino acid sequence of FAS, and a CDR3 with an amino acid sequence of QQHFSTPPT. The light chain variable region of the monoclonal antibody 1E1 of the present invention includes a CDR1 with an amino acid sequence of QKCQYIWLSL, a CDR2 with an amino acid sequence of SCI, and a CDR3 with an amino acid sequence of SAHSGAYT.

[0016] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F10 of the present invention is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E1 of the present invention is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0017] The DNA sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 13, and the DNA sequence encoding the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 14; the DNA sequence encoding the heavy chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO: 15, and the DNA sequence encoding the light chain variable region of monoclonal antibody 1E1 is shown in SEQ ID NO: 16.

[0018] The heavy chain constant region of the monoclonal antibody to the PEDV N protein of the present invention is of IgG1 type. The light chain constant region of the monoclonal antibody to the PEDV N protein of the present invention is of Kappa type.

[0019] Preferably, the preparation method of the conjugate pad is:

[0020] The conjugate pad is blocked with a conjugate pad blocking solution;

[0021] The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended with MES buffer, and fully dispersed with ultrasound, followed by adding TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10, incubated at room temperature, and then blocked with latex microsphere marker blocking solution, incubated at room temperature, and centrifuged; the precipitate was resuspended with a latex microsphere marker protective agent to obtain a latex microsphere marker mixed solution of TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10;

[0022] The prepared latex microsphere labeling mixed solution is evenly sprayed on the sealed conjugate pad by an instrument;

[0023] The conjugate pad blocking solution is an ultrapure aqueous solution of 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40.

[0024] The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA;

[0025] The latex microsphere marker protective agent is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40, and 1% v / v Tween-20.

[0026] The latex microsphere labeled mixed solution of the monoclonal antibody 1B7 for the TGEV N protein and the monoclonal antibody 1F10 for the PEDV N protein is composed of: each 1 mL of the latex microsphere solution contains 40 μg of the monoclonal antibody 1B7 for the TGEV N protein and 60 μg of the monoclonal antibody 1F10 for the PEDV N protein.

[0027] Preferably, the detection line T1 is coated with 1 mg / mL PEDV N protein monoclonal antibody 1E1.

[0028] Preferably, the detection line T2 is coated with 1 mg / mL TGEV N protein monoclonal antibody 2A9.

[0029] Preferably, the quality control line C is coated with 1 mg / mL of goat anti-mouse IgG.

[0030] Preferably, the sample pad is immersed in a sample pad blocking solution; the sample pad blocking solution is a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

[0031] In a sixth aspect, the present invention provides a method for preparing the above-mentioned latex double immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis and porcine epidemic diarrhea virus. The method comprises the following steps:

[0032] (1) preparing a binding pad coated with latex microspheres labeled with monoclonal antibody 1B7 against TGEV N protein and monoclonal antibody 1F10 against PEDV N protein;

[0033] (2) The monoclonal antibody 1E1 (1 mg / mL) against PEDV N protein and goat anti-mouse IgG antibody (1 mg / mL) were sprayed on the nitrocellulose membrane at an interval of 3 mm, as the detection line T1 and the quality control line C, respectively; the monoclonal antibody 2A9 (1 mg / mL) against TGEV N protein was sprayed 5 mm away from the monoclonal antibody 1E1 (1 mg / mL) against PEDV N protein, as the detection line T2.

[0034] (3) The sample pad, the binding pad, the nitrocellulose membrane and the absorbent pad are overlapped on the PVC bottom plate in sequence to obtain a latex double immunochromatographic test strip for identifying and detecting porcine transmissible gastroenteritis and porcine epidemic diarrhea viruses.

[0035] In a seventh aspect, the detection method of the test strip of the present invention comprises the following steps:

[0036] (1) Treatment of TGEV negative and positive control samples

[0037] TGEV was inoculated into a monolayer of PK15 cells. After 60 hours of infection, the cells were placed in a -80°C refrigerator and repeatedly frozen and thawed three times. The cells were centrifuged at 12,000 rpm and 4°C for 10 minutes to remove cell debris and the supernatant was used as the antigen to be tested. Uninoculated PK15 cells were taken and treated in the same way as negative controls.

[0038] (2) Treatment of PEDV negative and positive control samples

[0039] PEDV was inoculated into a monolayer of VeroE6 cells. After 60 hours of infection, the cells were placed in a -80°C refrigerator and repeatedly frozen and thawed for 3 times. The cells were centrifuged at 12000 rpm and 4°C for 10 minutes to remove cell debris and the supernatant was used as the antigen to be tested. Uninoculated VeroE6 cells were taken and treated in the same way as negative controls.

[0040] (3) Result determination

[0041] After the reaction is completed, the positive and negative properties are qualitatively judged by the naked eye.

[0042] The beneficial effects of the present invention are:

[0043] The latex double immunochromatographic test strip for identifying and detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus provided by the present invention has no nonspecific reaction when detecting TGEV or PEDV alone, and does not cross-react with other porcine enteric coronaviruses SADS-CoV and PDCoV with similar clinical symptoms. At the same time, the test strip can detect the virus in the positive control of TGEV and PEDV with a minimum limit of 10 4.69 TCID 50 / 0.1mL and 10 3.09 TCID 50 / 0.1mL, the prepared test strip has the advantages of good specificity, sensitivity, simple operation and short time, and provides a rapid detection method for the rapid identification and detection of the two diarrhea viruses on-site or in the laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The results of PCR amplification of TGEV N gene. M: DL2000 relative molecular mass standard; 1: control water; 2: N gene, with a size of about 1000 bp.

[0045] Figure 2 The results of TGEV pET32a-N recombinant protein expression and purification. (A) M: protein marker; 1: before pET32a-N induction; 2: after pET32a-N induction; 3: pET32a-N lysis supernatant; 4: pET32a-N lysis precipitate. (B) M: protein marker; 1: purified pET32a-N protein.

[0046] Figure 3 ELISA was used to detect the antibody titer of mice after TGEV N protein immunization. 1-3: mice immunized with No. 1, 2 and 3, NC: negative control.

[0047] Figure 4The results of SDS-PAGE identification of TGEV N protein antibody purification. M: protein marker, 1: 1B7 monoclonal antibody; 2: 2A9 monoclonal antibody.

[0048] Figure 5 Identification of the reactivity of monoclonal antibodies against TGEVN protein for IFA. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) SP2 / 0 cell culture medium.

[0049] Figure 6 Western blot was used to identify the reactivity of the monoclonal antibody against TGEVN protein. M: protein marker, Mock: PK-15 cell control; TGEV: TGEV-infected PK-15 cells.

[0050] Figure 7 This is the result of identification of the subclasses of monoclonal antibodies against TGEV N protein.

[0051] Figure 8 The results of PCR amplification of the variable region of the monoclonal antibody against the TGEV N protein. (A) PCR amplification results of the heavy chain variable region. M: DL2000 Marker; 1: water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody; (B) PCR amplification results of the light chain K variable region. M: DL2000 Marker; 1: water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody.

[0052] Fig. 9 The results of PCR amplification of PEDV N gene and restriction enzyme digestion of pET32a-N recombinant plasmid. (A) M: DL2000 relative molecular mass standard; 1: water control; 2: N gene, about 1300bp in size; (B) M: DL5000 relative molecular mass standard; 1: pET32a-N recombinant plasmid was double digested with BamHI and XhoI.

[0053] Fig.10 The results of pET32a-N recombinant protein expression and purification. (A) M: protein marker; 1: before pET32a-N induction; 2: after pET32a-N induction; 3: pET32a-N lysis supernatant; 4: pET32a-N lysis precipitate; (B) M: protein marker; 1: supernatant; 2: flow-through; 3-4: 25 μM imidazole elution; 5-6: 50 μM imidazole elution; 7-8: 75 μM imidazole elution; 9-10: 100 μM imidazole elution; 11: 150 μM imidazole elution; 12: 200 μM imidazole elution; 13: 300 μM imidazole elution; 14: 500 μM imidazole elution.

[0054] Fig.11This is a graph showing the test results of ELISA testing the titer of anti-mouse PEDV N protein polyclonal antibodies.

[0055] Fig.12 The PEDV N protein monoclonal antibody was purified and then identified by SDS-PAGE. M: protein marker; 1: 1F10 monoclonal antibody purification; 2: 1E1 monoclonal antibody purification.

[0056] Fig.13 IFA was used to verify the reactivity of the monoclonal antibody against PEDV N protein. (A) 1F10 hybridoma cell supernatant; (B) 1E1 hybridoma cell supernatant; (C) SP2 / 0 cell supernatant.

[0057] Fig.14 Western blot was used to identify the reactivity of the monoclonal antibody against PEDV N protein; M: protein marker, Mock VeroE6 cell control; PEDV: PEDV infected VeroE6 cells.

[0058] Fig.15 This is the result of subclass identification of monoclonal antibodies against PEDV N protein.

[0059] Fig.16 The results of PCR amplification of the variable region of the monoclonal antibody against the PEDV N protein. (A) PCR amplification of the heavy chain variable region. M: DL2000 Marker; 1F10: PCR amplification of the heavy chain variable region; 1E1: PCR amplification of the heavy chain variable region; (B) PCR amplification of the light chain K variable region. M: DL2000 Marker; 1F10: PCR amplification of the light chain variable region; 1E1: PCR amplification of the light chain variable region.

[0060] Fig.17 Schematic diagram of TGEV-PEDV double latex test strip.

[0061] Fig.18 Schematic diagram of TGEV-PEDV double latex test strip result determination.

[0062] Fig.19 TGEV-PEDV double latex test strip sensitivity test results. 1-9: First, PEDV and TGEV positive controls were both 1 Dilute to 2 9 , and then dilute each of the above samples as an independent sample at a ratio of 1:5, and finally test them one by one with test strips. 10: NC

[0063] Fig. 20Specific detection results of TGEV-PEDV double latex test strips; 1: simultaneous detection of PEDV and TGEV positive controls; 2: only detection of PEDV positive controls; 3: only detection of TGEV positive controls; 4: detection of SADS positive antigen; 5: detection of PDCoV positive antigen; 6: NC. DETAILED DESCRIPTION

[0064] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0065] Example 1 Preparation of monoclonal antibodies against TGEV N protein

[0066] 1. Construction, expression and purification of TGEV N protein recombinant plasmid

[0067] 1.1 Construction of pET32a-N recombinant plasmid

[0068] The TGEV strain was HLJ-17 (Genbank accession number: MT522161.1). The N protein of this strain was used as a reference to design specific primers, upstream P1: 5′-CGC GGATCC ATGGCCAACCAGGGACAACG-3′, downstream P2: 5′-CCG CTCGAG TTAGTTCGTTACCTCATCAATTATC-3′, BamHI and XhoI restriction sites (underlined parts) were introduced at the 5′ end of the upstream and downstream primers. After primer synthesis, RNA from the supernatant of TGEV cells infected with PK15 cells was extracted and reverse transcribed into cDNA, which was then used as a template for PCR amplification. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25μL, P1 and P2 1μL each, cDNA 1μL, ddH 2 O to 50 μL. The reaction procedure was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min. The PCR product was detected by 1% agarose gel electrophoresis. The results were as follows: Figure 1 As shown, the size of the N fragment of TGEV is about 1000 bp. Then, gel recovery was performed according to the instruction manual of EZNAGel Extraction Kit of Omega Company.

[0069] The TGEV N gene PCR gel recovery product and pET32a vector were double-digested with BamHI and XhoI, then connected and transformed into DH5α competent cells. The constructed recombinant plasmid was sequenced and identified, and the positive recombinant plasmid was named pET32a-N.

[0070] 1.2 Inducible expression and purification of TGEV N recombinant protein

[0071] The recombinant plasmid pET32a-N was transformed into Escherichia coli BL21 (DE3), and a single colony was picked and placed in 5 mL of ampicillin-resistant LB medium. 600 When the concentration of 1mmol / L IPTG was about 0.8, the expression was induced by adding IPTG at a final concentration of 1mmol / L. 5h after induction, the precipitate was collected by centrifugation at 12000rpm for 2min, and the precipitate was resuspended with an appropriate amount of PBS and ultrasonicated for 5min (ultrasonication for 3s, stop for 3s). After ultrasonication, centrifugation was performed at 4℃ and 12000rpm for 10min, and the supernatant and precipitate were collected respectively. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10min, and identified by SDS-PAGE. The results showed that pET32a-N was expressed in both the supernatant and the precipitate ( Figure 2 A), indicating that part of TGEV N protein is soluble.

[0072] According to the above method, the induced expression of pET32a-N protein was expanded, and the supernatant after ultrasonic lysis was taken for nickel column purification. The specific steps of purification are as follows:

[0073] (1) Loading the resin: Take an empty column and add 2 mL of nickel column NTA resin. When the storage solution drops to the surface of the resin, wash the column once with 5 column volumes of distilled water, and then equilibrate the column with 5 column volumes of equilibration solution (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.4);

[0074] (2) Sample loading: When the equilibrium solution drops to the resin surface, add 3 mL of lysate containing the recombinant protein. Repeat the sample loading 2 to 3 times, each time for 2 minutes, and collect the sample flow-through; add 5 column volumes of equilibrium solution to rinse the column once;

[0075] (3) Protein elution: The protein sample was eluted with different concentrations of imidazole (25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM and 300 mM) prepared with the equilibrium solution, 1 mL each time, and each concentration was repeated twice. The purpose was to determine the optimal concentration of imidazole for washing and protein elution;

[0076] (4) Column cleaning and storage: Rinse the column once with 5 volumes of 0.5M NaOH, then rinse once with distilled water, then add an appropriate amount of 70% anhydrous ethanol and store in a refrigerator at 4°C. Each sample collected was identified by SDS-PAGE. Figure 2 As shown in B, TGEV N protein with high purity was obtained.

[0077] 2. Animal Immunization

[0078] Three female BALB / c mice aged 6 weeks were selected, and the purified N recombinant protein was used to immunize the mice at a dose of 30 μg / mouse. For the first immunization, the N protein was mixed with an equal volume of Freund's complete adjuvant and emulsified, and multiple subcutaneous injections were performed on the mice (one point on the back and two points on the abdomen). Reinforcement immunization was performed every 2 weeks, for a total of four immunizations. The reinforcement immunization was to mix an equal volume of N recombinant protein and Freund's incomplete adjuvant and emulsify them. The immunization method was the same as the first immunization. Blood was collected from the tail 7 days after the four immunizations to determine the antibody titer.

[0079] 3. ELISA method to detect polyclonal antibody titer

[0080] The TGEV virus solution and the coating solution were diluted 1:1 and coated on the ELISA plate, 50 μL / well, overnight at 4°C, washed four times with PBST, and blocked with 2% trehalose at 4°C for 10 hours. The positive serum and negative serum were diluted with PBST in multiple ratios, with a dilution gradient from 1:100 to 1:12800, 8 dilution gradients, at 37°C for 1 hour, washed four times with PBST, and HRP-labeled goat anti-mouse IgG (1:20000 dilution) was added. After washing four times with PBST, TMB was used for color development, and the OD was measured on an enzyme reader. 450 .

[0081] The results are as follows Figure 3 As shown, the serum of non-immunized mice was used as negative control (NC). When the serum was diluted 1:102400, the OD of immunized mice No. 1, 2 and 3 was 450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 1:102400.

[0082] 4. Preparation of Monoclonal Antibodies

[0083] The specific steps are as follows:

[0084] (1) Preparation of feeder layer cells: HAT culture medium was injected into the peritoneal cavity of female BALB / c mice, and the liquid was slowly and repeatedly aspirated. After the liquid was aspirated, it was plated in a 96-well plate.

[0085] (2) Cell fusion: Splenocytes and an appropriate amount of SP20 cells were fused under the action of the fusion agent PEG. The fused cells were plated in a 96-well plate containing feeder cells.

[0086] (3) Screening of positive clones:

[0087] a) Indirect ELISA detection method: the purified TGEV N protein was coated on the ELISA plate to detect the secretion of antibodies by the fusion cells.

[0088] b) Indirect immunofluorescence (IFA) detection method: the supernatant in the ELISA antibody-positive cell wells was added to TGEV-infected PK15 cells, and then FITC-labeled goat anti-mouse IgG was added. After the reaction was completed, the results were observed under a fluorescence microscope.

[0089] (4) Subcloning of positive hybridoma cells: Select ELISA and IFA positive wells and use the limiting dilution method to subclone the screened positive hybridoma cells. Observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant, and use the above ELISA and IFA methods for antibody detection. Positive cells enter the next round of subcloning, which is performed three times in total.

[0090] (5) Preparation of ascites: 10-12 week old Balb / c mice were taken and each mouse was intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After 1 week, each mouse was intraperitoneally injected with 5 × 10 5 hybridoma cells (0.2 mL). After 7 to 10 days, the abdominal cavity of the mouse was obviously bulging. The ascites was collected and its titer was tested by ELISA. The cells were packaged and stored at -80°C.

[0091] (6) Purification of ascites: Affinity chromatography purification was performed using the PIERCE NAbTM Protein G Spin Purification Kit, and the purity was then determined by SDS-PAGE electrophoresis. The heavy and light chains of the purified 1B7 and 2A9 monoclonal antibodies were approximately 55 kDa and 25 kDa, respectively. Figure 4 ) This indicates that the antibody bands after purification by this method are correct and the purity is high.

[0092] 5. Identification of Monoclonal Antibodies

[0093] (1) Specific identification: including IFA and western blot verification.

[0094] a) IFA verification: PK15 cells were infected with TGEV. 36 hours after virus infection, the cells were fixed with paraformaldehyde. After cell fixation, the ascites was diluted with PBS (500 times) and added to the virus-infected cells. Then, FITC-anti-mouse secondary antibody (100 times dilution) was incubated and observed under a fluorescence microscope after the reaction was completed. The results are shown in Figure 2. Figure 5 As shown in A and 5B, monoclonal antibodies 1B7 and 2A9 were able to detect specific green fluorescence signals in PK15 cells infected with TGEV, while no fluorescence signals were observed in the supernatant of SP2 / 0 cells infected with TGEV ( Figure 5 C).

[0095] b) Western blot verification: Purified TGEV N protein or virus-infected and uninfected PK15 cells were collected for SDS-PAGE, and then the protein gel was transferred to NC membrane for western blot verification. The primary antibody was monoclonal antibody 1B7 or 2A9, and the secondary antibody was HRP-anti-mouse IgG. The results are shown in Figure 6 As shown, both monoclonal antibodies 1B7 and 2A9 recognized TGEV N protein in virus-infected cells.

[0096] (2) Subclass identification: The obtained monoclonal antibodies were identified according to the instructions of the SBA ClonotypingTM System / HRP Antibody Subclass Identification Kit of Southern Biotech. The results showed that Figure 7 The heavy chain constant regions of 1B7 monoclonal antibody are of IgG1 type, and those of 2A9 are of IgG2b type, and their light chain constant regions are of Kappa type.

[0097] 6. PCR amplification and sequence determination of monoclonal antibody variable region genes

[0098] First, RNA of monoclonal antibody hybridoma cells was extracted and reverse transcribed into cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0099] The antibody variable region gene was amplified by nested PCR. First, the above cDNA was used as a template to amplify the antibody variable region gene using the first round of mouse antibody IgG1 and κ light chain primers. Then, the first round product was used as a template to amplify the antibody variable region gene using the second round of mouse antibody IgG1 and κ light chain primers. The PCR reaction system was: PrimeSTAR Max Premix (2×) 25μL, P1 and P2 1μL each, cDNA 1μL, ddH 2O to 50 μL. The reaction procedure was: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. The primers for antibody variable region gene amplification refer to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.).

[0100] After amplification, 1% agarose gel electrophoresis was performed, and the heavy chain and κ light chain variable region gene sizes of monoclonal antibodies 1B7 and 2A9 were both approximately 300 bp ( Figure 8 A and 8B), gel excision to recover the target fragment, and the recovered target fragment was inserted into the pMD-19T vector for sequence determination.

[0101] The DNA sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO:5, and the DNA sequence of the light chain variable region is shown in SEQ ID NO:6; the DNA sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO:7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO:8.

[0102] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1B7 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 2A9 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0103] The heavy chain variable region of monoclonal antibody 1B7 includes CDR1 with an amino acid sequence of GFNIKDTY, CDR2 with an amino acid sequence of IDPADGYT, and CDR3 with an amino acid sequence of ARPGTLDY; the heavy chain variable region of monoclonal antibody 2A9 includes CDR1 with an amino acid sequence of GYTFTDFN, CDR2 with an amino acid sequence of INPNNGRS, and CDR3 with an amino acid sequence of ARRHWDWYFDV.

[0104] The light chain variable region of monoclonal antibody 1B7 includes CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS, and CDR3 with an amino acid sequence of QHIRELTR; the light chain variable region of monoclonal antibody 2A9 includes CDR1 with an amino acid sequence of KSLLHSN, CDR2 with an amino acid sequence of QMS, and CDR3 with an amino acid sequence of AQNLEFPWT.

[0105] Example 2 Preparation of monoclonal antibodies against PEDV N protein

[0106] 1. Construction, expression and purification of PEDV N protein recombinant plasmid

[0107] 1.1 Construction of pET32a-N recombinant plasmid

[0108] Using the N protein of PEDV CV777 strain (Genbank accession number: AF353511.1) as a reference, specific primers were designed, upstream P1: 5′-CGC GGATCC ATGGCTTCTGTCAGCTTTCA-3′, downstream P2: 5′-CCG CTCGAG TTAATTTCCTGTATCGAAGATC-3', BamHI and XhoI restriction sites (underlined parts) were introduced at the 5' ends of the upstream and downstream primers. Amplification, restriction digestion and ligation were carried out according to 1.1 in Example 1.

[0109] The results of PCR amplification of PEDV N gene are as follows Fig. 9 As shown in A, the double enzyme digestion identification results are as follows Fig. 9 As shown in B.

[0110] 1.2 Induced expression and purification of PEDV N recombinant protein

[0111] The induction, expression and purification of PEDV N recombinant protein refer to 1.2 of Example 1. Fig.10 As shown in A and 10B, a PEDV N protein with high purity was obtained.

[0112] 2. Animal Immunization

[0113] The method is the same as that of step 2 in Example 1.

[0114] 3. ELISA method to detect polyclonal antibody titer

[0115] The detection method is the same as that in Example 1, step 3.

[0116] The results showed that when the serum was diluted 1:12800, the OD of mice immunized with No. 1, 2 and 3 was 450 / NC>2.0, indicating that the antibody titer can reach 1:12800 or above ( Fig.11).

[0117] 4. Preparation of Monoclonal Antibodies

[0118] The preparation of monoclonal antibody and the purification of ascites were the same as in Example 1. After the ascites was purified, it was identified by SDS-PAGE. Fig.12 shown.

[0119] 5. Identification of Monoclonal Antibodies

[0120] After PEDV infected VeroE6 cells, the reactivity of the PEDV N protein monoclonal antibody was verified by IFA and western blot. The IFA verification results are shown in Fig.13 As shown in A and 13B, the monoclonal antibodies 1F10 and 1E1 were able to detect specific green fluorescence signals when used on PEDV-infected VeroE6 cells, while no fluorescence signals were observed when the supernatant of SP2 / 0 cells was used on PEDV-infected cells ( Fig.13 C) Western blot results Fig.14 As shown, both monoclonal antibodies 1F10 and 1E1 recognized the PEDV N protein in virus-infected cells.

[0121] The antibody subclass identification kit was used to identify the obtained monoclonal antibody. The results showed that Fig.15 As shown, the heavy chain constant regions of 1F10 and 1E1 mAbs are of IgG1 type, and their light chain constant regions are of Kappa type.

[0122] 6. PCR amplification and sequence determination of monoclonal antibody variable region genes

[0123] Referring to the method 6 in Example 1, the variable region sequences of the PEDV N protein monoclonal antibodies 1F10 and 1E1 were amplified. Fig.16 A and 16B. The recovered target fragment was inserted into the pMD-19T vector and sequenced.

[0124] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E1 of the present invention is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0125] The DNA sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO: 13, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 14; the DNA sequence of the heavy chain variable region of the monoclonal antibody 1E1 of the present invention is shown in SEQ ID NO: 15, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0126] The heavy chain variable region of monoclonal antibody 1F10 includes CDR1 with an amino acid sequence of GYTFTTYY, CDR2 with an amino acid sequence of IYPGNINT, and CDR3 with an amino acid sequence of ARISSALPY. The heavy chain variable region of monoclonal antibody 1E1 of the present invention includes CDR1 with an amino acid sequence of GYTFTAYV, CDR2 with an amino acid sequence of INPYNDRT, and CDR3 with an amino acid sequence of ARDDDDYEEGFAY.

[0127] The light chain variable region of monoclonal antibody 1F10 includes CDR1 with an amino acid sequence of RSLLNTSSQKSY, CDR2 with an amino acid sequence of FAS, and CDR3 with an amino acid sequence of QQHFSTPPT. The light chain variable region of monoclonal antibody 1E1 of the present invention includes CDR1 with an amino acid sequence of QKCQYIWLSL, CDR2 with an amino acid sequence of SCI, and CDR3 with an amino acid sequence of SAHSGAYT.

[0128] Example 3 Preparation of a latex double immunochromatographic test strip for detecting porcine transmissible gastroenteritis coronavirus

[0129] 1. Preparation of the Conjugate Pad

[0130] 1.1 Preparation of latex microsphere labeled mixed solution of TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10

[0131] Take 975 μL of 0.1M pH 5.6MES buffer and add it to a 2mL centrifuge tube, then add 25 μL of latex microspheres with a solid content of 4% (Suzhou Weidu Biotechnology Co., Ltd.) and mix gently; then add 10 μL of 20 mg / mL EDC (purchased from sigma, product number: 03449) and 5 μL of 20 mg / mL NHS (purchased from sigma, product number: 8045180025), mix thoroughly, activate at room temperature for 20 minutes, and centrifuge at 17000r for 20 minutes; remove the supernatant solution, resuspend it with 1mL MES buffer, and centrifuge it again at 17000r for 20 minutes; at the same time, add 40 μg of TGEV N protein monoclonal antibody 1B7 and 60 μg of PEDV N protein monoclonal antibody 1F10, and incubate at room temperature for 2 hours; add 200 μL of 10w / v% BSA solution for blocking, incubate at room temperature for 1 hour, and centrifuge at 17000r After 10 minutes of centrifugation, discard the supernatant; finally, resuspend with 2 mL of latex microsphere marker protective agent; if there is a precipitate that is obviously difficult to mix, sonicate at 100W for 3 seconds, stop for 3 seconds, and sonicate for a total of 1 minute, and store at 4°C for later use.

[0132] 1.2 Preparation of latex microsphere binding pad

[0133] At 37°C to 38°C, the conjugate pad was completely immersed in the conjugate pad blocking solution (5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, 1.5% m / v PVP-40 and 0.05% Krovin 300 ultrapure aqueous solution) for 2 hours, and then taken out and dried in an oven for 2 to 4 hours. The prepared latex microsphere labeled mixed solution of TGEV N protein monoclonal antibody 1B7 and PEDV N protein monoclonal antibody 1F10 was evenly sprayed on the conjugate pad using a film sprayer, placed in a 37°C oven to dry for 2 to 3 hours, and sealed for storage.

[0134] 2. Preparation of Sample Pad

[0135] At 37°C to 38°C, soak the sample pad in sample pad blocking solution (Tris-HCl solution of 2% BSA, 0.5% S17, 2% PEG200000, 0.5% sodium caseinate and 0.05% Krovin 300) for 2 hours, take it out and place it in an oven to dry for 2 to 4 hours, and then place it in an aluminum foil bag to dry for later use.

[0136] 3. Preparation of Nitrocellulose Membrane

[0137] The monoclonal antibody 1E1 (1 mg / mL) against PEDV N protein and goat anti-mouse IgG antibody (1 mg / mL) were sprayed on the nitrocellulose membrane at an interval of 3 mm, as the detection line T1 and the quality control line C, respectively; the monoclonal antibody 2A9 (1 mg / mL) against TGEV protein was sprayed 5 mm away from the monoclonal antibody 1E1 (1 mg / mL) against PEDV N protein as the detection line T2.

[0138] 4. Assembly of test strips

[0139] The sample pad, the binding pad, the nitrocellulose membrane, and the absorbent pad are sequentially connected on the PVC bottom plate to obtain a test strip, and the test strip is cut into strips according to the size of the test strip card shell, and then the cut test strips are loaded into the test strip card shell. The structural schematic diagram of the test strip is shown in FIG. Fig.17 shown.

[0140] Example 4 Application of a latex double immunochromatographic test strip for detecting porcine transmissible gastroenteritis and porcine epidemic diarrhea coronavirus

[0141] 1. How to use the test strips

[0142] (1) Detection: Take out the test strip and equilibrate it to room temperature. Add the sample to be tested to the sample adding area, let it stand at room temperature, and determine the result.

[0143] (2) Result determination: Dilute the sample to be tested with sample diluent (PBS, pH 7.4) at a ratio of 1:5. At room temperature, drop 100 μL of the sample on the sample pad and observe the result within 15 minutes. If the quality control line C and the detection line T1 are colored, but the detection line T2 is not colored, it means that PEDV is positive and TGEV is negative. If the quality control line C and the detection line T2 are colored, but the detection line T1 is not colored, it means that TGEV is positive and PEDV is negative. If the quality control line C, the detection lines T1 and T2 are all colored, it means that both PEDV and TGEV are positive. If the quality control line C is colored and the detection lines T1 and T2 are not colored, it is negative. If the quality control line C is not colored, it can be determined that the test strip is invalid. Fig.18 shown.

[0144] 2. Sensitivity of the test strip

[0145] TGEV (TCID 50 For 10 7.8 / 0.1mL) and PEDV (TCID 50 For 10 6.2 / 0.1mL) of virus solution was diluted 1:5, and then 2-fold dilution was performed to test the sensitivity of the established test strip. The results showed that when the virus was diluted 1:2 8 The test value is still positive after dilution, 1:2 9The test value after dilution was negative. Therefore, the sensitivity of the test strip prepared in this study can reach 1:2 8 , that is, the minimum TGEV and PEDV virus detection amounts of the method of the present invention are 10 4.69 TCID 50 / 0.1mL and 10 3.09 TCID 50 / 0.1mL, such as Fig.19 shown.

[0146] 3. Specificity of the test strip

[0147] When TGEV, PEDV, SADS-CoV and PDCoV were tested with test strips, it was found that when only PEDV was tested, T1 was positive, while T2 was negative; when only TGEV was tested, T2 was positive, while T1 was negative; the above showed that there was no cross reaction between the two. SADS-CoV and PDCoV did not react, indicating that the method established by the present invention can specifically identify TGEV and PEDV, and has no nonspecific cross reaction with other easily confused porcine diarrhea coronaviruses. The results are as follows Fig. 20 shown.

[0148] 4. Stability test

[0149] Stability is an important factor in field applications. The test strips were stored at room temperature (18-25°C) and 4°C, and stability tests were performed on the test strips at 0, 1, 3, 5, 7, 9, 12 and 15 months. As shown in Table 1, the test strips can be stably stored for 7 months at room temperature and for 12 months at 4°C.

[0150] Table 1 Test strip stability test results

[0151]

[0152] Note: -: negative; +, ++, +++: the degree of positivity increases in sequence.

[0153] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Rapid detection of porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus latex double immunochromatographic test strip, characterized in that: The test strip comprises a PVC bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad; a quality control line C, a detection line T1 and a detection line T2 are arranged on the nitrocellulose membrane; a monoclonal antibody A of porcine transmissible gastroenteritis virus N protein and a monoclonal antibody A of porcine epidemic diarrhea virus N protein as capture antibodies and labeled with latex microspheres are sprayed on the conjugation pad, a monoclonal antibody B of porcine epidemic diarrhea virus N protein as detection antibody is coated at the detection line T1, a monoclonal antibody B of porcine transmissible gastroenteritis virus N protein as detection antibody is coated at the detection line T2, and a goat anti-mouse IgG is coated at the quality control line C; Porcine epidemic diarrhea virus N protein monoclonal antibody A: The heavy chain variable region includes CDR1 with an amino acid sequence of GYTFTTYY, CDR2 with an amino acid sequence of IYPGNINT, and CDR3 with an amino acid sequence of ARISSALPY; the light chain variable region includes CDR1 with an amino acid sequence of RSLLNTSSQKSY, CDR2 with an amino acid sequence of FAS, and CDR3 with an amino acid sequence of QQHFSTPPT; Monoclonal antibody B against porcine epidemic diarrhea virus N protein: The heavy chain variable region includes CDR1 with the amino acid sequence of GYTFTAYV, CDR2 with the amino acid sequence of INPYNDRT, and CDR3 with the amino acid sequence of ARDDDDYEEGFAY; the light chain variable region includes CDR1 with the amino acid sequence of QKCQYIWLSL, CDR2 with the amino acid sequence of SCI, and CDR3 with the amino acid sequence of SAHSGAYT.

2. The test strip according to claim 1, characterized in that: Porcine epidemic diarrhea virus N protein monoclonal antibody A: the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; Porcine epidemic diarrhea virus N protein monoclonal antibody B: the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

3. The test strip according to claim 2, characterized in that: The gene sequences encoding the heavy chain variable region and light chain variable region of porcine epidemic diarrhea virus N protein monoclonal antibody A are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; The gene sequences encoding the heavy chain variable region and light chain variable region of porcine epidemic diarrhea virus N protein monoclonal antibody B are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

4. The test strip according to claim 1, characterized in that: The porcine transmissible gastroenteritis virus N protein monoclonal antibody is specifically as follows: Monoclonal antibody A against porcine transmissible gastroenteritis virus N protein: the heavy chain variable region includes CDR1 with an amino acid sequence of GFNIKDTY, CDR2 with an amino acid sequence of IDPADGYT, and CDR3 with an amino acid sequence of ARPGTLDY; the light chain variable region includes CDR1 with an amino acid sequence of KSVSTSGYSY, CDR2 with an amino acid sequence of LVS, and CDR3 with an amino acid sequence of QHIRELTR; Monoclonal antibody B against porcine transmissible gastroenteritis virus N protein: the heavy chain variable region includes CDR1 with the amino acid sequence of GYTFTDFN, CDR2 with the amino acid sequence of INPNNGRS, and CDR3 with the amino acid sequence of ARRHWDWYFDV; the light chain variable region includes CDR1 with the amino acid sequence of KSLLHSN, CDR2 with the amino acid sequence of QMS, and CDR3 with the amino acid sequence of AQNLEFPWT.

5. The test strip according to claim 4, characterized in that: Porcine transmissible gastroenteritis virus N protein monoclonal antibody A: the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region are shown in SEQ ID NO: 1 and SEQ ID NO: 2; Monoclonal antibody B against porcine transmissible gastroenteritis virus N protein: the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region are shown in SEQ ID NO:3 and SEQ ID NO:

4.

6. The test strip according to claim 5, characterized in that: The DNA sequences encoding the heavy chain variable region and light chain variable region of monoclonal antibody A against porcine transmissible gastroenteritis virus N protein are shown in SEQ ID NO:5 and SEQ ID NO:6; The DNA sequences encoding the heavy chain variable region and light chain variable region of monoclonal antibody B against the N protein of porcine transmissible gastroenteritis virus are shown in SEQ ID NO:7 and SEQ ID NO:

8.

7. The method for preparing the test strip according to claim 1, characterized in that: include: (1) preparing a binding pad coated with latex microspheres labeled with monoclonal antibody A against porcine transmissible gastroenteritis virus N protein and monoclonal antibody A against porcine epidemic diarrhea virus N protein; (2) Spray porcine epidemic diarrhea virus N protein monoclonal antibody B and goat anti-mouse IgG antibody on the nitrocellulose membrane at intervals, as the detection line T1 and quality control line C, respectively; spray porcine transmissible gastroenteritis virus N protein monoclonal antibody B 0.5 mm next to porcine epidemic diarrhea virus N protein monoclonal antibody B, as the detection line T2; (3) The sample pad, the binding pad, the nitrocellulose membrane and the absorbent pad are overlapped on the PVC bottom plate in sequence to obtain a latex double immunochromatographic test strip for identifying and detecting porcine transmissible gastroenteritis virus and porcine epidemic diarrhea virus.

8. The method according to claim 7, characterized in that The preparation method of the conjugate pad is as follows: The conjugate pad is blocked with a conjugate pad blocking solution; The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, centrifuged, resuspended with MES buffer, and fully dispersed with ultrasound, followed by adding monoclonal antibody A against N protein of porcine transmissible gastroenteritis coronavirus and monoclonal antibody A against N protein of porcine epidemic diarrhea virus, incubated at room temperature, and then blocked with latex microsphere marker blocking solution, incubated at room temperature, and centrifuged; the precipitate was resuspended with a latex microsphere marker protective agent to obtain a latex microsphere marker mixed solution of monoclonal antibody A against TGEV N protein and monoclonal antibody A against PEDV N protein; The prepared latex microsphere labeling mixed solution is evenly sprayed on the sealed conjugate pad.

9. The method according to claim 8, characterized in that The conjugate pad blocking solution is an ultrapure aqueous solution of 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40; The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA; The latex microsphere marker protective agent is a Tris-HCl solution containing 3% m / v sucrose, 1% m / v PVP-40, and 1% v / v Tween-20.

10. The method according to claim 8, characterized in that The sample pad was immersed in a sample pad blocking solution; the sample pad blocking solution was a Tris-HCl solution containing 2% BSA, 0.5% S17, 2% PEG20000 and 0.5% sodium caseinate.

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