A latex double immunochromatographic test strip for rapid identification and detection of SADS-CoV and TGEV and its preparation method

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a latex dual immunochromatographic test strip for rapid identification and detection of porcine acute diarrhea syndrome coronavirus (SADS-CoV) and porcine transmissible gastroenteritis coronavirus (TGEV), and its preparation method. The test strip comprises a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane is provided with a quality control line C, test lines T1, and test lines T2. The conjugation pad is sprayed with latex microspheres labeled with monoclonal antibodies 1B7 against the TGEV N protein and 6E8 against the SADS-CoV N protein. The test line T1 is coated with the anti-SADS-CoV N protein monoclonal antibody 6E8, the test line T2 is coated with the anti-TGEV N protein monoclonal antibody 2A9, and the quality control line C is coated with goat anti-mouse IgG. This test strip has the advantage of being a dual-link test strip, specifically enabling differential diagnosis of TGEV and SADS-CoV, whose clinical symptoms are easily confused.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a latex double immunochromatographic test strip for rapid identification and detection of porcine acute diarrhea syndrome coronavirus (SADS-CoV) and porcine transmissible gastroenteritis coronavirus (TGEV) and a preparation method thereof. Background Art

[0002] In the absence of an effective vaccine, it is particularly important to establish a diagnostic method that can quickly and conveniently identify and detect multiple viruses simultaneously.

[0003] Swine acute diarrhea syndrome coronavirus (SADS-CoV) and porcine transmissible gastroenteritis coronavirus (TGEV) are two enteric coronaviruses of pigs. Both present with clinical symptoms of acute diarrhea, vomiting, and neonatal death due to severe dehydration and weight loss. Commonly used methods for pathogen detection include virus isolation and identification, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), or polymerase chain reaction (PCR) and real-time quantitative PCR. However, these methods are time-consuming and complex, requiring specialized or expensive large-scale equipment and specialized technicians, making them difficult to meet the requirements of rapid, cost-effective, and efficient surveillance at the grassroots level. Compared with these methods, immunochromatographic detection technology can maintain high sensitivity and strong specificity with a strictly controlled reaction time of 10-15 minutes, significantly shortening the operation time and improving detection efficiency, making it ideal for rapid screening at the grassroots level or in the field.

[0004] Commonly used markers in immunochromatographic detection technology include colloidal gold, latex, quantum dots, and time-resolved fluorescent microspheres. Among them, colloidal gold is the most commonly used, but it is easily affected by pH and salt ions during the reaction process, so its stability is poor. Based on the carboxyl groups linked to the surface of latex microspheres, they are activated by water-soluble diimide (EDC) and can covalently bind to the amino groups of proteins or antibodies to form stable and strong chemical bonds. They are not easily affected by pH and high concentrations of salt ions during the reaction process. Therefore, the stability of latex microspheres is stronger than colloidal gold. Compared with colloidal gold, latex microspheres are filled with oil-soluble color dyes, which have high contrast, deep color, and high visualization, and are also better than colloidal gold in color development effect.

[0005] The coronavirus nucleocapsid (N) protein is well conserved and expressed in high amounts throughout the infection process, stimulating the body to produce specific antibodies. The N protein has important applications in immunological diagnosis and the development of new vaccines. Summary of the Invention

[0006] Based on the characteristics of the coronavirus nucleocapsid protein (N protein) and combining the advantages of latex microspheres, the present invention provides a latex double immunochromatographic test strip that can quickly identify and detect SADS-CoV and TGEV. The test strip is time-saving, easy to operate, can be directly observed by the naked eye without the aid of any instrument, has obvious color development, and is highly sensitive. It can be used in clinical testing of samples mixed with TGEV and SADS-CoV infection, providing new technical support for the prevention and control of TGEV and SADS-CoV. At the same time, this is also the first double latex test strip that can simultaneously detect TGEV and SADS-CoV.

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

[0008] In a first aspect, the present invention provides a latex double immunochromatographic test strip for rapid detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome. The test strip comprises a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad; the conjugation pad is sprayed with latex microspheres labeled with monoclonal antibodies 1B7 against TGEV N protein and 6E8 against SADS-CoV N protein. The nitrocellulose membrane is provided with a quality control line C, a detection line T1, and a detection line T2. Detection line T1 is coated with monoclonal antibody 6E8 against SADS-CoV N protein, detection line T2 is coated with monoclonal antibody 2A9 against TGEV N protein, and quality control line C is coated with goat anti-mouse IgG.

[0009] Among them, the heavy chain variable region of the anti-TGEV N protein 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 anti-TGEV N protein 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 anti-TGEV N protein 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 anti-TGEV N protein 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 anti-TGEV N protein 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 anti-TGEV N protein 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 of the heavy chain variable region of the anti-TGEV N protein monoclonal antibody 1B7 of the present invention 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 the anti-TGEV N protein monoclonal antibody 2A9 of the present invention is shown in SEQ ID NO: 7, and the DNA sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0013] The heavy chain constant region of the anti-TGEV N protein monoclonal antibody of the present invention is of IgG1 or IgG2b type. The light chain constant region of the anti-TGEV N protein monoclonal antibody of the present invention is of Kappa type.

[0014] In addition, the monoclonal antibody 6E8 against SADS-CoV N protein of the present invention is prepared by the patented method of the invention with publication number CN113956353A. The heavy chain variable region of the monoclonal antibody 6E8 includes a CDR1 with an amino acid sequence of GYTFTDYA, a CDR2 with an amino acid sequence of FSTYYGNA, and a CDR3 with an amino acid sequence of ARGGDYYGSSNVDYAMDY. The light chain variable region of the monoclonal antibody 6E8 includes a CDR1 with an amino acid sequence of KSVSTSGYSY, a CDR2 with an amino acid sequence of LVS, and an amino acid sequence of QHIRELTRCDR3.

[0015] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6E8 against SADS-CoV N protein 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.

[0016] 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, wherein S17 is a surfactant.

[0017] Preferably, the preparation method of the conjugate pad is:

[0018] Block the conjugate pad with conjugate pad blocking solution;

[0019] The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), centrifuged, resuspended in MES buffer, and fully dispersed by sonication. Anti-TGEV N protein monoclonal antibody 1B7 and anti-SADS-CoV N protein monoclonal antibody 6E8 were then added and incubated at room temperature. Blocking solution was then added for blocking, incubated at room temperature, and centrifuged. The precipitate was resuspended in a latex microsphere marker protective agent to obtain a latex microsphere labeled mixed solution of anti-TGEV N protein monoclonal antibody 1B7 and anti-SADS-CoV N protein monoclonal antibody 6E8.

[0020] The latex microsphere-labeled mixed solution of the monoclonal antibody 1B7 against TGEV N protein and the monoclonal antibody 6E8 against SADS-CoV N protein prepared above was evenly sprayed on the blocked conjugate pad.

[0021] The conjugate pad blocking solution is an ultrapure aqueous solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20 and 1.5% m / v PVP-40.

[0022] The latex microsphere marker blocking solution is an ultrapure aqueous solution containing 10% m / v BSA.

[0023] 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.

[0024] The latex microsphere labeled mixed solution of the monoclonal antibody 1B7 against TGEV N protein and the monoclonal antibody 6E8 against SADS-CoV N protein is composed of: each 1 ml of latex microsphere solution contains 80 μg of TGEV N protein monoclonal antibody 1B7 and 80 μg of SADS-CoV N protein monoclonal antibody 6E8.

[0025] Preferably, the test line T1 is coated with 1 mg / ml SADS-CoV N protein monoclonal antibody 6E8.

[0026] Preferably, the detection line T2 is coated with 1 mg / ml of TGEV N protein monoclonal antibody 2A9.

[0027] Preferably, the quality control line C is coated with 1 mg / ml goat anti-mouse IgG.

[0028] In a second aspect, the present invention provides a method for preparing the aforementioned latex dual immunochromatographic test strip for rapid detection of TGEV and SADS-CoV. The method comprises the following steps:

[0029] (1) preparing a sample pad and a binding pad labeled with latex microspheres coated with monoclonal antibody 1B7 against TGEV N protein and monoclonal antibody 6E8 against SADS-CoV N protein;

[0030] (2) Anti-SADS-CoV N protein monoclonal antibody 6E8 (1 mg / ml) and goat anti-mouse IgG antibody (1 mg / ml) were sprayed on the nitrocellulose membrane at a distance of 3 mm, as the detection line T1 and quality control line C, respectively; anti-TGEV protein monoclonal antibody 2A9 (1 mg / ml) was sprayed 5 mm away from anti-SADS-CoV N protein monoclonal antibody 6E8 (1 mg / ml) as the detection line T2.

[0031] (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were overlapped on the PVC base plate in sequence to obtain a latex double immunochromatographic test strip for identifying and detecting TGEV and SADS-CoV.

[0032] In a third aspect, the detection method of the test strip of the present invention comprises the following steps:

[0033] (1) Prepare samples to be tested.

[0034] (2) Detection: Take out the test strip and equilibrate it to room temperature. Add the sample to be tested to the sample addition area and let it stand at room temperature for no more than 15 minutes.

[0035] (3) Result determination: After the reaction is completed, the positive and negative properties are qualitatively determined by the naked eye.

[0036] The beneficial effects of the present invention are:

[0037] The latex double immunochromatographic test strip provided by the present invention for distinguishing and detecting TGEV and SADS-CoV has no nonspecific reaction when detecting TGEV or SADS-CoV alone, and does not cross-react with other porcine enteric coronaviruses PEDV and PDCoV with similar clinical symptoms. At the same time, the test strip can detect the virus in the positive control of TGEV and SADS-CoV at a minimum of 10 4.69 TCID 50 / 0.1mL and 10 3.39 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 two diarrhea viruses on-site or in the laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of PCR amplification of TGEV N gene are shown.

[0039] M: DL2000 relative molecular mass standard; 1: control water; 2: N gene, approximately 1000 bp in size.

[0040] Figure 2 The expression and purification results of the pET32a-N recombinant protein are shown. (A) M: Protein marker; 1: pET32a-N before induction; 2: pET32a-N after induction; 3: pET32a-N lysis supernatant; 4: pET32a-N lysis precipitate. (B) M: Protein marker; 1: Purified pET32a-N protein.

[0041] Figure 3 ELISA test of mouse antibody titers after N protein immunization. 1-3: mice immunized with No. 1, 2, and 3, NC: negative control.

[0042] Figure 4 The results of antibody purification SDS-PAGE identification are shown. M: protein marker, 1: 1B7 monoclonal antibody; 2: 2A9 monoclonal antibody.

[0043] Figure 5 Identification of monoclonal antibody reactivity for IFA. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) SP2 / 0 cell culture medium.

[0044] Figure 6 Western blot analysis of monoclonal antibody reactivity. (A) 1B7 monoclonal antibody; (B) 2A9 monoclonal antibody; (C) internal control.

[0045] Figure 7 The results of subclass identification.

[0046] Figure 8 Variable region PCR amplification results. (A) Heavy chain variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody. (B) Light chain K variable region PCR amplification results. M: DL2000 marker; 1: Water control; 2: 1B7 monoclonal antibody; 3: 2A9 monoclonal antibody.

[0047] Figure 9 This is a schematic diagram of the SADS-CoV / TGEV double latex test strip.

[0048] Figure 10 Schematic diagram of the SADS-CoV / TGEV double latex test strip result determination.

[0049] Figure 11The results of the sensitivity test of SADS-CoV / TGEV double latex test strips are shown in Table 1. 1 to 9: First, the SADS-CoV and TGEV positive controls were tested from 2 -1 Dilute to 2 -9 , 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 a test strip. 10: NC.

[0050] Figure 12 The specificity test results of the SADS-CoV / TGEV dual latex test strip are as follows: 1: simultaneous detection of SADS-CoV and TGEV positive controls; 2: detection of SADS-CoV positive controls only; 3: detection of TGEV positive controls only; 4: detection of PEDV positive antigens; 5: detection of PDCoV positive antigens; 6: NC. DETAILED DESCRIPTION

[0051] The present invention is described in more detail below through specific implementation methods to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

[0052] Example 1 Preparation of monoclonal antibodies against the N protein of porcine infectious diarrhea coronavirus

[0053] 1. Construction, expression, and purification of TGEV N protein recombinant plasmid

[0054] 1.1 Construction of pET32a-N recombinant plasmid

[0055] 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. The upstream P1: 5ʹ- CGC GGATCC ATGGCCAACCAGGGACAACG-3ʹ, downstream P2: 5ʹ- CCG CTCGA G TTAGTTCGTTACCTCATCAATTATC-3ʹ, the 5ʹ ends of the upstream and downstream primers introduced BamH I and XhoI restriction enzyme cutting site (underlined part). 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: 25 μL of PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O was added to 50 μL. The reaction procedure was: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, 30 cycles; extension at 72 °C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, and the results are shown in the figure. Figure 1 As shown, the size of the TGEV N fragment is approximately 1000 bp. Subsequently, gel recovery was performed according to the instructions of the EZNAGel Extraction Kit from Omega.

[0056] The PCR gel recovery product of TGEV N gene was used to separate the two products. BamH Ⅰ and Xho After double enzyme digestion with I, ligation and transformation into DH5α competent cells were performed. The constructed recombinant plasmid was sequenced and identified, and the positive recombinant plasmid was named pET32a-N.

[0057] 1.2 Inducible expression and purification of N recombinant protein

[0058] 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 p-value was about 0.8, IPTG was added to a final concentration of 1 mmol / L to induce expression. 5 hours after induction, the precipitate was collected by centrifugation at 12,000 rpm for 2 minutes. The precipitate was resuspended in an appropriate amount of PBS and sonicated for 5 minutes (supersonication for 3 seconds, on-off for 3 seconds). After sonication, the supernatant and precipitate were centrifuged at 4°C for 10 minutes, and the supernatant and precipitate were collected separately. The supernatant and precipitate were added to 5× loading buffer, boiled in boiling water for 10 minutes, 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 the N protein is soluble.

[0059] The pET32a-N protein was induced to express in amplified according to the above method. The supernatant was collected after sonication and purified by nickel column. The specific purification steps are as follows:

[0060] (1) Resin loading: Take an empty column and add 2 mL of nickel column NTA resin. When the preservation 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);

[0061] (2) Sample loading: When the equilibrium solution drops to the resin surface, add 3 mL of lysate containing recombinant protein, repeat the sample loading 2 to 3 times, each time for 2 minutes, collect the sample flow-through; add 5 times the column volume of equilibrium solution to rinse the column once;

[0062] (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) using 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.

[0063] (4) Column cleaning and storage: Rinse the column once with 5 volumes of 0.5 M 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 collected sample was identified by SDS-PAGE. Figure 2 As shown in B, a high-purity N protein was obtained.

[0064] 2. Animal immunization

[0065] Three 6-week-old female BALB / c mice were immunized with 30 μg of purified N recombinant protein per mouse. For the first immunization, the N protein was mixed with equal volumes of Freund's complete adjuvant and emulsified. The mice were then injected subcutaneously at multiple sites (one on the back and two on the abdomen). Booster immunizations were performed every two weeks for a total of four immunizations. Booster immunizations consisted of an equal volume of N recombinant protein mixed with incomplete Freund's adjuvant and emulsified. The immunization method was the same as for the first immunization. Seven days after the fourth immunization, tail blood was collected to determine antibody titers.

[0066] 3. ELISA method to detect polyclonal antibody titer

[0067] The TGEV virus solution and 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 h. Positive and negative sera were diluted with PBST in a series of 8 dilutions from 1:100 to 1:12800, incubated at 37°C for 1 h, 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 a microplate reader.450 .

[0068] 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 values ​​of immunized mice No. 1, 2 and 3 were 450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 1:102400.

[0069] 4. Preparation of Monoclonal Antibodies

[0070] The specific steps are as follows:

[0071] (1) Preparation of feeder layer cells: Inject HAT culture medium into the abdominal cavity of female BALB / c mice, slowly and repeatedly aspirate the liquid, and then plate it in a 96-well plate.

[0072] (2) Cell fusion: Splenocytes and an appropriate amount of SP20 cells were fused under the action of PEG, a fusion agent. The fused cells were plated in a 96-well plate containing feeder cells.

[0073] (3) Screening of positive clones:

[0074] a) Indirect ELISA detection method: the purified N protein is coated on the ELISA plate to detect the secretion of antibodies by the fused cells.

[0075] b) Indirect immunofluorescence (IFA) detection method: the supernatant from 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.

[0076] (4) Subcloning of positive hybridoma cells: Select ELISA and IFA positive wells and use the limiting dilution method to subclone the positive hybridoma cells screened. Observe under an inverted microscope, mark the wells where only a single clone grows, take the supernatant, and perform antibody detection using the above ELISA and IFA methods. Positive cells enter the next round of subcloning, and this process is repeated three times.

[0077] (5) Preparation of ascites: 10-12 W Balb / c mice were taken and 0.5 mL of Freund's incomplete adjuvant was injected intraperitoneally into each mouse. 1 W later, 5 × 10 5 After 7 to 10 days, the abdominal cavity of the mouse will be obviously bulging. The ascites is collected and its titer is detected by ELISA. The cells are then aliquoted and stored at -80℃.

[0078] (6) Purification of ascites: Affinity chromatography purification was performed using the PIERCE NAbTM Protein G Spin Purification Kit, and the purity was 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.

[0079] 5. Identification of Monoclonal Antibodies

[0080] (1) Specificity identification: including IFA and Western blot verification.

[0081] a) IFA verification: PK15 cells were infected with TGEV. 36 hours after virus infection, the cells were fixed with paraformaldehyde. After cell fixation, ascites was diluted 500-fold with PBS and added to the virus-infected cells. FITC-anti-mouse secondary antibody (100-fold dilution) was then incubated. After the reaction was complete, the cells were observed under a fluorescence microscope. The results are shown in Figure 2. Figure 5 As shown in A and 5B, monoclonal antibodies 1B7 and 2A9 can detect specific green fluorescence signals when used on TGEV-infected PK15 cells, while no fluorescence signals were observed when the supernatant of SP2 / 0 cells was used on TGEV-infected cells ( Figure 5 C).

[0082] b) Western blot verification: Purified 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 2. Figure 6 As shown in A and 6B, monoclonal antibodies 1B7 and 2A9 can both recognize the N protein in virus-infected cells. Figure 6 C is the detection of the intracellular reference β-actin.

[0083] (2) Subclass identification: The antibody subclass identification of the monoclonal antibody was performed according to the operating instructions of Southern Biotech's SBA ClonotypingTM System / HRP Antibody Subclass Identification Kit. 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.

[0084] 6. PCR amplification and sequence determination of monoclonal antibody variable region genes

[0085] First, RNA was extracted from the monoclonal antibody hybridoma cells and reverse transcribed into cDNA using Oligo-dT or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A).

[0086] The antibody variable region genes were amplified using nested PCR. First, the variable region genes were amplified using the aforementioned cDNA as a template using the first round of mouse IgG1 and κ light chain primers. Then, the first round product was used as a template for the second round of mouse IgG1 and κ light chain primers. The PCR reaction system consisted of 25 μL of PrimeSTAR Max Premix (2×), 1 μL each of P1 and P2, 1 μL of cDNA, and ddH2O to 50 μL. The reaction program was as follows: pre-denaturation at 98°C for 2 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and finally, extension at 72°C for 10 min. Primers for antibody variable region gene amplification refer to the literature (vonBoehmer, 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.).

[0087] After amplification, 1% agarose gel electrophoresis was performed, and the gene sizes of the heavy chain and κ light chain variable regions were approximately 300 bp ( Figure 8 A and 8B), gel excision was performed to recover the target fragment. The recovered target fragment was inserted into the pMD-19T vector for sequence determination.

[0088] 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.

[0089] 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.

[0090] The heavy chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of GFNIKDTY, CDR2 with the amino acid sequence of IDPADGYT, and CDR3 with the amino acid sequence of ARPGTLDY; the heavy chain variable region of monoclonal antibody 2A9 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.

[0091] The light chain variable region of monoclonal antibody 1B7 includes CDR1 with the amino acid sequence of KSVSTSGYSY, CDR2 with the amino acid sequence of LVS, and CDR3 with the amino acid sequence of QHIRELTR; the light chain variable region of monoclonal antibody 2A9 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.

[0092] Example 2 Preparation of Latex Double Immunochromatographic Test Strips for Detecting Porcine Acute Diarrhea Syndrome Coronavirus and Porcine Transmissible Gastroenteritis Coronavirus

[0093] 1. Preparation of the conjugate pad

[0094] 1.1 Preparation of the mixed solution of TGEV N protein monoclonal antibody 1B7 and SADS-CoV N protein monoclonal antibody 6E8 latex microspheres

[0095] 975 μl of 0.1M pH 5.6 MES buffer was added to a 2 mL centrifuge tube, and 25 μl of latex microspheres with a solid content of 4% (Suzhou Weidu Biotechnology Co., Ltd.) were added and gently mixed; 10 μl of 20 mg / mL EDC (purchased from Sigma, Catalog No.: 03449) and 5 μl of 20 mg / mL NHS (purchased from Sigma, Catalog No.: 8045180025) were added, mixed thoroughly, activated at room temperature for 20 min, and centrifuged at 17000 r for 20 min; the supernatant was removed, resuspended with 1 mL of MES buffer, and centrifuged again at 17000 r for 20 min; 80 μg of TGEV N protein monoclonal antibody 1B7 and 80 μg of SADS-CoV N protein monoclonal antibody 6E8 were added at the same time, and incubated at room temperature for 2 h; 200 μl of 10 w / v% Block with BSA solution, incubate at room temperature for 1 hour, centrifuge at 17000r for 10 minutes, and discard the supernatant; finally, resuspend with 2 mL of latex microsphere marker protective agent; if there is obviously a precipitate that cannot be mixed evenly, sonicate at 100W for 3 seconds, stop for 3 seconds, and sonicate for a total of 1 minute, and store at 4°C until use.

[0096] 1.2 Preparation of latex microsphere conjugate pad

[0097] Completely immerse the conjugate pad in a blocking solution (an ultrapure solution of 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) at 37°C–38°C for 2 hours. Then, remove the pad and dry it in an oven for 2–4 hours. Evenly spray the conjugate pad with a mixed solution of the prepared latex microspheres labeled with the anti-TGEV N protein monoclonal antibody 1B7 and the anti-SADS-CoV N protein monoclonal antibody 6E8 using a film sprayer. Dry the mixture in a 37°C oven for 2–3 hours and seal for storage.

[0098] Monoclonal antibody 6E8 against SADS-CoV N protein was prepared using the patented method of invention publication number CN113956353A. The amino acid sequence of the heavy chain variable region of monoclonal antibody 6E8 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.

[0099] 2. Preparation of the Sample Pad

[0100] 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) at 37℃~38℃ for 2 hours, take it out and place it in an oven to dry for 2~4 hours, and then place it in an aluminum foil bag to dry for later use.

[0101] 3. Preparation of Nitrocellulose Membrane

[0102] Anti-SADS-CoV N protein monoclonal antibody 6E8 (1 mg / ml) and goat anti-mouse IgG antibody (1 mg / ml) were sprayed on the nitrocellulose membrane at an interval of 3 mm, serving as the detection line T1 and quality control line C, respectively; anti-TGEV protein monoclonal antibody 2A9 (1 mg / ml) was sprayed 5 mm away from anti-SADS-CoV N protein monoclonal antibody 6E8 (1 mg / ml) as the detection line T2.

[0103] 4. Assembly of the test strips

[0104] The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are connected on the PVC base plate in sequence to obtain a test strip. The test strip is cut into strips according to the size of the test strip card, and then the cut test strips are loaded into the test strip card. The structural diagram of the test strip is shown in FIG. Figure 9 shown.

[0105] Example 3 Application of latex double immunochromatographic test strips for detecting SADS-CoV / TGEV

[0106] 1. How to use the test strips

[0107] (1) Detection: Take out the test strip and equilibrate it to room temperature. Dilute the sample to be tested with sample diluent 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.

[0108] (2) Result determination: If the quality control line C and the test line T1 are colored, but the test line T2 is not colored, it means that SADS-CoV is positive and TGEV is negative; if the quality control line C and the test line T2 are colored, but the test line T1 is not colored, it means that TGEV is positive and SADS-CoV is negative; if the quality control line C, the test lines T1 and T2 are all colored, it means that SADS-CoV and TGEV are both positive; if the quality control line C is colored, but the test 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. Figure 10 shown.

[0109] 2. Sensitivity of the test strip

[0110] TGEV (TCID 50 is 10 7.8 / 0.1mL) of virus solution and SADS-CoV (TCID 50 is 10 6.5 / 0.1mL)TCID 50After the virus was diluted to 1:5, the sensitivity of the established test strip was tested by diluting it to 2. 8 The test value is still positive after dilution, 1:2 9 The test value after dilution is negative. Therefore, the sensitivity of the test strip prepared by the present invention can reach 1: 2 8 , that is, the minimum virus detection amount TGEV of the method of the present invention is 10 4.69 TCID 50 / 0.1mL, SADS-CoV is 10 3.39 TCID 50 / 0.1mL, such as Figure 11 shown.

[0111] 3. Specificity of the test strip

[0112] The test strips were used to detect TGEV, SADS-CoV, porcine epidemic diarrhea coronavirus (PEDV) and porcine delta coronavirus (PDCoV). It was found that when only SADS was detected, T1 showed positive color, while T2 did not show color and was negative; similarly, when only TGEV was detected, T2 showed positive color, while T1 did not show color and was negative; the above shows that there was no cross reaction between the two. There was no reaction when detecting PEDV and PDCoV, indicating that the method established by the present invention can specifically identify TGEV and SADS-CoV, and has no nonspecific cross reaction with other easily confused porcine diarrhea coronaviruses. The results are as follows Figure 12 shown.

[0113] 4. Stability test

[0114] 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, 2, 4, 5, 6, and 7 months. As shown in Table 1, the test strips can be stably stored for 4 months at room temperature and 6 months at 4°C.

[0115] Table 1 Test strip stability test results

[0116]

[0117] Note: -: negative; +, ++, ++++: the positive degree increases in sequence.

[0118] The embodiments described above are only preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics 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. A latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome, the test strip comprising a PVC base, a sample pad, a conjugation pad, a nitrocellulose membrane, and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane, and the water-absorbing pad are sequentially overlapped on the PVC base; characterized in that: The conjugate pad is sprayed with latex microsphere-labeled monoclonal antibodies 1B7 against TGEV N protein and monoclonal antibodies against SADS-CoV N protein; a quality control line C, a detection line T1, and a detection line T2 are provided on the nitrocellulose membrane, the detection line T1 is coated with a monoclonal antibody against SADS-CoV N protein, the detection line T2 is coated with a monoclonal antibody 2A9 against TGEV N protein, and the quality control line C is coated with goat anti-mouse IgG; The heavy chain variable region of the monoclonal antibody 1B7 against TGEV N protein 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 light chain variable region of the monoclonal antibody 1B7 against TGEV N protein 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 heavy chain variable region of the monoclonal antibody 2A9 against TGEV N protein 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 of the monoclonal antibody 2A9 against TGEV N protein 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.

2. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, characterized in that: The monoclonal antibody against SADS-CoV N protein is the monoclonal antibody 6E8 against SADS-CoV N protein. The heavy chain variable region of the monoclonal antibody 6E8 against SADS-CoV N protein includes a CDR1 with an amino acid sequence of GYTFTDYA, a CDR2 with an amino acid sequence of FSTYYGNA, and a CDR3 with an amino acid sequence of ARGGDYYGSSNVDYAMDY. The light chain variable region of the monoclonal antibody 6E8 against SADS-CoV N protein 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 QHIRELTRCDR3.

3. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the anti-TGEV N protein 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 the anti-TGEV N protein 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.

4. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, characterized in that: The DNA sequence of the heavy chain variable region of the anti-TGEV N protein 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 the anti-TGEV N protein 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.

5. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6E8 against SADS-CoV N protein 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.

6. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, 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.

7. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, characterized in that: The preparation method of the conjugate pad is as follows: The conjugate pad was blocked with a conjugate pad blocking solution; the conjugate pad blocking solution was an ultrapure aqueous solution containing 5% m / v sucrose, 1.5% m / v BSA, 2% m / v Tween-20, and 1.5% m / v PVP-40; The latex microspheres were activated with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, centrifuged, resuspended with MES buffer, and fully dispersed by sonication. Subsequently, monoclonal antibodies 1B7 against TGEV N protein and monoclonal antibodies against SADS-CoV N protein were added, incubated at room temperature, and then blocked with blocking solution, incubated at room temperature, and centrifuged. The precipitate was resuspended with a latex microsphere marker protective agent to obtain a latex microsphere labeled mixed solution of monoclonal antibodies 1B7 against TGEV N protein and monoclonal antibodies against SADS-CoV N protein. The latex microsphere-labeled mixed solution of the monoclonal antibody 1B7 against TGEV N protein and the monoclonal antibody against SADS-CoV N protein prepared above was evenly sprayed on the blocked conjugate pad.

8. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 7, characterized in that: The 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.

9. The latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome according to claim 1, characterized in that: The test line T1 is coated with 1 mg / ml of monoclonal antibody against SADS-CoV N protein; The test line T2 is coated with 1 mg / ml of the monoclonal antibody 2A9 against TGEV N protein; The quality control line C was coated with 1 mg / ml goat anti-mouse IgG.

10. A method for preparing a latex double immunochromatographic test strip for rapid identification and detection of porcine transmissible gastroenteritis and porcine acute diarrhea syndrome, characterized in that: The method comprises the following steps: (1) preparing a sample pad and a conjugate pad labeled with latex microspheres coated with the monoclonal antibody 1B7 against TGEV N protein according to claim 1 and the monoclonal antibody 6E8 against SADS-CoV N protein according to claim 2; (2) 1 mg / ml of anti-SADS-CoV N protein monoclonal antibody 6E8 and 1 mg / ml of goat anti-mouse IgG antibody were sprayed on the nitrocellulose membrane at a distance of 3 mm, as the detection line T1 and the quality control line C, respectively; 1 mg / ml of the anti-TGEV N protein monoclonal antibody 2A9 according to claim 1 was sprayed 5 mm away from the anti-SADS-CoV N protein monoclonal antibody 6E8, as the detection line T2; the concentration of the anti-SADS-CoV N protein monoclonal antibody 6E8 was 1 mg / ml; (3) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were overlapped on the PVC base plate in sequence to obtain a latex double immunochromatographic test strip for identifying and detecting TGEV and SADS-CoV.

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