Fluorescence joint detection test paper card for swine fever, porcine reproductive and respiratory syndrome and African swine fever virus antibodies as well as preparation method and use method of fluorescence joint detection test paper card

Abstract

The invention discloses a swine fever, porcine reproductive and respiratory syndrome and African swine fever virus antibody fluorescence joint detection test paper card, a preparation method and a use method, a PVC backing (9) is installed on a card shell (10), and 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 porcine reproductive and respiratory syndrome virus N1 protein, a first detection line (3) coated with African hog cholera virus P30 protein and a quality control line (6) of an anti-his tag protein antibody are sequentially marked on the nitrocellulose membrane (9). The kit has the advantages that the time-resolved immunofluorescent microspheres are used as tracers, the antigens of the three pig diseases are marked at the same time at a time, and meanwhile, the detection sensitivity and the detection efficiency are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of animal immunization application, and more specifically to a fluorescent joint detection test paper card for antibodies of swine fever, blue ear and African swine fever viruses, and a preparation method and a use method thereof. Background Art

[0002] Classical swine fever, 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 the Classical swine fever virus (CSFV). It mainly infects domestic pigs and wild boars and can cause serious infectious diseases in pigs with high mortality. It is an RNA virus with very small virus particles and high variability. It is divided into seven different subtypes. Blue ear disease is a highly contagious pig disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which usually manifests as respiratory diseases and reproductive disorders. Due to the many subtypes of PRSV and the high variability of the virus, blue ear disease is still one of the most serious viral diseases in the pork industry around the world. African swine fever is an acute, hemorrhagic, highly contagious disease caused by the African swine fever virus (ASFV) infecting domestic pigs and various wild boars. Pigs of all breeds and ages can be infected, with morbidity and mortality rates as high as 100%. The World Organization for Animal Health lists it as a notifiable animal disease, and China also lists it as a Class I animal disease. These three common swine infectious diseases have seriously affected the development and stability of the pork industry. The existence of these viruses directly threatens the health and production of pigs, and may even lead to the death of a large number of pigs, causing considerable economic losses to animal husbandry and social economy. In addition, there are no effective specific treatments for these viruses, and prevention and control are also the main means of combating these diseases. At present, my country has adopted a purification strategy combining compulsory immunization with epidemic monitoring for these swine fever and blue ear. Vaccine immunization has become the key to influencing the prevention and control of the disease. It is of great significance to establish appropriate serological antibody evaluation methods for vaccines. Although there is currently no effective vaccine for African swine fever in the world, after the current local epidemic trend of African swine fever, supplemented by serological antibody testing, it is conducive to the diagnosis of suspected cases and invisible cases.

[0003] At present, the main detection method for serological testing is enzyme-linked immunosorbent assay (ELISA). ELISA has high sensitivity and can detect multiple samples in parallel, but this method is time-consuming. Indirect ELISA takes about 2 hours, and blocking ELISA takes 2-3 hours. It is not convenient for real-time and rapid diagnosis, so it cannot be widely promoted and used at the grassroots level. Therefore, the development of rapid detection methods for viral antibodies is of great significance for the clinical diagnosis and immune monitoring of swine diseases. The most common form of rapid detection technology for disease antibodies is immunochromatographic test strip technology, which combines the characteristics of chromatography technology and immunotechnology. It is simple and rapid, and the results can be directly observed. The current immunochromatographic rapid detection strips mostly use colloidal gold, colored latex microspheres or fluorescein as markers. Rapid detection products developed based on colloidal gold labeling technology have problems such as low sensitivity and large batch differences, and are often used for qualitative analysis; although colored latex microspheres have improved in sensitivity and batch differences, they are also often used for qualitative analysis or semi-quantitative analysis; immunochromatography based on fluorescein labeling technology has greatly improved sensitivity and can also perform quantitative detection, but because the sample contains a high fluorescence background signal and the Stock displacement is small, it will have a greater impact on the detection. Time-resolved fluorescence is a marker marked with lanthanide rare earth ions with unique fluorescence characteristics. Compared with ordinary fluorescence, it has the characteristics of large Stock displacement and long fluorescence lifetime. It can effectively avoid the background fluorescence in the sample and the influence of stray light such as excitation light. Therefore, it has higher sensitivity and anti-interference ability than ordinary fluorescence. Time-resolved fluorescence immunochromatography combines the two technologies of time-resolved fluorescent latex microsphere labeling and immunochromatography, which can perform multi-label immunoassays. Combined with instruments, rapid immunochromatographic test strips that can be accurately quantified can be developed. It has been widely used in immunotechnology, clinical diagnosis, cytology and other biomedical fields.

[0004] Based on the multi-link rapid detection technology for multiple swine diseases, Chinese invention patent 201610544386.1 discloses a multi-link rapid test strip that uses Staphylococcus aureus protein A as a capture antigen to simultaneously detect antibodies to swine fever, blue ear disease, and type O foot-and-mouth virus.

[0005] Chinese utility model patent 201220258469.1 discloses a multiple rapid test strip that uses a double antibody sandwich method to simultaneously detect O-type foot-and-mouth virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus antigens. The above patents all use colloidal gold as a labeled tracer for simple qualitative detection. However, the detection sensitivity of colloidal gold is low and the detection color is single, making it difficult to judge the results of different test items and unable to perform quantitative analysis. Summary of the invention

[0006] The first purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and propose a fluorescent joint detection test card for swine fever, blue ear disease and African swine fever virus antibodies based on fluorescent microsphere labeling technology. The test card uses time-resolved immunofluorescent microspheres as tracers and simultaneously labels the antigens of the three swine diseases, which can not only improve the sensitivity of detection, but also can perform quantitative analysis of the three items at the same time.

[0007] The first object of the present invention is implemented by the following technical scheme: a fluorescent joint detection test paper card for swine fever, blue ear disease and African swine fever virus antibodies, which includes a sample pad, a binding pad of three swine disease antigens labeled with time-resolved immunofluorescent microspheres, a first detection line coated with African swine fever virus P30 protein, a second detection line coated with blue ear disease virus N1 protein, a third detection line coated with swine fever virus E2a protein, a quality control line of anti-his tag protein antibody, a nitrocellulose membrane, a water absorbent pad, a PVC backing and a card shell.

[0008] The PVC backing is installed on the card shell, and a nitrocellulose membrane is laid on the PVC backing. The nitrocellulose membrane is sequentially marked with a third detection line coated with classical swine fever virus E2a protein, a second detection line coated with porcine reproductive and respiratory syndrome virus N1 protein, a first detection line coated with African swine fever virus P30 protein and a quality control line of anti-his tag protein antibody. A sample pad and a binding pad of three swine disease antigens labeled with time-resolved immunofluorescence microspheres are sequentially installed on one end of the PVC backing, and a water absorbent pad is installed on the other end.

[0009] The second purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and to propose a method for preparing a fluorescent joint detection test paper card for classical swine fever, blue ear and African swine fever virus antibodies.

[0010] The second object of the present invention is implemented by the following technical scheme: a method for preparing a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, which comprises the following steps;

[0011] ① The preparation method of the binding pad of three swine disease antigens labeled with time-resolved immunofluorescence microspheres is as follows;

[0012] Take 1ml of activated time-resolved immunofluorescent microsphere suspension, add three kinds of mixed antigens of African swine fever P30 protein + porcine blue ear virus N2 protein + classical swine fever virus E2 protein while stirring, the labeling dosage is 50-100ug, 50-100ug, 50-100ug respectively, mix at room temperature for 2 hours; add 10% BSA for blocking for 1 hour, centrifuge to remove the supernatant, take 1ml of preservation solution and add it to the microspheres after centrifugation, mix well, refrigerate and keep away from light for standby use. Dilute the prepared time-resolved immunofluorescent microspheres labeled with mixed antigens with working solution 5-10 times, spray evenly on the binding pad with a gold sprayer, the spray volume is 3.0-10.0μl / cm, place in a 37℃ incubator to dry for 12h-18h, and prepare the binding pad of three kinds of swine disease antigens labeled with time-resolved immunofluorescent microspheres for standby use.

[0013] ②. The method for labeling and activating time-resolved immunofluorescent microspheres is as follows: take out the time-resolved immunofluorescent microspheres, disperse them in an ultrasonic cleaner for 10 seconds, add 900ul of 0.03M MES buffer in a 2mL EP tube, then add 100ul of the dispersed microspheres, vortex mix, add 5ul of solution A to the microsphere suspension, solution A is a 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix solution A, then add 5ul of solution B, solution B is a 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix solution B again, place at room temperature to react for 30min, 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, and then disperse it evenly by ultrasound, which is the activated time-resolved immunofluorescent microspheres.

[0014] ③. The preparation method of the first test line coated with African swine fever virus P30 protein includes: using a continuous film spotting instrument to evenly streak 0.05-1 mg / mL African swine fever virus P30 protein onto a nitrocellulose membrane, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0015] ④. The preparation method of the second test line coated with the porcine reproductive and respiratory syndrome virus N1 protein includes: using a continuous film spotting instrument to evenly streak 0.2-1.5 mg / mL of porcine reproductive and respiratory syndrome virus N1 protein onto a nitrocellulose membrane at a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0016] ⑤. The preparation method of the third test line coated with the classical swine fever virus E2a protein includes: using a continuous film spotter to evenly streak 0.2-1.5 mg / mL classical swine fever virus E2a protein onto a nitrocellulose membrane at a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0017] ⑥. The quality control line preparation method of the anti-his tag protein antibody includes: using a continuous film spotter to evenly streak 0.2-0.8 mg / mL of the anti-his tag protein antibody onto a nitrocellulose membrane at a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0018] ⑦. Soak the sample pad in the blocking solution for 30 minutes, dry it at 37℃ and set aside.

[0019] ⑧. The third detection line coated with classical swine fever virus E2a protein is 5 mm away from the binding pad end of three swine disease antigens labeled with time-resolved immunofluorescence microspheres. The second detection line coated with porcine reproductive and respiratory syndrome virus N1 protein is 4 mm away from the third detection line end of classical swine fever virus E2a protein. The first detection line coated with African swine fever virus P30 protein is 4 mm away from the second detection line end of porcine reproductive and respiratory syndrome virus N1 protein. The quality control line of anti-his tag protein antibody is 3 mm away from the absorbent pad end.

[0020] The third purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and to propose a method for using a fluorescent joint detection test paper card for classical swine fever, blue ear disease and African swine fever virus antibodies.

[0021] The third object of the present invention is implemented by the following technical scheme: a method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, which comprises the following steps;

[0022] ①. When the sample to be tested contains antibodies to classical swine fever virus / or antibodies to porcine reproductive and respiratory syndrome virus / or antibodies to African swine fever virus, the antibodies to African swine fever virus / or antibodies to porcine reproductive and respiratory syndrome virus / or antibodies to classical swine fever virus in the sample migrate to the binding pad of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres and react with the P30 antigen / or N2 antigen / or E2 antigen labeled with time-resolved immunofluorescent microspheres.

[0023] ②. 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 reproductive and respiratory syndrome virus N1 protein, and the third detection line coated with classical swine fever virus E2a protein react again with the antibodies in the antigen-antibody complex, thereby forming a "fluorescently labeled antigen-antibody-coated antigen" double antigen sandwich complex at the detection line position, and corresponding fluorescent bands appear on the first detection line coated with African swine fever virus P30 protein, the second detection line coated with porcine reproductive and respiratory syndrome virus N1 protein, and the third detection line coated with classical swine fever virus E2a protein, respectively.

[0024] ③. At the same time, the excess labeled antigens in the binding pad of the three swine disease antigens labeled with time-resolved immunofluorescence microspheres will react with the anti-his tag protein antibody in the quality control line area of ​​the anti-his tag protein antibody, forming a fluorescent strip at the quality control line position of the anti-his tag protein antibody.

[0025] ④. When the antibody content in the sample is higher, the fluorescence signal in the test reaction area detection line area on the nitrocellulose membrane will be stronger, and vice versa.

[0026] ⑤. The change in fluorescence intensity of the test line is positively correlated with the content of the antibody to be tested. The content level of the test object can be analyzed by automatically identifying and interpreting the fluorescence signal of the reaction strip by the immunofluorescence rapid tester, thereby realizing quantitative detection and analysis.

[0027] In the above technical scheme: 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 with a final concentration of 0.6 mM IPTG, and the expression product is purified to obtain the African swine fever virus P30 recombinant antigen,

[0028] The preparation method of the recombinant strain Escherichia coli BL21 / pET-P30 comprises:

[0029] The genome sequence of the African swine fever virus P30 antigen protein was searched in the NCBI database, and GenBank: JX524222.1 was selected as the target gene. The target gene was selected, analyzed and optimized.

[0030] The optimized P30 protein gene sequence was synthesized by a gene synthesis company and connected to the pET-21a(+) vector to construct the recombinant plasmid pET-21a-P30.

[0031] The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-P30.

[0032] In the above technical scheme: the preparation method of porcine reproductive and respiratory syndrome virus N1 and N2 recombinant antigens is as follows: use final concentration 0.2mM / 0.8mM IPTG to induce recombinant strains of Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2, respectively, and after purification of the expression products, obtain porcine reproductive and respiratory syndrome virus N1 recombinant antigen and porcine reproductive and respiratory syndrome virus N2 recombinant antigen, respectively.

[0033] In the above technical solution: the preparation method of the recombinant strain Escherichia coli BL21 / pET-N1 includes:

[0034] The genomic sequence of classical swine fever virus N antigen protein was searched in the NCBI database, and a partial sequence of GenBank:EU926974.1 was selected as the target gene. The target gene was selected, analyzed and optimized, and the optimized N1 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct a recombinant plasmid pET-28a-N1. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-N1.

[0035] In the above technical scheme: the preparation method of the recombinant strain Escherichia coli BL21 / pMAL-N2 includes: searching the genome sequence of the classical swine fever virus N antigen protein in the NCBI database, selecting a partial sequence of GenBank:EU926974.1 as the target gene, and selecting, analyzing and optimizing the target gene.

[0036] The optimized N2 protein gene was synthesized by a gene synthesis company and connected to the pMAL-c5x vector to construct the recombinant plasmid pMAL-c5x-N2. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pMAL-N2.

[0037] In the above technical scheme: the preparation method of classical swine fever virus E2 and E2a recombinant antigens is as follows: use a final concentration of 0.1mM IPTG to induce recombinant strains of Escherichia coli BL21 / pE2 and Escherichia coli BL21 / pE2a, respectively, and after purification of the expression products, obtain classical swine fever virus E2 recombinant antigen and classical swine fever virus E2a recombinant antigen, respectively.

[0038] In the above technical solution: the preparation method of the recombinant strain Escherichia coli BL21 / pE2 includes:

[0039] Search the genomic sequence of the classical swine fever virus E2 antigen protein in the NCBI database and select GenBank: FJ598611.1 as the target gene. Analyze and optimize the target gene.

[0040] The optimized E2 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2.

[0041] In the above technical solution: the preparation method of the recombinant strain Escherichia coli BL21 / pE2a includes:

[0042] The genome sequence of the E2 antigen protein of classical swine fever virus was searched in the NCBI database, and part of the gene of GenBank: FJ598611.1 was selected as the E2a target gene. The target gene was selected, analyzed and optimized.

[0043] The optimized E2a protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2a. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2a.

[0044] Compared with the prior art, the present invention has the following outstanding advantages:

[0045] Conventional testing generally uses the same antigen as both a labeled antigen and a coated antigen. For example, the detection of African swine fever virus antibodies in the present invention uses this mode.

[0046] 1. However, the detection of swine fever blue ear virus antibody and swine fever antibody in the present invention uses two antigens with different molecular weights but the same antigenic site as the labeling antigen and the coating antigen respectively.

[0047] The detection of porcine reproductive and respiratory syndrome virus antibody uses N2 protein and N1 protein as latex microsphere marker protein and detection line coating protein respectively; the detection of classical swine fever virus antibody uses E2 protein and E2a protein as latex microsphere marker protein and detection line coating protein respectively;

[0048] The larger N2 protein (molecular weight of 57.2KD) / E2 protein (molecular weight of 33.3KD) has more spatial binding sites and is easy to bind to the tested porcine reproductive and respiratory syndrome virus antibodies / classical swine fever virus antibodies; the N2 protein (molecular weight of 17.9KD) / E2a protein (molecular weight of 26KD) is smaller and is an immune-specific protein of porcine reproductive and respiratory syndrome virus / classical swine fever virus. It can specifically bind to the latex microsphere-labeled porcine reproductive and respiratory syndrome virus / classical swine fever virus antigen-antibody complex chromatographed to the detection line position, thereby improving the sensitivity of antibody detection, providing a scientific and effective technical support for the prevention and control of the occurrence of porcine reproductive and respiratory syndrome virus / classical swine fever, and also providing a reference for the rapid and sensitive detection of antibodies for other diseases.

[0049] 2. The test paper card of the present invention uses time-resolved immunofluorescent microspheres as tracers to simultaneously label antigens of three swine diseases at one time, thereby simplifying the preparation process of the test paper card, saving preparation costs, and significantly improving the sensitivity and efficiency of detection. At the same time, the chromatography test paper card is used as a primary sensor, that is, the fluorescent microsphere-labeled antigen first combines with the antibody to be tested to form a fluorescently labeled antigen-antibody complex, and then combines with the capture antigen fixed on the test paper strip after swimming, so that the detection strip produces a fluorescent signal change, and the intensity change of the fluorescent signal is positively correlated with the content of the antibody to be tested. The content level of the object to be tested is analyzed after automatic recognition and interpretation of the fluorescent signal value of the reaction strip by a computer, and quantitative detection and analysis of the three swine disease antibodies is achieved at the same time.

[0050] 3. The present invention combines three kinds of swine disease antibody fluorescence joint detection reagent cards with immunofluorescence rapid tester to form swine fever, blue ear and African swine fever virus antibody fluorescence joint detection test paper cards, which realizes real-time, rapid and quantitative monitoring of three kinds of swine disease antibodies. The application of the present invention can reduce the error rate caused by human visual judgment when using qualitative test paper cards such as colloidal gold for detection and judgment in the past, which not only improves the timeliness of detection, but also improves the objectivity and accuracy of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 : Schematic diagram of the assembly of the test strip of the present invention.

[0052] In the figure: sample pad 1, binding pad of three swine disease antigens labeled with time-resolved immunofluorescence microspheres 2, first detection line coated with African swine fever virus P30 protein 3, second detection line coated with porcine reproductive and respiratory syndrome virus N1 protein 4, third detection line coated with classical swine fever virus E2a protein 5, quality control line of anti-his tag protein antibody 6, nitrocellulose membrane 7, water absorbent pad 8, PVC backing 9, card shell 10.

[0053] Figure 2 : Schematic diagram of the test result determination of the test card of the present invention (under ultraviolet light irradiation).

[0054] In the figure: 11: indicates that the detection results of ASFV antibody, PRRSV antibody and CSFV antibody are all positive; 12: indicates that the detection results of ASFV antibody are positive, and the results of PRRSV antibody and CSFV antibody are both negative; 13: indicates that the detection results of PRRSV antibody are positive, and the results of ASFV antibody and CSFV antibody are both negative; 14: indicates that the detection results of CSFV antibody are positive, and the results of ASFV antibody and PRRSV antibody are both negative; 15: indicates that the detection results of ASFV antibody, PRRSV antibody and CSFV antibody are all negative; 16, 17: indicate that the test card is invalid.

[0055] Figure 3 : Rapid immunofluorescence test instrument. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the present invention are described clearly and completely below. The embodiments of the present invention and all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0057] Reference Figure 1-3As shown: a fluorescent joint detection test paper card for swine fever, porcine reproductive and respiratory syndrome and African swine fever virus antibodies, which includes a sample pad 1, a binding pad 2 for three swine disease antigens labeled with time-resolved immunofluorescent microspheres, a first detection line 3 coated with African swine fever virus P30 protein, a second detection line 4 coated with porcine reproductive and respiratory syndrome 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 absorbent pad 8, a PVC backing 9 and a card shell 10.

[0058] The PVC backing 9 is installed on the card shell 10, and a nitrocellulose membrane 7 is laid on the PVC backing 9. The nitrocellulose membrane 9 is sequentially marked with a third detection line 5 coated with classical swine fever virus E2a protein, a second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein, a first detection line 3 coated with African swine fever virus P30 protein and a quality control line 6 of anti-his tag protein antibody. A sample pad 1 and a binding pad 2 of three swine disease antigens labeled with time-resolved immunofluorescence microspheres are sequentially installed on one end of the PVC backing 9, and a water absorbent pad 8 is installed on the other end.

[0059] The present invention also includes a method for preparing a test paper card: a method for preparing a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, which comprises the following steps;

[0060] ① The preparation method of the binding pad 2 of three swine disease antigens labeled with time-resolved immunofluorescence microspheres is as follows;

[0061] Take 1ml of activated time-resolved immunofluorescent microsphere suspension, add three kinds of mixed antigens of African swine fever P30 protein + porcine blue ear virus N2 protein + swine fever virus E2 protein while stirring, the labeling dosage is 50-100ug, 50-100ug, 50-100ug respectively, mix at room temperature for 2 hours; add 10% BSA for blocking for 1 hour, centrifuge to remove the supernatant, take 1ml of preservation solution and add it to the microspheres after centrifugation, mix, refrigerate and keep away from light for standby use. Dilute the prepared time-resolved immunofluorescent microsphere labeled mixed antigen with working solution 5-10 times, spray it evenly on the binding pad with a gold sprayer, the spraying amount is 3.0-10.0μl / cm, place it in a 37℃ incubator to dry for 12h-18h, and prepare the binding pad 2 of three kinds of swine disease antigens labeled by time-resolved immunofluorescent microspheres for standby use.

[0062] ②. The method for labeling and activating the time-resolved immunofluorescent microspheres is as follows: take out the time-resolved immunofluorescent microspheres, disperse them in an ultrasonic cleaner for 10 seconds, add 900ul of 0.03M MES buffer in a 2mL EP tube, then add 100ul of the dispersed microspheres, vortex mix, add 5ul of solution A to the microsphere suspension, solution A is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix solution A, then add 5ul of solution B, solution B is 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix solution B again, place at room temperature to react for 30min, 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, and then disperse it evenly by ultrasound to obtain the activated time-resolved immunofluorescent microspheres.

[0063] ③. The preparation method of the first detection line 3 coated with African swine fever virus P30 protein includes: using a continuous film spotting instrument to evenly streak 0.05-1 mg / mL African swine fever virus P30 protein onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0064] ④. The preparation method of the second detection line 4 coated with the porcine reproductive and respiratory syndrome virus N1 protein includes: using a continuous film spotting instrument to evenly streak 0.2-1.5 mg / mL of porcine reproductive and respiratory syndrome virus N1 protein onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0065] ⑤. The preparation method of the third detection line 5 coated with the classical swine fever virus E2a protein includes: using a continuous film spotter to evenly streak 0.2-1.5 mg / mL classical swine fever virus E2a protein onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0066] ⑥. The preparation method of the quality control line 6 of the anti-his tag protein antibody includes: using a continuous film spotter to evenly streak 0.2-0.8 mg / mL of the anti-his tag protein antibody onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0067] ⑦. Soak the sample pad 1 in the blocking solution for 30 minutes, dry it at 37℃ and set aside.

[0068] ⑧. The third detection line 5 coated with classical swine fever virus E2a protein is 5 mm away from the end of the binding pad 2 of the three swine disease antigens labeled with time-resolved immunofluorescence microspheres. The second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein is 4 mm away from the third detection line 5 coated with classical swine fever virus E2a protein. The first detection line 3 coated with African swine fever virus P30 protein is 4 mm away from the second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein. The quality control line 6 of the anti-his tag protein antibody is 3 mm away from the end of the absorbent pad 8.

[0069] The present invention also includes a method for using a test paper card: a method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, which comprises the following steps;

[0070] ①. When the sample to be tested contains antibodies to classical swine fever virus / or antibodies to porcine reproductive and respiratory syndrome virus / or antibodies to African swine fever virus, the antibodies to African swine fever virus / or antibodies to porcine reproductive and respiratory syndrome virus / or antibodies to classical swine fever virus in the sample migrate to the binding pad 2 of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres and react with the P30 antigen / or N2 antigen / or E2 antigen labeled with time-resolved immunofluorescent microspheres.

[0071] ②. 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 African swine fever virus P30 protein, the second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein, and the third detection line 5 coated with classical swine fever virus E2a protein react again with the antibodies in the antigen-antibody complex, thereby forming a "fluorescently labeled antigen-antibody-coated antigen" double antigen sandwich complex at the detection line position, and corresponding fluorescent bands appear on the first detection line 3 coated with African swine fever virus P30 protein, the second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein, and the third detection line 5 coated with classical swine fever virus E2a protein, respectively.

[0072] ③. At the same time, the excess labeled antigens in the binding pad 2 of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres will react with the anti-his tag protein antibody in the quality control line 6 area of ​​the anti-his tag protein antibody, forming a fluorescent strip at the quality control line 6 position of the anti-his tag protein antibody.

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

[0074] ⑤. The change in fluorescence intensity of the test line is positively correlated with the content of the antibody to be tested. The content level of the test object can be analyzed by automatically identifying and interpreting the fluorescence signal of the reaction strip by the immunofluorescence rapid tester, thereby realizing quantitative detection and analysis.

[0075] 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 with a final concentration of 0.6 mM IPTG, and the expression product is purified to obtain the African swine fever virus P30 recombinant antigen,

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

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

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

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

[0080] The preparation method of recombinant porcine reproductive and respiratory syndrome virus N1 and N2 antigens is as follows: the recombinant strains of Escherichia coli BL21 / pET-N1 and Escherichia coli BL21 / pMAL-N2 are induced with a final concentration of 0.2mM / 0.8mM IPTG, respectively, and the expression products are purified to obtain recombinant porcine reproductive and respiratory syndrome virus N1 antigen and recombinant porcine reproductive and respiratory syndrome virus N2 antigen, respectively.

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

[0082] The genomic sequence of classical swine fever virus N antigen protein was searched in the NCBI database, and a partial sequence of GenBank:EU926974.1 was selected as the target gene. The target gene was selected, analyzed and optimized, and the optimized N1 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct a recombinant plasmid pET-28a-N1. The recombinant plasmid was 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: searching the genome sequence of the classical swine fever virus N antigen protein in the NCBI database, selecting a partial sequence of GenBank: EU926974.1 as a target gene, and selecting, analyzing and optimizing the target gene.

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

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

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

[0087] Search the genomic sequence of the classical swine fever virus E2 antigen protein in the NCBI database and select GenBank: FJ598611.1 as the target gene. Analyze and optimize the target gene.

[0088] The optimized E2 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2. The recombinant plasmid was 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:

[0090] The genome sequence of the E2 antigen protein of classical swine fever virus was searched in the NCBI database, and part of the gene of GenBank: FJ598611.1 was selected as the E2a target gene. The target gene was selected, analyzed and optimized.

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

[0092] Embodiment 1

[0093] The present invention provides a fluorescent joint detection test paper card for swine fever, blue ear disease and African swine fever virus antibodies based on fluorescent microsphere labeling technology. The PVC backing 9 is installed on a card shell 10, and a nitrocellulose membrane 7 is laid on the PVC backing 9. 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 disease virus N1 protein, a first detection line 3 coated with African swine fever virus P30 protein and a quality control line 6 of anti-his tag protein antibody. A sample pad 1 and a binding pad 2 of three swine disease antigens labeled with time-resolved immunofluorescent microspheres are sequentially installed on one end of the PVC backing 9, and a water absorbent pad 8 is installed on the other end.

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

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

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

[0097] The optimized P30 protein gene sequence was synthesized by a gene synthesis company and connected to the pET-21a(+) vector to construct the recombinant plasmid pET-21a-P30.

[0098] The above-mentioned recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-P30 (Escherichia coli BL21 / pET-P30). The recombinant strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on June 8, 2023, with the deposit number CCTCC NO: M2023955.

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

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

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

[0102] The optimized N1 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-N1.

[0103] The above-mentioned recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-N1 (Escherichia coli BL21 / pET-N1). The recombinant strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on June 8, 2023, with the deposit number CCTCC NO: M2023956.

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

[0105] The genomic sequence of classical swine fever virus N antigen protein was searched in the NCBI database, and a partial sequence of GenBank:EU926974.1 (Porcine respiratory and reproductive syndrome virus N gene) was selected as the target gene, and the target gene was selected, analyzed and optimized.

[0106] The optimized N2 protein gene was synthesized by a gene synthesis company and connected to the pMAL-c5x vector to construct the recombinant plasmid pMAL-c5x-N2.

[0107] The above recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pMAL-N2. The recombinant strain was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 8, 2023, with the deposit number CCTCC NO: M2023957

[0108] The preparation method of the classical swine fever virus E2 and E2a recombinant antigens is as follows: the recombinant strains of Escherichia coli BL21 / pE2 (Escherichia coli BL21 / pE2) and Escherichia coli BL21 / pE2a (Escherichia coli BL21 / pE2a) are induced respectively with a final concentration of 0.1 mM IPTG, and the expression products are purified to obtain the classical swine fever virus E2 recombinant antigen and the classical swine fever virus E2a recombinant antigen respectively.

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

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

[0111] The optimized E2 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2.

[0112] The above recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2. The recombinant strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on January 25, 2018, with the deposit number of CCTCC NO: M 2018060.

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

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

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

[0116] The above recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2a. The recombinant strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on January 25, 2018, with the deposit number of CCTCC NO: M2018059.

[0117] Among them, a preparation method of a fluorescent joint detection test paper card for swine fever, porcine reproductive and respiratory syndrome and African swine fever virus antibodies based on fluorescent microsphere labeling technology is as follows: the PVC backing 9 is installed on the card shell 10, and a nitrocellulose membrane 7 is laid on the PVC backing 9. 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 porcine reproductive and respiratory syndrome virus N1 protein, a first detection line 3 coated with African swine fever virus P30 protein and a quality control line 6 of anti-his tag protein antibody; a sample pad 1 and a binding pad 2 of three swine disease antigens labeled with time-resolved immunofluorescent microspheres are sequentially installed on one end of the PVC backing 9, and a water absorbent pad 8 is installed on the other end.

[0118] Specifically, the preparation method of the binding pad of African swine fever P30 protein / polysaccharide blue ear virus N2 protein / classic swine fever virus E2 protein labeled with time-resolved immunofluorescent microspheres is as follows: take 1 ml of the activated time-resolved immunofluorescent microsphere suspension, add three kinds of swine disease mixed antigens of African swine fever P30 protein +polysaccharide blue ear virus N2 protein +classic swine fever virus E2 protein dropwise while stirring, the labeled dosage is 80ug, 100ug, 100ug (0.2mg / ml, diluted with ultrapure water), respectively, at room temperature

[0119] Mix at room temperature for 2 hours (Speed ​​20, vertical); add 10% BSA (final concentration of 0.5%) for blocking for 1 hour (Speed ​​20, vertical), centrifuge to remove supernatant (12000r, 4°C, 15min); take 1ml preservation solution (African swine fever P30 protein latex labeling preservation solution) and add it to the microspheres after centrifugation, mix well, refrigerate and keep away from light for later use. Dilute the prepared time-resolved immunofluorescence microspheres labeled with mixed antigen working solution 10 times, spray it evenly on the binding pad with a gold sprayer, the spray volume is 8.0μl / cm, and place it in a 37°C incubator to dry for 12h-18h to prepare the African swine fever P3 protein / porcine blue ear virus N2 protein / swine fever virus E2 protein binding pad labeled with time-resolved immunofluorescence microspheres for later use.

[0120] Furthermore, the method for labeling and activating the time-resolved immunofluorescent microspheres is as follows: take out the time-resolved immunofluorescent microspheres (solid content 1%), disperse them in an ultrasonic cleaner (180w) for 10s; add 900u1 of 0.03M MES buffer (pH6.0) to a 2mL EP tube, then add 100u1 of the 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 place at room temperature for reaction for 30min (Speed 20, vertical), centrifuge and remove the supernatant (12000r, 4℃, 15min); add 2ml of cross-linking buffer to resuspend and wash the precipitate, centrifuge and remove the supernatant (12000r, 4℃, 15min); add 1ml of cross-linking buffer to the precipitate, and then ultrasonically disperse it evenly (180W, 2min), which is the activated time-resolved immunofluorescent microspheres.

[0121] Specifically, the preparation method of the first detection line 3 coated with African swine fever virus P30 protein includes: using a continuous film dot meter to evenly streak 0.5 mg / mL African swine fever virus P30 protein onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0122] Specifically, the second detection line 4 coated with the porcine reproductive and respiratory syndrome virus N1 protein has a preparation method comprising: using a continuous dot-film apparatus to evenly streak 1.2 mg / mL of porcine reproductive and respiratory syndrome virus N1 protein onto a nitrocellulose membrane 9 at a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0123] Specifically, the preparation method of the third detection line 5 coated with the classical swine fever virus E2a protein includes: using a continuous film dot meter to evenly streak 1 mg / mL classical swine fever virus E2a protein onto a nitrocellulose membrane 9, with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0124] Specifically, the preparation method of quality control line 6 of anti-his tag protein antibody includes: using a continuous spotting instrument to evenly streak 0.5 mg / mL anti-his tag protein antibody onto a nitrocellulose membrane with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside.

[0125] Specifically, the absorbent pad 8 is soaked in the blocking solution for 30 minutes, dried at 37°C, and set aside.

[0126] The third detection line 5 coated with classical swine fever virus E2a protein is 5 mm away from the end of the binding pad 2 of the three swine disease antigens labeled with time-resolved immunofluorescence microspheres, the second detection line 4 coated with porcine reproductive and respiratory syndrome virus N1 protein is 4 mm away from the third detection line 5 coated with classical 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 reproductive and respiratory syndrome 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 absorbent pad 8.

[0127] Embodiment 2

[0128] The performance test paper cards for the swine fever, blue ear and African swine fever virus antibody fluorescence joint detection prepared in Example 1 were tested as follows:

[0129] (1) Determination of the critical value (judgment point) of the test paper card

[0130] The test paper cards were prepared using the preparation process established above, and 189 African swine fever serum sample plates with known results were tested (101 of them were positive serum and 88 were negative serum), 196 porcine blue ear serum sample plates were tested (125 of them were positive serum and 71 were negative serum in the neutralization test), and 205 swine fever serum sample plates with known results were tested (133 of them were positive serum and 72 were negative serum). The ROC curves were drawn using Prism6.0 software, and the point closest to the upper left corner of the curve was selected. The Youden index was calculated based on the sensitivity and specificity of each possible cut point in the statistical results. The cut point with the largest Youden index was used as the critical point to determine the optimal critical values ​​of the three swine disease antibodies. The area under the ROC curve can reflect the accuracy of the diagnostic test. When the area under the ROC curve is above 0.9, the diagnosis has a high accuracy.

[0131] The results are as follows: 189 African swine fever serum sample plates with known results were tested and the ROC curve was drawn. The area under the ROC curve was 0.9823. The ROC curve confirmed that the optimal critical point for African swine fever antibody diagnosis was 0.4004, with a sensitivity of 85.56% and a specificity of 91.65%. Therefore, the critical point of the swine fever antibody detection of the present invention was set to 0.4. A detection value ≥ 0.4 indicates that the ASFV antibody test is positive; a detection value < 0.4 indicates that the ASFV antibody test is negative

[0132] 196 porcine blue ear serum sample plates with known results were tested and the ROC curve was drawn. The area under the ROC curve was 0.9141. The ROC curve confirmed that the optimal critical point for the diagnosis of African blue ear antibodies was 0.4164, with a sensitivity of 82.59% and a specificity of 87.34%. Therefore, the critical point of the porcine blue ear antibody detection of the present invention was set to 0.4. If the detection value is ≥0.4, the PRRSV antibody detection is positive; if the detection value is <0.4, the PRRSV antibody detection is negative

[0133] 205 swine fever serum sample plates with known results were tested and the ROC curve was drawn. The area under the ROC curve was 0.9468. The ROC curve confirmed that the optimal critical point for swine fever antibody diagnosis was 0.4963, with a sensitivity of 83.46% and a specificity of 90.28%. Therefore, the critical point of the swine fever antibody detection item of the test paper card of the present invention was set to 0.5. If the detection value is ≥0.5, the CSFV antibody test is positive; if the detection value is <0.5, the CSFV antibody test is negative

[0134] (2) Sensitivity test

[0135] The positive serum of African swine fever virus (ASFV) was diluted from 1:100 to 1:100, 1:200, 1:400, ... 1:6400 series of positive serum. 50 μL of the diluted serum was taken respectively and tested with the test paper card of the present invention. The positive serum of each dilution was tested 5 times. After reacting for 15 minutes, the test paper card was placed in the immunofluorescence rapid tester to read the test results.

[0136] The positive serum of porcine reproductive and respiratory syndrome virus (PRRSV) was diluted from 1:100 to 1:100, 1:200, 1:400, ... 1:3200 series of positive serum. 50 μL of the diluted serum was taken respectively and tested with the test paper card of the present invention. The positive serum of each dilution was tested 5 times. After reacting for 15 minutes, the test paper card was placed in an immunofluorescence rapid tester to read the test result.

[0137] The national reference sample of swine fever virus (CSFV) positive serum was diluted from 1:16 to 1:16, 1:32, 1:64 ... 1:512 series of positive serum. 50 μL of the diluted serum was taken respectively and tested with the test paper card of the present invention. The positive serum of each dilution was tested 5 times. After reacting for 15 minutes, the test paper card was placed in an immunofluorescence rapid tester to read the test results.

[0138] Specifically, African swine fever virus-positive serum was purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, specification: 1 mL.

[0139] Specifically, the porcine reproductive and respiratory syndrome virus (PRRSV) positive serum was purchased from the China Veterinary Microbiological Culture Collection Center, number: CVCC Z230, specification: 5 mL.

[0140] Specifically, the national reference product of swine fever positive serum was purchased from China Veterinary Microbiological Culture Collection Center, No.: CVCC Z102, specification: 1 mL.

[0141] Test results: ASFV / PRRSV / CSFV positive sera of different dilutions were measured using the test paper card of the present invention. When the ASFV positive serum was diluted to 1:3200, the detection value of the ASFV detection item was still ≥0.4, showing a positive result; when the PRRSV positive serum was diluted to 1:1600, the detection value of the PRRSV detection item was still ≥0.4, showing a positive result; when the CSFV positive serum was diluted to 1:256, the detection value of the CSFV detection item was still ≥0.5, showing a positive result; indicating that the test paper card of the present invention has high sensitivity.

[0142] Table 1 Sensitivity test results of the test card of the present invention for detecting three standard antigens

[0143]

[0144] (2) Specificity experiment

[0145] Take 50 μL each of African swine fever virus (ASFV) positive serum, porcine blue ear (PRRSV) positive serum, classical swine fever virus (CSFV) positive serum national reference, swine foot-and-mouth disease virus (FMDV) antibody positive serum, pseudorabies virus (PRV) positive serum reference, porcine circovirus (PCV) CAP protein antibody, and recombinant porcine parvovirus (PPV) VP2 protein antibody, and detect them with the test paper card of the present invention respectively, react for 15 minutes, put the test paper card into the immunofluorescence rapid tester, and read the test results.

[0146] Specifically, African swine fever virus (ASFV) positive serum was purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, specification: 1 mL.

[0147] Specifically, the porcine reproductive and respiratory syndrome virus (PRRSV) positive serum was purchased from the China Veterinary Microbiological Culture Collection Center, number: CVCC Z230, specification: 5 mL.

[0148] Specifically, the national reference product of swine fever virus (CSFV) positive serum was purchased from China Veterinary Microbiological Culture Collection Center, No.: CVCC Z102, Specification: 1 mL

[0149] Specifically, the swine foot-and-mouth disease virus (FMDV) antibody-positive serum was taken from a swine foot-and-mouth disease antibody ELISA kit, purchased from Beijing Biodi Biotechnology Co., Ltd., brand: Cusabio, item number: CSB-EQ027745PI.

[0150] Specifically, the pseudorabies virus (PRV) positive serum reference material was purchased from the China Veterinary Microbiological Culture Collection Center, number: CVCC Z289, specification: 1 mL.

[0151] Specifically, porcine circovirus (PCV) CAP protein antibody was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., brand: Biosen, product number: bs-20021R.

[0152] Specifically, the porcine recombinant porcine parvovirus (PPV) VP2 protein antibody was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., brand: Biosen, product number: bs-2309R.

[0153] Table 2: Specificity test results of the test card of the present invention

[0154]

[0155]

[0156] Test results: When testing ASFV, PRRSV, and CSFV antibodies, the test paper card should be placed in the immunofluorescence rapid tester after 15 minutes to read the test results. When testing ASFV positive serum, the corresponding ASFV test value is positive (2.3431), and the test values ​​of the other two items are negative; when testing PRRSV positive serum, the corresponding PRRSV test value is positive (1.8932), and the test values ​​of the other two items are negative; when testing CSFV positive serum, the corresponding CSFV test value is positive (1.2467), and the test values ​​of the other two items are negative; when testing FMDV, PRV, PCV, and PP antibodies, after 15 minutes of reaction, the test paper card should be placed in the immunofluorescence rapid tester to read the test results. The test results of ASFV items are all <0.4, the test results of PRRSV items are all <0.4, and the test results of CSFV items are all <0.5. The test results of the three swine disease test items and other swine disease items are all negative.

[0157] It can be seen from this that the test paper card of the present invention has high specificity and has no cross-reaction with antibodies of other important disease viruses of pigs.

[0158] (3) Compliance test

[0159] A total of 246 pig sera were collected from Hubei, Henan, Jiangxi, Shanghai and other places using the test paper card of the present invention, and the test paper card of the present invention and the African swine fever\blue ear\swine fever ELISA antibody detection kit were used for detection. The test results compared between the two are shown in Tables 3-5. As can be seen from Table 3, when detecting ASFV antibodies, the positive compliance rate of the test paper card of the present invention and the African swine fever ELISA kit is 90.83% and the negative compliance rate is 92.06%, and the total compliance rate is 91.46%. As can be seen from Table 4, when detecting PRRSV antibodies, the positive compliance rate of the test paper card of the present invention and the blue ear ELISA kit is 87.88% and the negative compliance rate is 83.33%, and the total compliance rate is 86.99%. And as can be seen from Table 5, when detecting CSFV antibodies, the positive compliance rate of the test paper card of the present invention and the swine fever ELISA kit is 95.12% and the negative compliance rate is 92.68%, and the total compliance rate is 94.31%. The results show that the test results of the kit of the present invention are basically consistent with those of ELISA, and the coincidence rate is good.

[0160] Specifically, the African swine fever virus ELISA antibody detection kit was purchased from INGENASA in Spain, batch number: 011019-B.

[0161] Specifically, the porcine blue ear virus ELISA antibody detection kit was purchased from IDEXX in the United States, product number 43220.

[0162] Specifically, the ELISA antibody detection kit for classical swine fever virus was purchased from IDEXX in the United States, catalog number 43222.

[0163] Table 3 Results of clinical detection of ASFV antibodies by the test paper card of the present invention and the ELISA kit

[0164]

[0165] Table 4 Clinical detection results of PRRSV antibodies by the test paper card of the present invention and the ELISA kit

[0166]

[0167] Table 5 Clinical test results of CSFV antibodies by the test paper card of the present invention and ELISA kit

[0168]

[0169] The parts not described in detail above are all prior art.

Claims

1. A fluorescent joint test paper card for swine fever, porcine reproductive and respiratory syndrome and African swine fever virus antibodies, comprising a sample pad (1), a binding pad (2) of three swine disease antigens labeled with time-resolved immunofluorescent microspheres, a first detection line (3) coated with African swine fever virus P30 protein, a second detection line (4) coated with porcine reproductive and respiratory syndrome 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 absorbent 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), and a nitrocellulose membrane (7) is laid on the PVC backing (9). The nitrocellulose membrane (9) is sequentially marked with a third detection line (5) coated with classical swine fever virus E2a protein, a second detection line (4) coated with porcine reproductive and respiratory syndrome virus N1 protein, a first detection line (3) coated with African swine fever virus P30 protein, and a quality control line (6) of anti-his tag protein antibody. A sample pad (1) and a binding pad (2) of three swine disease antigens labeled with time-resolved immunofluorescence microspheres are sequentially installed on one end of the PVC backing (9), and a water absorbent pad (8) is installed on the other end.

2. The method for preparing a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 1, characterized in that: It includes the following steps; ① The preparation method of the binding pad (2) of three swine disease antigens labeled with time-resolved immunofluorescence microspheres is as follows; Take 1ml of activated time-resolved immunofluorescent microsphere suspension, add three kinds of mixed antigens of African swine fever P30 protein + porcine blue ear virus N2 protein + classical swine fever virus E2 protein while stirring, the labeling dosage is 50-100ug, 50-100ug, 50-100ug respectively, mix at room temperature for 2 hours; add 10% BSA for blocking for 1 hour, centrifuge to remove the supernatant, take 1ml of preservation solution and add it to the microspheres after centrifugation, mix well, refrigerate and keep away from light for standby use. Dilute the prepared time-resolved immunofluorescent microsphere labeled mixed antigen with working solution 5-10 times, spray it evenly on the binding pad with a gold sprayer, the spray volume is 3.0-10.0μl / cm, place it in a 37℃ incubator to dry for 12h-18h, and prepare the binding pad (2) of three kinds of swine disease antigens labeled by time-resolved immunofluorescent microspheres, set aside, ②. The method for labeling and activating the time-resolved immunofluorescent microspheres is as follows: take out the time-resolved immunofluorescent microspheres, disperse them in an ultrasonic cleaner for 10 seconds, add 900u1 of 0.03MMES buffer to a 2mL EP tube, then add 100u1 of the dispersed microspheres, vortex mix, add 5ul of solution A to the microsphere suspension, solution A is a 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix the solution A, then add 5ul of solution B, solution B is a 10mg / ml N-hydroxysuccinimide ethanol solution, vortex mix the solution B again, place at room temperature to react 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, and then disperse it evenly by ultrasound to obtain the activated time-resolved immunofluorescent microspheres. ③. The preparation method of the first detection line (3) coated with African swine fever virus P30 protein comprises: using a continuous film spotting instrument to evenly streak 0.05-1 mg / mL of African swine fever virus P30 protein onto a nitrocellulose membrane (9), with a streaking volume of 1 μL / cm, drying in a 37° C. incubator for 12-18 hours, and setting aside. ④. The preparation method of the second detection line (4) coated with the porcine reproductive and respiratory syndrome virus N1 protein comprises: using a continuous film spotting instrument to evenly streak 0.2-1.5 mg / mL of porcine reproductive and respiratory syndrome virus N1 protein onto a nitrocellulose membrane (9) at a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside. ⑤. The preparation method of the third test line (5) coated with the classical swine fever virus E2a protein comprises: using a continuous film spotter to evenly streak 0.2-1.5 mg / mL classical swine fever virus E2a protein onto a nitrocellulose membrane (9) at a streaking volume of 1 μL / cm, drying in a 37° C. incubator for 12-18 hours, and setting aside. ⑥. The preparation method of the quality control line (6) of the anti-his tag protein antibody comprises: using a continuous spotting instrument to evenly streak 0.2-0.8 mg / mL of the anti-his tag protein antibody onto a nitrocellulose membrane (9), with a streaking volume of 1 μL / cm, drying in a 37°C incubator for 12-18 hours, and setting aside. ⑦. Soak the sample pad (1) in the blocking solution for 30 minutes, dry it at 37°C and set aside. ⑧. The third detection line (5) coated with classical swine fever virus E2a protein is 5 mm away from the end of the binding pad (2) of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres. The second detection line (4) coated with porcine reproductive and respiratory syndrome virus N1 protein is 4 mm away from the third detection line (5) coated with classical 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 reproductive and respiratory syndrome virus N1 protein. The quality control line (6) of the anti-his tag protein antibody is 3 mm away from the end of the absorbent pad (8).

3. A method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies, characterized in that: It includes the following steps; ①. When the sample to be tested contains antibodies to classical swine fever virus / antibodies to porcine reproductive and respiratory syndrome virus / antibodies to African swine fever virus, the antibodies to African swine fever virus / antibodies to porcine reproductive and respiratory syndrome virus / antibodies to classical swine fever virus in the sample migrate to the binding pad (2) of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres and react with the P30 antigen / or the N2 antigen / or the E2 antigen labeled with the time-resolved immunofluorescent microspheres. ②. 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 reproductive and respiratory syndrome virus N1 protein, and the third detection line (5) coated with the classical swine fever virus E2a protein react again with the antibodies in the antigen-antibody complex, thereby forming a "fluorescently labeled antigen-antibody-coated antigen" double antigen sandwich complex at the detection line position, and corresponding fluorescent bands are respectively presented on the first detection line (3) coated with the African swine fever virus P30 protein, the second detection line (4) coated with the porcine reproductive and respiratory syndrome virus N1 protein, and the third detection line (5) coated with the classical swine fever virus E2a protein. ③. The excess labeled antigens in the binding pad (2) of the three swine disease antigens labeled with time-resolved immunofluorescent microspheres will react with the anti-his tag protein antibody in the quality control line (6) of the anti-his tag protein antibody to form a fluorescent strip at the quality control line (6) of the anti-his tag protein antibody. ④. When the antibody content in the sample is higher, the fluorescence signal in the test reaction zone detection line area on the nitrocellulose membrane (9) is stronger, and vice versa. ⑤. The change in fluorescence intensity of the test line is positively correlated with the content of the antibody to be tested. The content level of the test object can be analyzed by automatically identifying and interpreting the fluorescence signal of the reaction strip by the immunofluorescence rapid tester, thereby realizing quantitative detection and analysis.

4. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies 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 with a final concentration of 0.6 mM 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 comprises: The genome sequence of the African swine fever virus P30 antigen protein was searched in the NCBI database, and GenBank: JX524222.1 was selected as the target gene. The target gene was selected, analyzed and optimized. The optimized P30 protein gene sequence was synthesized by a gene synthesis company and connected to the pET-21a(+) vector to construct the recombinant plasmid pET-21a-P30. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-P30.

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

6. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 5, characterized in that: The preparation method of the recombinant strain Escherichia coli BL21 / pET-N1 comprises: The genomic sequence of classical swine fever virus N antigen protein was searched in the NCBI database, and a partial sequence of GenBank:EU926974.1 was selected as the target gene. The target gene was selected, analyzed and optimized, and the optimized N1 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct a recombinant plasmid pET-28a-N1. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pET-N1.

7. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 6, characterized in that: The preparation method of the recombinant strain Escherichia coli BL21 / pMAL-N2 comprises: searching the genome sequence of the classical swine fever virus N antigen protein in the NCBI database, selecting a partial sequence of GenBank: EU926974.1 as a target gene, and selecting, analyzing and optimizing the target gene. The optimized N2 protein gene was synthesized by a gene synthesis company and connected to the pMAL-c5x vector to construct the recombinant plasmid pMAL-c5x-N2. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pMAL-N2.

8. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 7, characterized in that: The preparation method of classical swine fever virus E2 and E2a recombinant antigens is as follows: the recombinant strains of Escherichia coli BL21 / pE2 and Escherichia coli BL21 / pE2a are induced with a final concentration of 0.1 mM IPTG, and the expression products are purified to obtain classical swine fever virus E2 recombinant antigen and classical swine fever virus E2a recombinant antigen, respectively.

9. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 8, characterized in that: The preparation method of the recombinant strain Escherichia coli BL21 / pE2 comprises: Search the genomic sequence of the classical swine fever virus E2 antigen protein in the NCBI database and select GenBank: FJ598611.1 as the target gene. Analyze and optimize the target gene. The optimized E2 protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2.

10. The method for using a fluorescent joint detection test paper card for swine fever, blue ear and African swine fever virus antibodies according to claim 9, characterized in that: The preparation method of the recombinant strain Escherichia coli BL21 / pE2a comprises: The genome sequence of the E2 antigen protein of classical swine fever virus was searched in the NCBI database, and part of the gene of GenBank: FJ598611.1 was selected as the E2a target gene. The target gene was selected, analyzed and optimized. The optimized E2a protein gene was synthesized by a gene synthesis company and connected to the pET-28a(+) vector to construct the recombinant plasmid pET-28a-E2a. The recombinant plasmid was transformed into Escherichia coli BL21 competent cells to obtain the recombinant strain Escherichia coli BL21 / pE2a.

Images