A fluorescent joint detection test paper card for antibodies of swine fever, blue ear and African swine fever virus and a preparation method and use method thereof

Abstract

The application discloses a fluorescent joint detection test paper card for antibodies of swine fever, blue ear and African swine fever virus and a preparation method and use method thereof. The PVC backing (9) is mounted on the card shell (10), the PVC backing (9) is paved with a nitrocellulose membrane (7), the nitrocellulose membrane (9) is sequentially marked with a third detection line (5) coated with swine fever virus E2a protein, a second detection line (4) coated with blue ear virus N1 protein, a first detection line (3) coated with African swine fever virus P30 protein and a quality control line (6) of an anti-his tag protein antibody. The application overcomes the shortcomings of high signal amplification cost, low sensitivity and single color of colloidal gold in the prior art, has the advantages of using time-resolved immunofluorescence microspheres as tracers, one-time simultaneous labeling of three kinds of pig disease antigens, and significantly improved detection sensitivity and detection efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal immunization, more particularly to a fluorescent co-detection test paper card for antibodies of classical swine fever, porcine blue ear and African swine fever virus and a preparation method and use method thereof. BACKGROUND

[0002] Classical swine fever, porcine blue ear disease and African swine fever are all highly contagious diseases caused by viruses. Classical swine fever is a highly contagious pig disease caused by classical swine fever virus (CSFV). It mainly infects domestic pigs and wild boars and can cause a highly infectious pig disease with high mortality. It is an RNA virus with very small virus particles and high variability, which is divided into seven different subtypes. Porcine reproductive and respiratory syndrome (PRRSV) is a highly contagious pig disease that usually manifests as respiratory disease and reproductive disorder. Due to the numerous subtypes of PRSV and the high variability of the virus, blue ear disease remains one of the most serious viral diseases in the pork industry worldwide. African swine fever is an acute, hemorrhagic and highly contagious disease caused by African swine fever virus (ASFV) infection in domestic pigs and various wild boars. All breeds and ages of pigs can be infected, and the morbidity and mortality can be as high as 100%. The World Organization for Animal Health has listed it as a legal reportable animal disease, and China has also listed it as a class A animal disease. These three common pig infectious diseases seriously affect the development and stability of the pork industry. The existence of these viruses directly threatens the health and production of pigs, and even causes a large number of pigs to die, causing significant economic losses to the livestock industry and the society and economy. In addition, there is no effective specific treatment method for these viruses, and prevention and control are currently the main means for these diseases. At present, China adopts a purification strategy combining compulsory immunization and epidemic monitoring for classical swine fever and porcine blue ear, and vaccine immunization becomes the key to the prevention and control of the epidemic, so it is of great significance to establish a suitable serological antibody evaluation method for the vaccine. Although there is no effective vaccine for African swine fever worldwide, the serological antibody detection is conducive to the diagnosis of suspected cases and hidden cases after the local prevalence of African swine fever.

[0003] Currently, the main detection method for serological detection is enzyme-linked immunosorbent assay (ELISA). ELISA has high sensitivity and can detect multiple samples in parallel, but the method takes a long time, indirect ELISA takes about 2h, and blocking ELISA takes 2-3h, which is not convenient for real-time and rapid diagnosis, so it cannot be widely used in primary level. Therefore, the development of rapid detection methods for viral antibodies is of great significance for clinical diagnosis and immune monitoring of swine diseases. The most common form of rapid detection technology for disease antibodies is the immunochromatography test strip technology, which combines the characteristics of chromatography and immunology, is simple, fast, and the results can be directly observed. The current immunochromatography rapid detection test strip uses colloidal gold, colored latex microspheres or fluorescent as a marker. The rapid detection product based on colloidal gold labeling technology has low sensitivity and large batch-to-batch variability, and is often used for qualitative analysis; although the sensitivity and batch-to-batch variability of colored latex microspheres have improved, they are often used for qualitative analysis or semi-quantitative analysis; the sensitivity of immunochromatography based on fluorescent labeling technology has been greatly improved, and it can also be used for quantitative detection, but due to the high background fluorescence signal in the sample and the small Stock displacement, it will have a great impact on detection. Time-resolved fluorescence is a marker with unique fluorescence characteristics of lanthanide rare earth ions, which has a large Stock displacement and a long fluorescence lifetime compared to ordinary fluorescence, which can effectively avoid the influence of background fluorescence in the sample and excitation light and other stray light, so it has higher sensitivity and anti-interference ability than ordinary fluorescence. Time-resolved fluorescence immunochromatography combines time-resolved fluorescence latex microspheres labeling and immunochromatography technology, which can be used for multi-label immunoassay, and combined with instruments, it can develop rapid immunochromatography test strips that can accurately quantify. It has been widely used in immunology, clinical diagnosis, cytology and other biological and medical fields.

[0004] Based on the multi-union rapid detection technology of various swine diseases, Chinese invention patent 201610544386.1 discloses a multi-union rapid detection test strip for simultaneously detecting swine fever, blue ear disease and O-type foot-and-mouth disease virus antibodies using staphylococcal A protein as a capture antigen.

[0005] Chinese utility model patent 201220258469.1 discloses a multi-union rapid detection test strip for simultaneously detecting O-type foot-and-mouth disease virus, swine fever virus and porcine blue ear virus antigen using double antibody sandwich method. The above-mentioned patents all use colloidal gold as a marker tracer for simple qualitative detection, and the detection sensitivity of colloidal gold is low, the detection color is single, which makes the result judgment of different detection items not intuitive, and cannot be quantitatively analyzed. SUMMARY

[0006] The first object of the present application is to overcome the deficiencies of the prior art and provide a fluorescent microsphere marker-based fluorescent joint detection test paper card for antibodies of hog cholera, porcine blue ear and African swine fever virus, which uses time-resolved immunofluorescent microspheres as tracers and simultaneously labels the antigens of the three pig diseases, thereby improving the detection sensitivity and enabling quantitative analysis of the three items at the same time.

[0007] The first object of the present application is achieved by the following technical solution: a fluorescent joint detection test paper card for antibodies of hog cholera, porcine blue ear and African swine fever virus, which comprises a sample pad, a time-resolved immunofluorescent microsphere-labeled combined pad of antigens of the three pig diseases, a first detection line coated with P30 protein of African swine fever virus, a second detection line coated with N1 protein of porcine blue ear disease virus, a third detection line coated with E2a protein of hog cholera virus, a quality control line of anti-his tag protein antibody, a nitrocellulose membrane, a water absorption pad, a PVC backing and a card shell,

[0008] The PVC backing is installed on the card shell, the PVC backing is laid with a nitrocellulose membrane, and the nitrocellulose membrane is sequentially marked with a third detection line coated with E2a protein of hog cholera virus, a second detection line coated with N1 protein of porcine blue ear disease virus, a first detection line coated with P30 protein of African swine fever virus and a quality control line of anti-his tag protein antibody, and one end of the PVC backing is sequentially installed with a sample pad and a time-resolved immunofluorescent microsphere-labeled combined pad of antigens of the three pig diseases, and the other end is installed with a water absorption pad.

[0009] The second object of the present application is to overcome the deficiencies of the prior art and provide a preparation method of a fluorescent joint detection test paper card for antibodies of hog cholera, porcine blue ear and African swine fever virus.

[0010] The second object of the present application is achieved by the following technical solution: a preparation method of a fluorescent joint detection test paper card for antibodies of hog cholera, porcine blue ear and African swine fever virus, which comprises the following steps:

[0011] ① The preparation method of the time-resolved immunofluorescent microsphere-labeled combined pad of antigens of the three pig diseases is as follows:

[0012] Take 1 ml of activated time-resolved immunofluorescence microspheres suspension, add African swine fever P30 protein + porcine blue ear virus N2 protein + porcine swine fever virus E2 protein mixed antigen, and mark the amount as 50-100 ug, 50-100 ug and 50-100 ug respectively, mix well at room temperature for 2 hours; add 10% BSA for blocking for 1 hour, centrifuge to remove the supernatant, take 1 ml of the preservation solution and add it to the centrifuged microspheres, mix well, store in cold storage and avoid light, and wait for use. The prepared time-resolved immunofluorescence microsphere labeled mixed antigen is diluted 5-10 times with working solution, and is uniformly sprayed on the conjugate pad by gold spraying instrument, the spraying amount is 3.0-10.0 μl / cm, and is placed in a 37°C incubator for drying for 12-18 hours to prepare a time-resolved immunofluorescence microsphere labeled three-pig-disease-antigen conjugate pad, which is ready for use,

[0013] ②, the activation method of the time-resolved immunofluorescence microspheres is as follows: take out the time-resolved immunofluorescence microspheres, disperse them in an ultrasonic cleaner for 10 seconds, add 900 ul of 0.03 mM MES buffer in a 2 mL EP tube, then add 100 ul of the above dispersed microspheres, vortex to mix, add 5 ul of A liquid, which is 10 mg / ml N-hydroxysuccinimide ethanol solution, vortex to mix, then add 5 ul of B liquid, which is 10 mg / ml N-hydroxysuccinimide ethanol solution, vortex to mix again, and react at room temperature for 30 minutes, centrifuge to remove the supernatant, add 2 ml of cross-linking buffer to resuspend and wash the precipitate, centrifuge to remove the supernatant, add 1 ml of cross-linking buffer to the precipitate, and then ultrasonically disperse it evenly to obtain the activated time-resolved immunofluorescence microspheres,

[0014] ③, the preparation method of the first detection line coated with African swine fever virus P30 protein includes: using a continuous dot membrane instrument to uniformly draw 0.05-1 mg / mL African swine fever virus P30 protein onto a nitrocellulose membrane, the amount of drawing is 1 μL / cm, and the membrane is dried in a 37°C incubator for 12-18 hours for standby,

[0015] ④, the preparation method of the second detection line coated with porcine blue ear virus N1 protein includes: using a continuous dot membrane instrument to uniformly draw 0.2-1.5 mg / mL porcine blue ear virus N1 protein onto a nitrocellulose membrane, the amount of drawing is 1 μL / cm, and the membrane is dried in a 37°C incubator for 12-18 hours for standby,

[0016] ⑤, the preparation method of the third detection line coated with porcine swine fever virus E2a protein includes: using a continuous dot membrane instrument to uniformly draw 0.2-1.5 mg / mL porcine swine fever virus E2a protein onto a nitrocellulose membrane, the amount of drawing is 1 μL / cm, and the membrane is dried in a 37°C incubator for 12-18 hours for standby,

[0017] The preparation method of the quality control line of the anti-his tag protein antibody comprises the following steps: uniformly drawing 0.2-0.8 mg / mL anti-his tag protein antibody on a nitrocellulose membrane by using a continuous dot film instrument, the drawing amount is 1 muL / cm, and the membrane is dried in a 37 DEG C incubator for 12-18 hours, and the membrane is ready for use,

[0018]

[0019]

[0020] The third purpose of the present application is to overcome the shortcomings of the background art, and to provide a use method of the porcine fever, porcine blue ear and African swine fever virus antibody fluorescence joint detection test paper card.

[0021] The third purpose of the present application is achieved by the following technical scheme: a use method of the porcine fever, porcine blue ear and African swine fever virus antibody fluorescence joint detection test paper card, which comprises the following steps:

[0022] When the sample to be detected contains porcine fever virus antibody, porcine blue ear virus antibody or African swine fever virus antibody, the African swine fever virus antibody, porcine blue ear virus antibody or porcine fever virus antibody in the sample moves to the position of the binding pad of the time-resolved immunofluorescence microsphere-labeled three pig disease antigens and reacts with the time-resolved immunofluorescence microsphere-labeled P30 antigen, N2 antigen or E2 antigen,

[0023] When the antigen-antibody complex is chromatographed to the detection line position, the P30 / N1 / E2a antigens on the first detection line coated with African swine fever virus P30 protein, the second detection line coated with porcine blue ear virus N1 protein and the third detection line coated with porcine fever virus E2a protein react with the antibodies in the antigen-antibody complex again, so as to form a “fluorescence-labeled antigen-antibody-coated antigen” double-antigen sandwich complex at the detection line position, and the first detection line coated with African swine fever virus P30 protein, the second detection line coated with porcine blue ear virus N1 protein and the third detection line coated with porcine fever virus E2a protein respectively present corresponding fluorescence bands,

[0024] ​​③ Simultaneously, excess labeled antigens in the binding pads of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres will react with the anti-His-tagged protein antibody in the control line region, forming a fluorescent band at the control line position of the anti-His-tagged protein antibody.

[0025] ④ The higher the antibody content in the sample, the stronger the fluorescence signal in the detection line area of ​​the test reaction zone on the nitrocellulose membrane, and vice versa.

[0026] ⑤ The change in fluorescence intensity of the detection line is positively correlated with the content of the antibody to be tested. After the fluorescence signal of the reaction band is automatically identified and interpreted by the immunofluorescence rapid assay instrument, the content level of the analyte can be analyzed, thereby realizing quantitative detection and analysis.

[0027] In the above technical solution, the preparation method of African swine fever virus P30 recombinant antigen is as follows:

[0028] Recombinant strain Escherichia coli BL21 / pET-P30 was induced with IPTG at a final concentration of 0.6 mM. The expression product was purified to obtain the African swine fever virus P30 recombinant antigen.

[0029] The preparation method of recombinant strain Escherichia coli BL21 / pET-P30 includes:

[0030] The genomic sequence of the African swine fever virus P30 antigen protein was searched in the NCBI database. GenBank: JX524222.1 was selected as the target gene, and the target gene was selected and optimized through analysis.

[0031] The optimized P30 protein gene sequence was synthesized by a gene synthesis company and ligated into the pET-21a(+) vector to construct the recombinant plasmid pET-21a-P30.

[0032] The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-P30.

[0033] In the above technical solution, the preparation methods of porcine reproductive and respiratory syndrome virus (PRRSV) N1 and N2 recombinant antigens are as follows: recombinant strains Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2 are induced with IPTG at final concentrations of 0.2mM / 0.8mM, respectively. After purification, the expression products are used to obtain porcine reproductive and respiratory syndrome virus (PRRSV) N1 recombinant antigen and porcine reproductive and respiratory syndrome virus (PRRSV) N2 recombinant antigen, respectively.

[0034] In the above technical solution, the preparation method of recombinant strain Escherichia coli BL21 / pET-N1 includes:

[0035] The genome sequence of the classical swine fever virus N antigen protein is searched in the NCBI database, partial sequence of GenBank: EU926974.1 is selected as a target gene, the target gene is selected, analyzed and optimized, the optimized N1 protein gene is synthesized by a gene synthesis company, the N1 protein gene is connected with a pET-28a(+) vector, a recombinant plasmid pET-28a-N1 is constructed, and the recombinant plasmid is transformed into competent cells of escherichia coli BL21 to obtain a recombinant strain escherichia coli BL21 / pET-N1.

[0036] In the technical scheme, the preparation method of the recombinant strain escherichia coli BL21 / pMAL-N2 includes the following steps: searching the genome sequence of the classical swine fever virus N antigen protein in the NCBI database, selecting partial sequence of GenBank: EU926974.1 as a target gene, and optimizing the target gene.

[0037] The optimized N2 protein gene is synthesized by a gene synthesis company, the N2 protein gene is connected with a pMAL-c5x vector, a recombinant plasmid pMAL-c5x-N2 is constructed, and the recombinant plasmid is transformed into competent cells of escherichia coli BL21 to obtain the recombinant strain escherichia coli BL21 / pMAL-N2.

[0038] In the technical scheme, the preparation method of the classical swine fever virus E2 and E2a recombinant antigen includes the following steps: using IPTG with a final concentration of 0.1 mM to induce the recombinant strain escherichia coli BL21 / pE2 and escherichia coli BL21 / pE2a respectively, and obtaining the classical swine fever virus E2 recombinant antigen and the classical swine fever virus E2a recombinant antigen respectively after the expression products are purified.

[0039] In the technical scheme, the preparation method of the recombinant strain escherichia coli BL21 / pE2 includes the following steps:

[0040] The genome sequence of the classical swine fever virus E2 antigen protein is searched in the NCBI database, GenBank: FJ598611.1 is selected as a target gene, the target gene is analyzed and optimized,

[0041] The optimized E2 protein gene is synthesized by a gene synthesis company, the E2 protein gene is connected with a pET-28a(+) vector, a recombinant plasmid pET-28a-E2 is constructed, and the recombinant plasmid is transformed into competent cells of escherichia coli BL21 to obtain the recombinant strain escherichia coli BL21 / pE2.

[0042] In the technical scheme, the preparation method of the recombinant strain escherichia coli BL21 / pE2a includes the following steps:

[0043] The E2 antigen protein genome sequence of the swine fever virus is searched in the NCBI database, part of the GenBank: FJ598611.1 gene is selected as the E2a target gene, the target gene is selected, analyzed and optimized,

[0044] The optimized E2a protein gene is synthesized by a gene synthesis company, is connected with the pET-28a(+) vector, the recombinant plasmid pET-28a-E2a is constructed, and the recombinant plasmid is transformed into the competent cell of the E.coli BL21, so that the recombinant strain E.coli BL21 / pE2a is obtained.

[0045] Compared with the prior art, the application has the following outstanding advantages:

[0046] In the conventional detection, the same antigen is used as the labeled antigen and the coated antigen, for example, the detection of the African swine fever virus antibody in the application adopts this mode,

[0047] 1. However, the detection of the porcine pestivirus antibody and the swine fever antibody in the application adopts two antigens with different molecular weights but the same antigen sites as the labeled antigen and the coated antigen,

[0048] The N2 protein and the N1 protein are used as the latex microsphere labeled protein and the detection line coated protein respectively for detecting the porcine blue ear virus antibody, and the E2 protein and the E2a protein are used as the latex microsphere labeled protein and the detection line coated protein respectively for detecting the swine fever virus antibody;

[0049] The larger N2 protein (with a molecular weight of 57.2KD) / E2 protein (with a molecular weight of 33.3KD) has more spatial binding sites and is easy to combine with the detected porcine blue ear virus antibody / swine fever virus antibody; the N1 protein (with a molecular weight of 17.9KD) / E2a protein (with a molecular weight of 26KD) is smaller, is the immune specificity protein of the porcine blue ear virus / swine fever virus, can be specifically combined with the latex microsphere labeled porcine blue ear virus / swine fever virus antigen antibody complex chromatographed to the detection line position, improves the sensitivity of the antibody detection, provides a scientific and effective technical support for preventing and controlling the occurrence of the porcine blue ear / swine fever, and also provides a reference basis for the rapid and sensitive detection of other disease antibodies.

[0050] 2、The test paper card in the application uses time-resolved immunofluorescence microspheres as tracers, labels three kinds of pig disease antigens at one time, simplifies the preparation process of the test paper card, saves the preparation cost, and significantly improves the detection sensitivity and detection efficiency. Meanwhile, the chromatographic test paper card is used as a primary sensor, that is, the fluorescence microsphere-labeled antigen is first combined with the antibody to be detected to form a fluorescence-labeled antigen-antibody complex, and then combined with the fixed capture antigen on the test paper strip to make the test strip produce a fluorescence signal change. The intensity change of the fluorescence signal is positively correlated with the content of the antibody to be detected. After automatic identification and interpretation of the fluorescence signal value of the reaction strip by a computer, the content level of the test substance is analyzed, and quantitative detection and analysis of the three kinds of pig disease antibodies are realized.

[0051] 3、The application combines the three kinds of pig disease antibody fluorescence joint detection reagent card with the immunofluorescence rapid detector to form a pig fever, blue ear and African swine fever virus antibody fluorescence joint detection test paper card, and real-time, rapid and quantitative monitoring of the three kinds of pig disease antibodies is realized. The application can reduce the error rate caused by human visual determination when using colloidal gold and other qualitative test paper cards for detection and determination in the past, improve the timeliness of detection, and improve the objectivity and accuracy of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0052] Fig. 1 is a schematic diagram of the assembly of the test paper strip of the application.

[0053] In the figure: sample pad 1, time-resolved immunofluorescence microsphere-labeled three kinds of pig disease antigen combination pad 2, first detection line 3 coated with African swine fever virus P30 protein, second detection line 4 coated with porcine blue ear disease virus N1 protein, third detection line 5 coated with porcine fever virus E2a protein, quality control line 6 of anti-his tag protein antibody, nitrocellulose membrane 7, water absorption pad 8, PVC backing 9, card shell 10.

[0054] Fig. 2 is a schematic diagram of the test result determination of the test card of the application (under ultraviolet lamp irradiation).

[0055] In the figure: 11 represents that the detection of ASFV antibody, PRRSV antibody and CSFV antibody is all positive results; 12 represents that the detection of ASFV antibody is positive, and the detection of PRRSV antibody and CSFV antibody is both negative results; 13 represents that the detection of PRRSV antibody is positive, and the detection of ASFV antibody and CSFV antibody is both negative results; 14 represents that the detection of CSFV antibody is positive, and the detection of ASFV antibody and PRRSV antibody is both negative results; 15 represents that the detection of ASFV antibody, PRRSV antibody and CSFV antibody is all negative results; 16 and 17 represent that the test card is invalid.

[0056] Fig. 3 is an immunofluorescence rapid detector. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present application will be described clearly and completely below. All other embodiments obtained by those skilled in the art without creative labor on the basis of the embodiments in the present application also belong to the protection scope of the present application.

[0058] Referring to FIGS. 1-3, a hog fever, blue ear and African swine fever virus antibody fluorescence joint test paper card includes a sample pad 1, a time-resolved immunofluorescence microsphere labeled three hog disease antigen combination pad 2, a first detection line 3 coated with African swine fever virus P30 protein, a second detection line 4 coated with blue ear disease virus N1 protein, a third detection line 5 coated with hog fever virus E2a protein, a quality control line 6 of anti-his tag protein antibody, a nitrocellulose membrane 7, a water absorption pad 8, a PVC backing 9 and a card shell 10.

[0059] The PVC backing 9 is installed on the card shell 10, the PVC backing 9 is laid with the nitrocellulose membrane 7, the nitrocellulose membrane 9 is sequentially marked with the third detection line 5 coated with the hog fever virus E2a protein, the second detection line 4 coated with the blue ear disease virus N1 protein, the first detection line 3 coated with the African swine fever virus P30 protein and the quality control line 6 of the anti-his tag protein antibody, one end of the PVC backing 9 is sequentially installed with the sample pad 1 and the time-resolved immunofluorescence microsphere labeled three hog disease antigen combination pad 2, and the other end is installed with the water absorption pad 8.

[0060] The present application also includes a preparation method of a test paper card: a preparation method of a hog fever, blue ear and African swine fever virus antibody fluorescence joint test paper card, which includes the following steps:

[0061] ①, the preparation method of the time-resolved immunofluorescence microsphere labeled three hog disease antigen combination pad 2 is as follows: take 1ml activated time-resolved immunofluorescence microsphere suspension, add three hog disease mixed antigens of African swine fever P30 protein+blue ear virus N2 protein+hog fever virus E2 protein dropwise while stirring, and the labeling amount is 50-100ug, 50-100ug and 50-100ug respectively, mix well at room temperature for 2 hours; add 10% BSA for blocking for 1 hour, centrifuge to remove the supernatant, take 1ml storage solution and add it to the centrifuged microspheres, mix well, store in cold storage and avoid light, and wait for use. After the prepared time-resolved immunofluorescence microsphere labeled mixed antigen is diluted 5-10 times with working solution, it is uniformly sprayed on the combination pad by using a gold spraying instrument, the spraying amount is 3.0-10.0ul / cm, and it is placed in a 37℃ incubator for drying for 12-18h to prepare the time-resolved immunofluorescence microsphere labeled three hog disease antigen combination pad 2 for standby,

[0062] ②、Time-Resolved Fluorescence Microspheres Activation Method: Take out the time-resolved immunofluorescence microspheres, disperse them in an ultrasonic cleaner for 10 seconds, add 900 ul of 0.03M MES buffer solution into a 2ml EP tube, then add 100ul of the above dispersed microspheres, vortex to mix, add 5ul of A liquid into the microsphere suspension, the A liquid is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex to mix, then add 5ul of B liquid, the B liquid is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex to mix again, react at room temperature for 30 minutes, centrifuge to remove the supernatant, add 2ml of cross-linking buffer to resuspend and wash the precipitate, centrifuge to remove the supernatant, add 1ml of cross-linking buffer to the precipitate, then ultrasonically disperse it evenly, which is the activated time-resolved immunofluorescence microspheres,

[0063] ③、The first detection line 3 coated with African swine fever virus P30 protein preparation method includes: using a continuous dot membrane instrument to uniformly draw 0.05-1mg / mL African swine fever virus P30 protein onto nitrocellulose membrane 9 at a draw amount of 1ul / cm, drying at 37℃ for 12-18h, standby,

[0064] ④、The second detection line 4 coated with porcine blue ear disease virus N1 protein preparation method includes: using a continuous dot membrane instrument to uniformly draw 0.2-1.5mg / mL porcine blue ear disease virus N1 protein onto nitrocellulose membrane 9 at a draw amount of 1ul / cm, drying at 37℃ for 12-18h, standby,

[0065] ⑤、The third detection line 5 coated with porcine swine fever virus E2a protein preparation method includes: using a continuous dot membrane instrument to uniformly draw 0.2-1.5mg / mL porcine swine fever virus E2a protein onto nitrocellulose membrane 9 at a draw amount of 1ul / cm, drying at 37℃ for 12-18h, standby,

[0066] ⑥、The quality control line 6 of anti-his tag protein antibody preparation method includes: using a continuous dot membrane instrument to uniformly draw 0.2-0.8mg / mL anti-his tag protein antibody onto nitrocellulose membrane 9 at a draw amount of 1ul / cm, drying at 37℃ for 12-18h, standby,

[0067] ⑦、The sample pad 1 is soaked in blocking solution for 30min and dried at 37℃, standby,

[0068] 8、coated with the third detection line 5 of the porcine epidemic diarrhea virus E2a protein, 5mm from the binding pad 2 of the three porcine disease antigen labeled with time-resolved immunofluorescence microspheres, 4mm from the end of the second detection line 4 coated with the porcine epidemic diarrhea virus N1 protein, 4mm from the end of the first detection line 3 coated with the African swine fever virus P30 protein, 3mm from the end of the quality control line 6 of the anti-his tag protein antibody, and 3mm from the end of the water absorption pad 8.

[0069] The application also includes a method for using the test paper card: a method for using a porcine epidemic diarrhea virus, porcine epidemic blue ear disease virus and African swine fever virus antibody fluorescence combined test paper card, which includes the following steps:

[0070] ①、when the sample to be tested contains porcine epidemic diarrhea virus antibodies / or porcine epidemic blue ear disease virus antibodies / or African swine fever virus antibodies, the African swine fever virus antibodies / or porcine epidemic blue ear disease virus antibodies / or porcine epidemic diarrhea virus antibodies in the sample move to the position of the binding pad 2 of the three porcine disease antigens labeled with time-resolved immunofluorescence microspheres and react with the time-resolved immunofluorescence microspheres labeled P30 antigen / or N2 antigen / or E2 antigen,

[0071] ②、when the antigen-antibody complex is chromatographed to the detection line position, the P30 / N1 / E2a antigen on the first detection line 3 coated with the African swine fever virus P30 protein, the second detection line 4 coated with the porcine epidemic blue ear disease virus N1 protein and the third detection line 5 coated with the porcine epidemic diarrhea virus E2a protein reacts again with the antibodies in the antigen-antibody complex, thereby forming a “fluorescence-labeled antigen-antibody-coated antigen” double-antigen sandwich complex at the detection line position, and the first detection line 3 coated with the African swine fever virus P30 protein, the second detection line 4 coated with the porcine epidemic blue ear disease virus N1 protein and the third detection line 5 coated with the porcine epidemic diarrhea virus E2a protein respectively present corresponding fluorescence bands,

[0072] ③、at the same time, the excess labeled antigen in the binding pad 2 of the three porcine disease antigens labeled with time-resolved immunofluorescence microspheres reacts with the anti-his tag protein antibody in the quality control line 6 region, and a fluorescence band is formed at the position of the anti-his tag protein antibody quality control line 6,

[0073] ④、the higher the content of the antibody in the sample, the stronger the fluorescence signal in the detection line region of the test reaction area on the nitrocellulose membrane 9, and vice versa,

[0074] 5. The fluorescence intensity of the detection line is positively correlated with the content of the antibody to be detected, and the content level of the to-be-detected substance is analyzed after the automatic recognition and interpretation of the fluorescence signal of the reaction strip by the immunofluorescence rapid detector, so that quantitative detection and analysis are realized.

[0075] The preparation method of the P30 recombinant antigen of African swine fever virus is as follows: the recombinant strain Escherichia coli BL21 / pET-P30 is induced by 0.6 mM IPTG, and the expression product is purified to obtain the P30 recombinant antigen of African swine fever virus,

[0076] The preparation method of the recombinant strain Escherichia coli BL21 / pET-P30 comprises the following steps:

[0077] The P30 antigen protein genome sequence of African swine fever virus is searched in the NCBI database, GenBank:JX524222.1 is selected as the target gene, the target gene is selected, analyzed and optimized,

[0078] The optimized P30 protein gene sequence is synthesized by a gene synthesis company, and is connected with a pET-21a(+) vector to construct a recombinant plasmid pET-21a-P30.

[0079] The recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-P30.

[0080] The preparation method of the N1 and N2 recombinant antigens of porcine blue ear virus is as follows: the recombinant strains Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2 are induced by 0.2 mM / 0.8 mM IPTG respectively, and the expression product is purified to obtain the N1 recombinant antigen of porcine blue ear virus and the N2 recombinant antigen of porcine blue ear virus respectively.

[0081] The preparation method of the recombinant strain Escherichia coli BL21 / pET-N1 comprises the following steps:

[0082] The N antigen protein genome sequence of porcine epidemic diarrhea virus is searched in the NCBI database, GenBank:EU926974.1 is selected as the target gene, the target gene is selected, analyzed and optimized, the optimized N1 protein gene is synthesized by a gene synthesis company, and is connected with a pET-28a(+) vector to construct a recombinant plasmid pET-28a-N1, and the recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-N1.

[0083] The preparation method of the recombinant strain Escherichia coli BL21 / pMAL-N2 comprises the following steps: searching for a classical swine fever virus N antigen protein genome sequence in an NCBI database, selecting a partial sequence of GenBank: EU926974.1 as a target gene, and performing selection analysis and optimization on the target gene.

[0084] The optimized N2 protein gene is synthesized by a gene synthesis company, connected with a pMAL-c5x vector to construct a recombinant plasmid pMAL-c5x-N2, and the recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pMAL-N2.

[0085] The preparation method of the classical swine fever virus E2 and E2a recombinant antigens is as follows: the recombinant strain Escherichia coli BL21 / pE2 and Escherichia coli BL21 / pE2a are respectively induced by using an IPTG with a final concentration of 0.1 mM, and the expression products are respectively obtained after purification to obtain the classical swine fever virus E2 recombinant antigen and the classical swine fever virus E2a recombinant antigen.

[0086] The preparation method of the recombinant strain Escherichia coli BL21 / pE2 comprises the following steps:

[0087] A classical swine fever virus E2 antigen protein genome sequence is searched for in an NCBI database, a partial gene of GenBank: FJ598611.1 is selected as an E2a target gene, and selection analysis and optimization are performed on the target gene,

[0088] The optimized E2 protein gene is synthesized by a gene synthesis company, connected with a pET-28a(+) vector to construct a recombinant plasmid pET-28a-E2, and the recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2.

[0089] The preparation method of the recombinant strain Escherichia coli BL21 / pE2a comprises the following steps:

[0090] A classical swine fever virus E2 antigen protein genome sequence is searched for in an NCBI database, a partial gene of GenBank: FJ598611.1 is selected as an E2a target gene, and selection analysis and optimization are performed on the target gene,

[0091] The optimized E2a protein gene is synthesized by a gene synthesis company, connected with a pET-28a(+) vector to construct a recombinant plasmid pET-28a-E2a, and the recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2a.

[0092] Example one

[0093] The application provides a fluorescent microsphere marking technology-based antibody fluorescent joint detection test paper card for hog cholera, blue ear and African swine fever virus, a PVC backing 9 is installed on a card shell 10, a nitrocellulose membrane 7 is laid on the PVC backing 9, a third detection line 5 coated with hog cholera virus E2a protein, a second detection line 4 coated with blue ear virus N1 protein, a first detection line 3 coated with African swine fever virus P30 protein and a quality control line 6 of an anti-his tag protein antibody are sequentially drawn on the nitrocellulose membrane 9, a sample pad 1 and a time-resolved immunofluorescence microsphere marking combined pad 2 of three pig disease antigens are sequentially installed at one end of the PVC backing 9, and a water absorption pad 8 is installed at the other end.

[0094] The preparation method of the African swine fever virus P30 recombinant antigen is as follows: a recombinant strain Escherichia coli BL21 / pET-P30 (BL21 / pET-P30) is induced by using 0.6 mM IPTG with a final concentration, and the expression product is purified to obtain the African swine fever virus P30 recombinant antigen. Escherichia coli BL21 / pET-P30.

[0095] Specifically, the preparation method of the recombinant strain Escherichia coli BL21 / pET-P30 includes the following steps.

[0096] The African swine fever virus P30 antigen protein genome sequence is searched in an NCBI database, GenBank:JX524222.1 (African swine fever virus strain ZAM 2001 6 phosphoprotein p30 (CP204L) gene, complete cds) is selected as a target gene, and the target gene is selected, analyzed and optimized.

[0097] The optimized P30 protein gene sequence is synthesized by a gene synthesis company, is connected with a pET-21a(+) vector, and a recombinant plasmid pET-21a-P30 is constructed.

[0098] The recombinant plasmid is transformed into Escherichia coli (E. coli) BL21 competent cells to obtain a recombinant strain Escherichia coli BL21 / pET-P30 (BL21 / pET-P30). Escherichia coli BL21 / pET-P30. Escherichia coli The recombinant strain is preserved in China. Wuhan. Wuhan University China Typical Culture Collection Center on June 15, 2023, and the preservation number is CCTCC NO: M 2023955.

[0099] The preparation method of the porcine blue ear virus N1 and N2 recombinant antigens is as follows: the recombinant strains Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2 are induced by IPTG with a final concentration of 0.2 mM / 0.8 mM, respectively, and the expression products are purified to obtain porcine blue ear virus N1 recombinant antigen and porcine blue ear virus N2 recombinant antigen, respectively.

[0100] Specifically, the preparation method of the recombinant strain Escherichia coli BL21 / pET-N1 comprises the following steps:

[0101] The porcine pestivirus N antigen protein genome sequence is searched in the NCBI database, and part of the sequence of GenBank:EU926974.1 (Porcine respiratory and reproductive syndrome virus N gene) is selected as a target gene, the target gene is selected, analyzed and optimized,

[0102] The optimized N1 protein gene is synthesized by a gene synthesis company, linked with a pET-28a(+) vector, and a recombinant plasmid pET-28a-N1 is constructed.

[0103] The recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-N1, which is preserved in the China Center of Typical Culture Collection of Wuhan University in Wuhan, China on June 15, 2023, and the preservation number is CCTCC NO: M2023956.

[0104] Specifically, the preparation method of the recombinant strain Escherichia coli BL21 / pMAL-N2 comprises the following steps:

[0105] The porcine pestivirus N antigen protein genome sequence is searched in the NCBI database, and part of the sequence of GenBank:EU926974.1 (Porcine respiratory and reproductive syndrome virus N gene) is selected as a target gene, the target gene is selected, analyzed and optimized,

[0106] The optimized N2 protein gene is synthesized by a gene synthesis company, linked with a pMAL-c5x vector, and a recombinant plasmid pMAL-c5x-N2 is constructed.

[0107] The above-mentioned recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain a recombinant strain Escherichia coli BL21 / pMAL-N2, and the recombinant strain is preserved in the China Center of Typical Culture Collection of Wuhan University, Wuhan, China on June 15, 2023, with a preservation number of CCTCC NO: M2023957.

[0108] The preparation method of the porcine pestivirus E2 and E2a recombinant antigens is as follows: the recombinant strains Escherichia coli BL21 / pE2 and Escherichia coli BL21 / pE2a are induced by 0.1 mM IPTG respectively, and the expression products are purified to obtain porcine pestivirus E2 recombinant antigen and porcine pestivirus E2a recombinant antigen respectively.

[0109] Specifically, the preparation method of the recombinant strain Escherichia coli BL21 / pE2 includes:

[0110] The porcine pestivirus E2 antigen protein genome sequence is searched in the NCBI database, and GenBank: FJ598611.1 (Classical swine fever virus strain C strain-ZJ E2 glycoprotein gene) is selected as the target gene. The target gene is analyzed and optimized,

[0111] The optimized E2 protein gene is synthesized by a gene synthesis company, linked with a pET-28a(+) vector, and a recombinant plasmid pET-28a-E2 is constructed.

[0112] The above-mentioned recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain a recombinant strain Escherichia coli BL21 / pE2, and the recombinant strain is preserved in the China Center of Typical Culture Collection of Wuhan University, Wuhan, China on January 7, 2018, with a preservation number of CCTCC NO: M 2018060.

[0113] Specifically, the preparation method of the recombinant strain Escherichia coli BL21 / pE2a includes:

[0114] The E2a target gene is selected and analyzed from the genome sequence of the classical swine fever virus E2 antigen protein in the NCBI database, and the partial gene of GenBank: FJ598611.1 (Classical swine fever virus strain C-strain-ZJ E2 glycoprotein gene) is selected as the E2a target gene,

[0115] The E2a protein gene is synthesized by a gene synthesis company, and is connected with a pET-28a(+) vector to construct a recombinant plasmid pET-28a-E2a.

[0116] The recombinant plasmid is transformed into Escherichia coli BL21 competent cells to obtain a recombinant strain Escherichia coli BL21 / pE2a, which is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on January 7, 2018, and the preservation number is CCTCC NO: M 2018059.

[0117] The preparation method of the pig fever, blue ear and African swine fever virus antibody fluorescent joint test paper card based on the fluorescent microsphere marking technology is as follows: the PVC backing 9 is installed on the card shell 10, the PVC backing 9 is laid with a nitrocellulose membrane 7, the nitrocellulose membrane 9 is sequentially marked with a third detection line 5 coated with a pig fever virus E2a protein, a second detection line 4 coated with a pig blue ear disease virus N1 protein, a first detection line 3 coated with an African swine fever virus P30 protein and a quality control line 6 coated with an anti-his tag protein antibody, one end of the PVC backing 9 is sequentially installed with a sample pad 1 and a time-resolved immunofluorescence microsphere marked three pig disease antigen binding pad 2, and the other end is installed with a water absorption pad 8.

[0118] Specifically, the preparation method of the time-resolved immunofluorescence microsphere-labeled African swine fever P30 protein / porcine blue ear virus N2 protein / porcine swine fever virus E2 protein binding pad is as follows: take 1ml of activated time-resolved immunofluorescence microsphere suspension, and add African swine fever P30 protein+porcine blue ear virus N2 protein+porcine swine fever virus E2 protein mixed antigens dropwise while stirring, and the labeling amounts are 80ug, 100ug and 100ug (0.2mg / ml, diluted with ultrapure water) respectively, mix well at room temperature for 2 hours (Speed 20, vertical); add 10% BSA (the final concentration is 0.5%) for blocking for 1 hour (Speed 20, vertical), and centrifuge to remove the supernatant (12000r, 4°C, 15min); take 1ml of storage solution (African swine fever P30 protein latex labeling storage solution) and add it to the centrifuged microspheres, mix well, and store in a refrigerator in the dark, ready for use. After diluting the prepared time-resolved immunofluorescence microsphere-labeled mixed antigens 10 times with the working solution, use a gold spraying instrument to uniformly spray them onto the binding pad at a spraying amount of 8.0ul / cm, and place it in a 37°C incubator for drying for 12-18h, to prepare a time-resolved immunofluorescence microsphere-labeled African swine fever P3 protein / porcine blue ear virus N2 protein / porcine swine fever virus E2 protein binding pad, ready for use.

[0119] Further, the method for activating the time-resolved immunofluorescence microspheres is as follows: take out the time-resolved immunofluorescence microspheres (solid content 1%), and disperse them in an ultrasonic cleaner (180w) for 10s; in a 2ml EP tube, add 900ul of 0.03M MES buffer (pH6.0), then add 100ul of the above dispersed microspheres, and vortex mix (3-5s); add 5ul of A solution (10mg / ml N-hydroxysuccinimide (NHS) ethanol solution) to the microsphere suspension, vortex mix (3-5s), then add 5ul of B solution (10mg / ml N-hydroxysuccinimide (NHS) ethanol solution), vortex mix again (3-5s), and react at room temperature for 30min (Speed 20, vertical), and centrifuge to remove the supernatant (12000r, 4°C, 15min); add 2ml of cross-linking buffer to resuspend and wash the precipitate, centrifuge to remove the supernatant (12000r, 4°C, 15min); add 1ml of cross-linking buffer to the precipitate, and then ultrasonically disperse it uniformly (180W, 2min), to obtain the activated time-resolved immunofluorescence microspheres.

[0120] Specifically, the preparation method of the first detection line 3 coated with African swine fever virus P30 protein comprises: using a continuous dot membrane instrument to uniformly draw 0.5 mg / mL African swine fever virus P30 protein on the nitrocellulose membrane 9 at a membrane amount of 1 µL / cm, drying at 37°C for 12-18 h in a drying oven, and reserving.

[0121] Specifically, the preparation method of the second detection line 4 coated with porcine blue ear virus N1 protein comprises: using a continuous dot membrane instrument to uniformly draw 1.2 mg / mL porcine blue ear virus N1 protein on the nitrocellulose membrane 9 at a membrane amount of 1 µL / cm, drying at 37°C for 12-18 h in a drying oven, and reserving.

[0122] Specifically, the preparation method of the third detection line 5 coated with porcine swine fever virus E2a protein comprises: using a continuous dot membrane instrument to uniformly draw 1 mg / mL porcine swine fever virus E2a protein on the nitrocellulose membrane 9 at a membrane amount of 1 µL / cm, drying at 37°C for 12-18 h in a drying oven, and reserving.

[0123] Specifically, the preparation method of the quality control line 6 of the anti-his tag protein antibody comprises: using a continuous dot membrane instrument to uniformly draw 0.5 mg / mL anti-his tag protein antibody on the nitrocellulose membrane at a membrane amount of 1 µL / cm, drying at 37°C for 12-18 h in a drying oven, and reserving.

[0124] Specifically, the water absorption pad 8 is soaked with the blocking solution for 30 min and dried at 37°C, and reserved.

[0125] The third detection line 5 coated with porcine swine fever virus E2a protein is 5 mm away from the binding pad 2 end of the time-resolved immunofluorescence microsphere-labeled three porcine disease antigens, the second detection line 4 coated with porcine blue ear virus N1 protein is 4 mm away from the end of the third detection line 5 coated with porcine swine fever virus E2a protein, the first detection line 3 coated with African swine fever virus P30 protein is 4 mm away from the end of the second detection line 4 coated with porcine blue ear virus N1 protein, and the quality control line 6 of the anti-his tag protein antibody is 3 mm away from the end of the water absorption pad 8. Example Two

[0126] The porcine swine fever, porcine blue ear, and African swine fever virus antibody fluorescence combined test strip card prepared in Example One is subjected to performance test, specifically as follows:

[0127] (1) Determination of critical value (determination point) of test strip card

[0128] The test paper card is prepared by using the established preparation process, 189 known result African swine fever serum sample plates (101 of which are positive serum and 88 of which are negative serum) are detected, 196 porcine blue ear serum sample plates (125 of which are positive serum and 71 of which are negative serum) are detected, 205 known result porcine fever serum sample plates (133 of which are positive serum and 72 of which are negative serum) are detected, and the Prism 6.0 software is used to draw the ROC curve respectively, the point closest to the upper left of the curve is selected, the sensitivity and specificity of each possible cut-off point in the statistical result are calculated, and the Youden index is calculated. The cut-off point with the maximum Youden index is the critical point, and the best critical value of each of the three pig disease antibodies is determined. The area under the ROC curve can reflect the accuracy of the diagnostic test, and when the area under the curve is above 0.9, the diagnosis has high accuracy.

[0129] The results are as follows: 189 known result African swine fever serum sample plates are detected, and the ROC curve is drawn, and the area under the ROC curve is 0.9823. The best critical point of African swine fever antibody diagnosis is confirmed by the ROC curve to be 0.4004, the sensitivity is 85.56%, and the specificity is 91.65%, so the critical point of the porcine fever antibody detection of the application is 0.4. The detection value is greater than or equal to 0.4, the ASFV antibody detection is positive; the detection value is less than 0.4, the ASFV antibody detection is negative,

[0130] 196 known result porcine blue ear serum sample plates are detected, and the ROC curve is drawn, and the area under the ROC curve is 0.9141. The best critical point of African blue ear antibody diagnosis is confirmed by the ROC curve to be 0.4164, the sensitivity is 82.59%, and the specificity is 87.34%, so the critical point of the porcine blue ear antibody detection of the application is 0.4. The detection value is greater than or equal to 0.4, the PRRSV antibody detection is positive; the detection value is less than 0.4, the PRRSV antibody detection is negative,

[0131] 205 known result porcine fever serum sample plates are detected, and the ROC curve is drawn, and the area under the ROC curve is 0.9468. The best critical point of porcine fever antibody diagnosis is confirmed by the ROC curve to be 0.4963, the sensitivity is 83.46%, and the specificity is 90.28%, so the critical point of the porcine fever antibody detection project of the test paper card of the application is 0.5. The detection value is greater than or equal to 0.5, the CSFV antibody detection is positive; the detection value is less than 0.5, the CSFV antibody detection is negative.

[0132] (2) Sensitivity experiment

[0133] The African swine fever virus (ASFV) positive serum is diluted by a multiple ratio from 1:100 to 1:100, 1:200, 1:400, …1:6400 series of positive serum. 50 μL of the above diluted serum is taken respectively, and the test paper card is detected, the positive serum of each dilution is repeated for 5 times, and the test paper card is placed into the immunofluorescence rapid detector after 15 min of reaction, and the detection result is read.

[0134] The porcine reproductive and respiratory syndrome virus (PRRSV) positive serum is diluted by a multiple ratio from 1:100 to 1:100, 1:200, 1:400, …1:3200 series of positive serum. 50 μL of the above diluted serum is taken respectively, and the test paper card is detected, the positive serum of each dilution is repeated for 5 times, and the test paper card is placed into the immunofluorescence rapid detector after 15 min of reaction, and the detection result is read.

[0135] The classical swine fever virus (CSFV) positive serum national reference is diluted by a multiple ratio from 1:16 to 1:16, 1:32, 1:64, …1:512 series of positive serum. 50 μL of the above diluted serum is taken respectively, and the test paper card is detected, the positive serum of each dilution is repeated for 5 times, and the test paper card is placed into the immunofluorescence rapid detector after 15 min of reaction, and the detection result is read.

[0136] Specifically, the African swine fever virus positive serum is purchased from the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and the specification is 1 mL.

[0137] Specifically, the porcine reproductive and respiratory syndrome virus (PRRSV) positive serum is purchased from the China Veterinary Microbiological Data Center, and the number is CVCC Z230, and the specification is 5 mL.

[0138] Specifically, the classical swine fever virus positive serum national reference is purchased from the China Veterinary Microbiological Data Center, and the number is CVCC Z102, and the specification is 1 mL.

[0139] The detection result is that the test paper card is used for respectively detecting the ASFV / PRRSV / CSFV positive serum of different dilutions, when the ASFV positive serum is diluted by a multiple ratio to 1:3200, the detection value of the ASFV detection item is still greater than or equal to 0.4, and the positive result is obtained; when the PRRSV positive serum is diluted by a multiple ratio to 1:1600, the detection value of the PRRSV detection item is still greater than or equal to 0.4, and the positive result is obtained; when the CSFV positive serum is diluted by a multiple ratio to 1:256, the detection value of the CSFV detection item is still greater than or equal to 0.5, and the positive result is obtained; and it is shown that the test paper card has high sensitivity.

[0140] Table 1 Sensitivity test result of the test card of the application for detecting three kinds of standard antigens

[0141]

[0142] (2) Specificity experiment

[0143] Take 50 μL of African swine fever virus (ASFV) positive serum, porcine reproductive and respiratory syndrome virus (PRRSV) positive serum, swine fever virus (CSFV) positive serum national reference, porcine foot-and-mouth disease virus (FMDV) antibody positive serum, pseudorabies virus (PRV) positive serum reference, porcine circovirus (PCV) CAP protein antibody, recombinant porcine parvovirus (PPV) VP2 protein antibody, respectively, use the test paper card of the application to detect, react for 15 min, put the test paper card into the immunofluorescence rapid tester, read the detection result.

[0144] Specifically, the African swine fever virus (ASFV) positive serum is purchased from the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and the specification is 1 mL.

[0145] Specifically, the porcine reproductive and respiratory syndrome virus (PRRSV) positive serum is purchased from the China Veterinary Microbiological Data Center, and the number is CVCC Z230, and the specification is 5 mL.

[0146] Specifically, the swine fever virus (CSFV) positive serum national reference is purchased from the China Veterinary Microbiological Data Center, and the number is CVCC Z102, and the specification is 1 mL.

[0147] Specifically, the porcine foot-and-mouth disease virus (FMDV) antibody positive serum is taken from the porcine foot-and-mouth disease antibody ELISA kit, which is purchased from Beijing Biodot Biotechnology Co., Ltd., the brand is Cusabio, and the article number is CSB-EQ027745PI.

[0148] Specifically, the pseudorabies virus (PRV) positive serum reference is purchased from the China Veterinary Microbiological Data Center, and the number is CVCC Z289, and the specification is 1 mL.

[0149] Specifically, the porcine circovirus (PCV) CAP protein antibody is purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., the brand is Boaosen, and the article number is bs-20021R.

[0150] Specifically, the porcine recombinant porcine parvovirus (PPV) VP2 protein antibody is purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., the brand is Boaosen, and the article number is bs-2309R.

[0151] Table 2: Specificity experiment results of the detection card of the application

[0152] positive sera ASFV PRRSV CSFV FMDV PRV PCV PPV ASFV project 2.3431 ＜0.4 ＜0.4 ＜0.4 ＜0.4 ＜0.4 ＜0.4 PRRSV project ＜0.4 1.8932 ＜0.4 ＜0.4 ＜0.4 ＜0.4 ＜0.4 ASFV project ASFV project ＜0.5 ＜0.5 1.2467 ＜0.5 ＜0.5 ＜0.5 ＜0.5

[0153] Detection results: when detecting ASFV, PRRSV and CSFV antibodies, the test paper card is placed into the immunofluorescence rapid detector after 15 minutes, and the detection results are read. When detecting ASFV positive serum, the corresponding ASFV detection value is positive (2.3431), and the detection values of the other two items are negative; when detecting PRRSV positive serum, the corresponding PRRSV detection value is positive (1.8932), and the detection values of the other two items are negative; when detecting CSFV positive serum, the corresponding CSFV detection value is positive (1.2467), and the detection values of the other two items are negative; when detecting FMDV, PRV, PCV and PP antibodies, the test paper card is placed into the immunofluorescence rapid detector after 15 minutes, and the detection results are read. The detection results of the ASFV item are all <0.4, the detection results of the PRRSV item are all <0.4, the detection results of the CSFV item are all <0.5, and the detection results of the three pig disease detection items are all negative results.

[0154] Therefore, it can be known that the test paper card has high specificity, and has no cross reaction with antibodies of other important disease viruses of pigs.

[0155] (3) coincidence rate test

[0156] The test paper card is used for detecting 246 pig serum samples collected from Hubei, Henan, Jiangxi and Shanghai, and the test paper card and the African swine fever / porcine blue ear / porcine pestilence ELISA antibody detection kit are used for detection. The comparison detection results between the two are shown in Tables 3-5. As shown in Table 3, when detecting ASFV antibodies, the positive coincidence rate of the test paper card of the application and the African swine fever ELISA kit is 90.83%, the negative coincidence rate is 92.06%, and the total coincidence rate is 91.46%. As shown in Table 4, when detecting PRRSV antibodies, the positive coincidence rate of the test paper card of the application and the porcine blue ear ELISA kit is 87.88%, the negative coincidence rate is 83.33%, and the total coincidence rate is 86.99%. As shown in Table 5, when detecting CSFV antibodies, the positive coincidence rate of the test paper card of the application and the porcine pestilence ELISA kit is 95.12%, the negative coincidence rate is 92.68%, and the total coincidence rate is 94.31%. The results show that the detection results of the kit of the application and the ELISA are basically consistent, and the coincidence rate is good.

[0157] Specifically, the African swine fever virus ELISA antibody detection kit is purchased from INGENASA in Spain, and the batch number is 011019-B.

[0158] Specifically, the porcine blue ear virus ELISA antibody detection kit is purchased from IDEXX in the United States, and the product number is 43220.

[0159] Specifically, the porcine pestivirus ELISA antibody detection kit was purchased from IDEXX, USA, item number 43222.

[0160] Table 3 Results of clinical detection of ASFV antibody by the test paper card and the ELISA kit of the present application

[0161]

[0162] Table 4 Results of clinical detection of PRRSV antibody by the test paper card and the ELISA kit of the present application

[0163]

[0164] Table 5 Results of clinical detection of CSFV antibody by the test paper card and the ELISA kit of the present application

[0165]

[0166] The above parts not described in detail are all prior art.

Claims

1. A fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, comprising a sample pad (1), a time-resolved immunofluorescence microsphere-labeled three-pig disease antigen combination pad (2), a first detection line (3) coated with African swine fever virus P30 protein, a second detection line (4) coated with porcine blue ear disease virus N1 protein, a third detection line (5) coated with swine fever virus E2a protein, a quality control line (6) of anti-his tag protein antibody, a nitrocellulose membrane (7), a water absorption pad (8), a PVC backing (9) and a card shell (10), characterized in that: The PVC backing (9) is installed on the card shell (10), the PVC backing (9) is laid with nitrocellulose membrane (7), the nitrocellulose membrane (7) is sequentially marked with the third detection line (5) coated with E2a protein of hog cholera virus, the second detection line (4) coated with N1 protein of blue ear disease virus of pig, the first detection line (3) coated with P30 protein of African swine fever virus and the quality control line (6) of anti his tag protein antibody, one end of the PVC backing (9) is sequentially installed with sample pad (1) and time-resolved immunofluorescence microsphere labeled three kinds of pig disease antigen binding pad (2), the other end is installed with water absorption pad (8); ​ The three kinds of pig disease antigens are P30 protein of African swine fever virus, N2 protein of blue ear virus of pig and E2 protein of hog cholera virus.

2. The preparation method of the hog cholera, blue ear and African swine fever virus antibody fluorescent joint test paper card according to claim 1, characterized in that: It comprises the following steps; ①, the preparation method of the time-resolved immunofluorescence microsphere labeled three kinds of pig disease antigen binding pad (2) is as follows: Take 1ml activated time-resolved immunofluorescence microsphere suspension, add three kinds of pig disease mixed antigen of P30 protein of African swine fever virus+ N2 protein of blue ear virus of pig+ E2 protein of hog cholera virus dropwise while stirring, the labeling amount is 50-100ug, 50-100ug and 50-100ug respectively, mix uniformly at room temperature for 2 hours, add 10% BSA for blocking for 1 hour, centrifuge to remove supernatant, take 1ml storage solution and add to the centrifuged microspheres, mix uniformly, store in cold storage and avoid light, and wait for use; the prepared time-resolved immunofluorescence microsphere labeled mixed antigen is diluted 5-10 times with working solution, uniformly sprayed on the binding pad by gold spraying instrument, the spraying amount is 3.0-10.0ul / cm, placed in 37℃ incubator for drying for 12-18h, prepared into time-resolved immunofluorescence microsphere labeled three kinds of pig disease antigen binding pad (2), standby, ②, the activation method of time-resolved immunofluorescence microsphere is as follows: take out time-resolved immunofluorescence microsphere, disperse in ultrasonic cleaning instrument for 10s, add 900ul of 0.03M MES buffer in 2ml EP tube, add 100ul of the above dispersed microspheres, vortex mix, add 5ul of A liquid in the microsphere suspension, A liquid is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix A liquid, then add 5ul of B liquid, B liquid is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix B liquid again, react at room temperature for 30min, centrifuge to remove supernatant, add 2ml crosslinking buffer to resuspend and wash the precipitate, centrifuge to remove supernatant, add 1ml crosslinking buffer to the precipitate, then ultrasonic dispersion, which is the activated time-resolved immunofluorescence microsphere, ③, the preparation method of the first detection line (3) coated with P30 protein of African swine fever virus includes: using a continuous dot membrane instrument to uniformly mark 0.05-1mg / mL P30 protein of African swine fever virus on the nitrocellulose membrane (7), the marking amount is 1ul / cm, 37℃ incubator drying for 12-18h, standby, The preparation method of the second detection line (4) coated with the porcine blue ear disease virus N1 protein comprises the following steps: The preparation method of the third detection line (5) coated with the porcine epidemic diarrhea virus E2a protein comprises the following steps: The preparation method of the quality control line (6) of the anti-his tag protein antibody comprises the following steps: The sample pad (1) is soaked in the blocking solution for 30 minutes and dried at 37 DEG C, and serves as standby. The third detection line (5) coated with the porcine epidemic diarrhea virus E2a protein is 5 mm away from the binding pad (2) of the three porcine disease antigens marked by the time-resolved immunofluorescence microspheres, the second detection line (4) coated with the porcine blue ear disease virus N1 protein is 4 mm away from the end of the third detection line (5) coated with the porcine epidemic diarrhea virus E2a protein, the first detection line (3) coated with the African swine fever virus P30 protein is 4 mm away from the end of the second detection line (4) coated with the porcine blue ear disease virus N1 protein, and the quality control line (6) of the anti-his tag protein antibody is 3 mm away from the end of the water absorption pad (8).

3. The method for using the porcine pestivirus, porcine blue ear and African swine fever virus antibody fluorescent joint test paper card of claim 1, characterized in that: It comprises the following steps. When the sample to be detected contains porcine epidemic diarrhea virus antibodies, porcine blue ear virus antibodies or African swine fever virus antibodies, the African swine fever virus antibodies, porcine blue ear virus antibodies or porcine epidemic diarrhea virus antibodies in the sample move to the position of the binding pad (2) of the three porcine disease antigens marked by the time-resolved immunofluorescence microspheres and react with the P30 antigen, N2 antigen or E2 antigen marked by the time-resolved immunofluorescence microspheres, When the antigen-antibody complex is chromatographed to the detection line position, the P30 / N1 / E2a antigens on the first detection line (3) coated with the African swine fever virus P30 protein, the second detection line (4) coated with the porcine blue ear disease virus N1 protein and the third detection line (5) coated with the porcine epidemic diarrhea virus E2a protein react with the antibodies in the antigen-antibody complex again, so that a "fluorescently labeled antigen-antibody-coated antigen" double-antigen sandwich complex is formed at the detection line position, and the first detection line (3) coated with the African swine fever virus P30 protein, the second detection line (4) coated with the porcine blue ear disease virus N1 protein and the third detection line (5) coated with the porcine epidemic diarrhea virus E2a protein respectively present corresponding fluorescent bands, Meanwhile, the excess labeled antigens in the binding pad (2) of the three porcine disease antigens marked by the time-resolved immunofluorescence microspheres react with the anti-his tag protein antibodies in the quality control line (6) region, and a fluorescent band is formed at the position of the quality control line (6) of the anti-his tag protein antibody. IV. When the content of the antibody in the sample is higher, the fluorescence signal of the detection line area of the test reaction zone on the nitrocellulose membrane (7) is stronger, and vice versa, V. The fluorescence intensity change of the detection line is positively correlated with the content of the antibody to be detected. The content level of the to-be-detected substance is analyzed after the automatic recognition and interpretation of the fluorescence signal of the reaction strip by the immunofluorescence rapid detector, so as to realize quantitative detection and analysis.

4. The use method of the hog cholera, blue ear and African swine fever virus antibody fluorescence joint detection test paper card according to claim 3, characterized in that: The preparation method of the African swine fever virus P30 recombinant antigen is as follows: the recombinant strain Escherichia coli BL21 / pET-P30 is induced by a final concentration of 0.6mM IPTG, and the expression product is purified to obtain the African swine fever virus P30 recombinant antigen, The preparation method of the recombinant strain Escherichia coli BL21 / pET-P30 includes: The P30 protein genome sequence optimized by the gene synthesis company is synthesized, linked with the pET-21a(+) vector, and the recombinant plasmid pET-21a-P30 is constructed. The recombinant plasmid is transformed into the competent cells of Escherichia coli BL21 to obtain the recombinant strain Escherichia coli BL21 / pET-P30.

5. The use method of the hog cholera, blue ear and African swine fever virus antibody fluorescence joint detection test paper card according to claim 4, characterized in that: The preparation method of the porcine reproductive and respiratory syndrome virus N1 and N2 recombinant antigens is as follows: the recombinant strains Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2 are induced by a final concentration of 0.2mM / 0.8mM IPTG, respectively, and the expression product is purified to obtain the porcine reproductive and respiratory syndrome virus N1 recombinant antigen and the porcine reproductive and respiratory syndrome virus N2 recombinant antigen, respectively.

6. The use method of the hog cholera, blue ear and African swine fever virus antibody fluorescence joint detection test paper card according to claim 5, characterized in that: The preparation method of the recombinant strain Escherichia coli BL21 / pET-N1 includes: The P30 protein genome sequence optimized by the gene synthesis company is synthesized, linked with the pET-21a(+) vector, and the recombinant plasmid pET-21a-P30 is constructed.

7. The use method of the hog cholera, blue ear and African swine fever virus antibody fluorescence joint detection test paper card according to claim 6, characterized in that: ​ The preparation method of the recombinant strain Escherichia coli BL21 / pMAL-N2 comprises the following steps: searching for a porcine pestivirus N antigen protein genome sequence in an NCBI database, selecting a partial sequence of GenBank: EU926974.1 as a target gene, and performing selection analysis and optimization on the target gene; The optimized N2 protein gene is synthesized by a gene synthesis company, is connected with a pMAL-c5x vector, a recombinant plasmid pMAL-c5x-N2 is constructed, and the recombinant plasmid is transformed into competent cells of Escherichia coli BL21 to obtain the recombinant strain Escherichia coli BL21 / pMAL-N2.

8. The use method of the hog cholera, blue ear and African swine fever virus antibody fluorescent joint test paper card according to claim 7, characterized in that: The preparation method of the porcine pestivirus E2 and E2a recombinant antigens is as follows: recombinant strain Escherichia coli BL21 / pE2 and Escherichia coli BL21 / pE2a are respectively induced by 0.1 mM IPTG with a final concentration, and porcine pestivirus E2 recombinant antigens and porcine pestivirus E2a recombinant antigens are respectively obtained after the expression products are purified.

9. The method according to claim 8, wherein the method is characterized by the following steps: 1) taking the test paper card out of the package and placing it on a clean, flat surface; 2) dipping the sample pad into the sample solution, and then placing the sample pad on the sample well; 3) placing the test paper card on a clean, flat surface, and then observing the test results after 10-15 minutes. The preparation method of the recombinant strain Escherichia coli BL21 / pE2 comprises the following steps: A porcine pestivirus E2 antigen protein genome sequence is searched for in an NCBI database, GenBank: FJ598611.1 is selected as a target gene, and the target gene is analyzed and optimized, The optimized E2 protein gene is synthesized by a gene synthesis company, is connected with a pET-28a(+) vector, a recombinant plasmid pET-28a-E2 is constructed, and the recombinant plasmid is transformed into competent cells of Escherichia coli BL21 to obtain the recombinant strain Escherichia coli BL21 / pE2.

10. The method according to claim 9, wherein the method is characterized by the following steps: 1) taking the test paper card out of the package and placing it on a clean, flat surface; 2) adding 2-3 drops of the sample to the sample well; 3) placing the test paper card into the sample well; 4) reading the results after 10-15 minutes. The preparation method of the recombinant strain Escherichia coli BL21 / pE2a comprises the following steps: A porcine pestivirus E2 antigen protein genome sequence is searched for in an NCBI database, GenBank: FJ598611.1 is selected as a target gene, and the target gene is analyzed and optimized, The optimized E2a protein gene is synthesized by a gene synthesis company, is connected with a pET-28a(+) vector, a recombinant plasmid pET-28a-E2a is constructed, and the recombinant plasmid is transformed into competent cells of Escherichia coli BL21 to obtain the recombinant strain Escherichia coli BL21 / pE2a.

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