1. An African swine fever serum antibody IPMA detection reagent, wherein an African swine fever virus attenuated strain (HLJ/18-7GD) is selected as a seed virus in the detection reagent.

2. The reagent for detecting serum antibody IPMA of African swine fever according to claim 1, wherein PK15 cell is used as inoculation cell of African swine fever attenuated strain (HLJ/18-7 GD).

3. The preparation method of the African swine fever serum antibody IPMA detection reagent of claim 1, the method comprises the following steps:

(1) selection of seed virus: selecting an African swine fever attenuated strain (HLJ/18-7GD strain) as an inoculum;

(2) and preparing a reaction plate: culturing PK15 cells in a cell plate, discarding culture solution when the cell density is 80-90%, inoculating HLJ/18-7GD strain, and culturing in an incubator to obtain the reaction plate.

(3) And observation of results: adopting a dilution multiple of 1: 10-1: 50 to incubate ASFV high-immune pig positive serum, diluting anti-pig secondary antibody at a ratio of 1:1000-1:5000, incubating, developing with a developing solution, and observing a developing result under a common optical microscope.

4. The method for preparing the African swine fever serum antibody IPMA detection reagent of claim 3, wherein the virus infection dose is 5 x10⁶TCID₅₀/mL-2×10⁷TCID₅₀/mL。

5. The method for preparing the African swine fever serum antibody IPMA detection reagent according to claim 3 or 4, wherein the serum dilution factor is 1:80-1: 160.

Background

African Swine Fever (ASF) is an acute, virulent and highly infectious disease of pigs caused by African Swine Fever Virus (ASFV), and no commercial vaccine for prevention and effective therapeutic drug exist at present. The African swine fever virus belongs to the genus of African swine fever virus of the family of African swine fever virus, and is the only member of the genus at present. ASFV mainly infects domestic pigs and wild pigs, the clinical symptoms are similar to Classical Swine Fever (CSF), the main symptoms are persistent fever, and the pigs only show anorexia or disappearance and dyspnea. Pigs usually die acutely after ASFV infection, and the death rate is up to 100%. From 2016 to 2020, 14327 of global co-occurrence of ASF epidemic, loss of 820 million pigs. According to incomplete statistics, since the epidemic situation outbreaks in China in 2018, the domestic reported ASF epidemic situation reaches 188 times, more than 120 thousands of live pigs are killed, and serious loss is caused to the pig industry in China. Nowadays, ASF seriously threatens pork production and food safety in China and even the world, is an infectious disease which is highly concerned and continuously monitored by the world animal health Organization (OIE), and is listed as an animal epidemic disease in China.

ASFV is the only double-stranded nucleoplasmic large DNA viruses (NCLDV) that have been found to transmit via arthropods as vectors. The virus particles are of a multilayer structure of a positive 20-face body, and are respectively nucleolus, protein nucleocapsid, lipid envelope and outermost protein capsid from inside to outside. The ASFV particle diameter is between 260-300 nm, the protein shell is composed of main capsid protein (P72) and four minor capsid proteins (M1249, P17, P49 and H240R). The ASFV genome length is about 170-190Kbp, and can encode 151-167 Open Reading Frames (ORF).

Commonly used detection methods for African swine fever virus include enzyme-linked immunosorbent assay (ELISA), serum neutralization assay, immunofluorescence technique, Polymerase Chain Reaction (PCR), nucleic acid probe, gene chip, and the like. These methods have good specificity, sensitivity and efficiency, but the cost is relatively high, the equipment requirement is also high, and the methods are not beneficial to basic level and clinical popularization.

The serological method is an important content of epidemiological monitoring and an important basis for establishing a prevention and control strategy, particularly has important significance for detecting ASF-resistant pigs and ASF attenuated strain infected pigs (the clinical symptoms are slight and are not easy to be found), and can also become a powerful means for immune monitoring once the commercialized ASF vaccine is put into the market. Meanwhile, an immunoperoxidase monolayer cell test (IPMA) has the characteristics of rapidness, sensitivity and specificity. The result can be observed and judged under an optical microscope. In addition, the IPMA reaction plate can be prepared in advance and stored for a long time at 4 ℃; the reaction result of IPMA can be stored for a long time, and is convenient to observe and analyze.

The domestic pig breeding is roughly divided into two types of free-range breeding and large-scale breeding, and the two types of free-range breeding and large-scale breeding have differences in breeding management, laboratory detection capability and epidemic prevention level. For scattered farmers and medium-sized and small enterprises with low feeding management level, an epidemic disease prevention and control system is often not perfect, and detection equipment is relatively lagged or deficient, so that the IPMA detection method is a good choice during spreading of African swine fever epidemic diseases.

However, the safety requirement of IPMA detection on antigen is not met by epidemic isolation strains (PIG/HLJ/18) of African swine fever virus in China, and the detection rate of IPMA is influenced by unobvious antibody reaction aiming at ASF resistant PIGs and ASF attenuated strains infected PIGs. The virus adopted in the foreign ASF-IPT (ASF-IPMA) detection method is an ASF cell adapted strain, the adopted cell is an MS or Vero cell, and the adapted strain has more mutations and may have unknown antigenic change.

Therefore, the construction and selection of the safe and effective African swine fever attenuated strain are the basis for establishing the IPMA method of the African swine fever, and the accuracy of the IPMA detection result is further improved by further improving the presentation degree of the antigen-antibody reaction result.

Disclosure of Invention

Important zoonosis and virulent exotic disease teams in the high headquarters of the Harbin veterinary research institute of Chinese agricultural academy of sciences construct and select safe and effective African swine fever attenuated strains (HLJ/18-7GD) by carrying out partial gene knockout on epidemic isolates (PIG/HLJ/18) of the African swine fever virus in China, and lay the foundation for establishing an IPMA method of the African swine fever. Meanwhile, PK15 cells are selected as inoculation cells of an African swine fever attenuated strain (HLJ/18-7GD), infected cells with uniform shapes and specific coloring are obtained, and the result misjudgment rate of IPMA is reduced.

The present invention has been completed based on the above scientific findings.

Firstly, the invention provides an African swine fever serum antibody IPMA detection reagent, wherein the African swine fever virus in the detection reagent is African swine fever attenuated strain (HLJ/18-7GD) (the preservation numbers are CCTCC NO: V201925 and CCTCC NO: V201924).

Further, PK15 cells are used as inoculation cells of an African swine fever attenuated strain (HLJ/18-7GD) in the detection reagent.

Secondly, the invention provides a preparation method of the African swine fever serum antibody IPMA detection reagent, which comprises the following steps:

1. selection of seed virus: selecting an African swine fever attenuated strain (HLJ/18-7GD strain) as an inoculum;

2. preparation of reaction plate: culturing PK15 cells in a cell plate, discarding culture solution when the cell density is 80-90%, inoculating HLJ/18-7GD strain, and culturing in an incubator to obtain the reaction plate.

3. Observation of the results: adopting a dilution multiple of 1: 10-1: 50 to incubate ASFV high-immune pig positive serum, diluting anti-pig secondary antibody at a ratio of 1:1000-1:5000, incubating, developing with a developing solution, and observing a developing result under a common optical microscope.

Further, it is preferable that the virus-infecting dose is 5X 10⁶TCID₅₀/mL-2×10⁷TCID₅₀mL, more preferably 1X 10⁷TCID₅₀and/mL. Preferably, the virus replication time is 60h to 80h, more preferably 72 h.

Further, the serum dilution factor is 1:80 to 1:160, and more preferably 1: 100.

Further, the secondary antibody dilution was 1:1000-1: 2000 fold dilution, more preferably 1:2000 fold dilution.

The African swine fever serum antibody IPMA detection reagent takes HLJ/18-7GD virus as seed virus and PK15 cell as infected cell, after staining, the cells infected with the HLJ/18-7GD virus are uniformly stained, uniform in shape, small and regular dark rose red round staining, and the negative control cell hole has clean background and no non-specific staining.

The antigen for detection is a whole virus infecting cells, so the detected antibody can relatively truly represent the serum reaction of the antibody or close to the antibody, and the IPMA has potential value of becoming a 'gold standard' of ASF serological detection under the condition that the African swine fever lacks effective neutralizing antibodies aiming at all strains. Compared with ELISA, the sensitivity of the detection is higher, the specificity and the detection time are equivalent to those of ELISA, but the requirements on instruments and equipment are lower, observation and judgment are carried out only by means of a common optical microscope, and meanwhile, the IPMA detection result can be stored for a long time, so that later-period experimental result arrangement and file retrieval are facilitated.

Description of the drawings:

FIG. 1: rASFV delta 360 and HLJ/18-7GD infection PK15 and Vero cell fluorescence and IPMA results (the position shown by an arrow and a circle is that partial cells are lighter after Vero cell staining and are easy to be regarded as non-staining to cause misjudgment)

FIG. 2: fluorescence results at different times after PK15 cells were infected with different doses of HLJ/18-7GD virus (the position shown by the arrow and the circle is a well-formed fluorescent clonal focus)

FIG. 3: ASFV positive serum and virus infected cell show rose red negative serum and cell contrast have no coloration

Detailed Description

The following examples further illustrate the significant effects of the african swine fever serum antibody IPMA detection reagent of the present invention and the preparation method.

Example 1: materials and methods

1.1 safe and effective 7 gene deletion African swine fever attenuated strain (HLJ/18-7GD strain, simultaneously expressing red and green fluorescent protein) and MGF360-505R deletion African swine fever attenuated strain (rASFV delta 360 strain, expressing green fluorescent protein) attenuated strain, which are prepared, identified and stored by Harbin veterinary research of Chinese academy of agricultural sciences.

1.2 cells African green monkey Kidney (Vero) cells, pig Kidney (Porcine Kidney, PK15) cells, preserved by Harbin veterinary research, Chinese academy of agricultural sciences.

1.3 the culture solution of the Vero cells in the culture medium is DMEM containing 10% FBS; the culture solution of PK15 cells was DMEM containing 5% FBS.

1.4 main materials and reagent AEC color developing solution purchased from Sigma Aldrich (Sigma-Aldrich); HRP-labeled murine anti-porcine IgG (H + L) secondary antibodies were purchased from fornedy (Frdbio) science and technology (wuhan) ltd; MEM liquid media was purchased from Hyclone (GE); 8% Paraformaldehyde was obtained from white shark Biotechnology (Biosharp Life Sciences) Ltd; selective stabilizers were purchased from sumodiqi (Surmodics) inc; the ASF-P30-iELISA antibody detection kit is purchased from Loyang Putai Biotechnology Co., Ltd; ASF-VP 72-blocking ELISA antibody detection kit was purchased from Ingenasa; ASF-P54-i-ELISA antibody detection kit was purchased from the Netherlands Baibiao (Biochek) company.

1.5 screening of strains and cells two alternative cells, PK15 and Vero, were selected and cultured with 5% DMEM culture solution until the cells were full of the whole monolayer, trypsinized, the cells were plated in 96-well cell plates at a ratio of 1:5, the culture solution was discarded when the cell density was 80-90%, and two ASFV strains, rASFV Δ 360 and HLJ/18-7GD, were diluted with 2% DMEM until the virus content was 3 × 10⁷Or 3X 10⁶TCID₅₀The cells were inoculated with each mL of the virus (rASFV. DELTA.360 infection was strong and the virus content was 3X 10⁶TCID₅₀Inoculation is carried out in a volume of one milliliter (mL); HLJ/18-7GD infectivity is weak, and the virus content is 3 x10⁷TCID₅₀/mL inoculation), placed in CO at 37 deg.C₂Culturing in an incubator for 72 hours, placing cells inoculated with rASFV delta 360 in an inverted fluorescence microscope for green fluorescence, and placing cells inoculated with HLJ/18-7GD in red fluorescence for observation. Inoculated cells were CO-incubated at 37 ℃ with non-inoculated empty cell control plates₂After culturing for 72 hours in an incubator, preparing a reaction plate, and incubating ASFV high-immune pig positive by adopting a dilution multiple of 1:50The anti-porcine secondary antibody was diluted at a ratio of 1:2000 in serum and incubated, and after development with 30ul of developing solution at 37 ℃ for 30min, the development results were observed under a normal optical microscope.

1.6 determination of viral infection dose and replication time based on viral content of 3X 10⁷、1×10⁷、3×10⁶TCID₅₀HLJ/18-7GD seeded cells in three infection gradients/mL, placed at 37 ℃ CO₂Culturing, and observing under an inverted fluorescence microscope at three infection times of 24h, 48h and 72h respectively.

1.7 preparation of IPMA reaction plate and storage of inoculated PK15 cells together with non-inoculated empty cell control plate at 37 deg.C CO₂The culture was carried out in an incubator for 72 hours, the culture medium was discarded, and 4% paraformaldehyde tissue fixing solution was added in a volume of 100ul per well of a 96-well plate. And (3) washing the plate for 5 times by PBST, adding 0.04% Triton-X100 membrane permeation solution, standing for 15min, continuously washing the plate for 5 times, and sealing the fish skin glue sealing solution in a 37 ℃ incubator for 2 h. And removing the sealing liquid after sealing, airing the reaction plate, and sealing and storing at 4 ℃.

1.8 determination of secondary antibody dilution ASFV standard positive serum and standard negative serum were diluted in four concentration gradients of 0, 1:100, 1:300, 1:1000 and incubated in one column (1, 2, 3, 4); and diluting the secondary antibody with a secondary antibody preservative solution by four concentration gradients of 1:1000, 1:2000, 1:4000 and 1:8000, incubating each concentration into one line (A, B, C, D) to perform a chessboard test of the dilution of the standard positive serum and the standard negative serum and the secondary antibody, observing the coloration shade of positive cells and the coloration shade of non-specific coloration and background coloration under a common optical microscope after AEC coloration, and judging the final dilution multiple of the secondary antibody according to the coloration of the standard negative serum and the standard positive serum.

1.9 determination of serum dilution factor, 26 known negative sera and four weak positive sera which are sampled from two different regions, have different hemolysis degrees and different ages in days are selected for determination of serum dilution factor. Performing gradient dilution on 30 parts of serum at four concentrations of 0, 1:10, 1:20, 1:40, 1:80 and 1:160, and performing 1: 100-fold dilution on standard positive serum and standard negative serum; the anti-porcine secondary antibody was diluted with a secondary antibody preservative at a ratio of 1:2000 to carry out a serum dilution factor determination test, and after AEC color development, nonspecific coloration of cells and background coloration were observed under a normal optical microscope, and the final dilution factor of serum was judged from the coloration of 30 portions of serum.

1.10 specific test selected from Classical Swine Fever Virus (CSFV), Porcine reproductive and respiratory syndrome virus (PPRSV), Porcine Parvovirus (PPV), Porcine circovirus type 2 (PCV 2), Bovine Viral Diarrhea Virus (BVDV), Porcine foot-and-mouth disease virus (FMDV), Porcine Delta Coronavirus (Porcine Delta Coronavir, PDCoV), Porcine Pseudorabies virus (Primula virus, Primula V), Porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), Porcine epidemic virus (Porcine epidemic viral hepatitis virus), Porcine infectious swine virus (Porcine seronegative seropositive seronegative Porcine influenza virus, Porcine influenza virus (Porcine infectious seronegative seropositive influenza virus, Porcine swine fever virus (Porcine reproductive negative seronegative influenza virus, Porcine influenza virus), Porcine influenza virus (Porcine infectious seronegative seropositive influenza virus, Porcine swine fever virus (Porcine swine fever virus), Porcine reproductive virus (Porcine swine fever virus), Porcine infectious seronegative swine influenza virus (Porcine influenza virus, Porcine swine fever virus), Porcine swine fever virus (Porcine swine fever virus, Porcine swine virus, Porcine reproductive virus negative seronegative seropositive swine virus, Porcine swine influenza virus, Porcine swine fever virus, Porcine swine virus (Porcine swine virus), Porcine seronegative swine virus, Porcine swine virus sample, Porcine swine virus positive swine virus sample, Porcine swine virus positive swine virus sample, Porcine swine virus positive swine virus sample, Porcine virus sample, 13 swine virus sample, Porcine, and (3) carrying out specificity test, wherein the sample to be tested, the ASFV high-immunity positive serum and the ASFV standard negative serum are diluted according to the proportion of 1:100, and the anti-swine secondary antibody is diluted according to the proportion of 1: 2000. After AEC color development, the experimental result is observed under a common optical microscope.

1.11 compared with the coincidence rate of the existing ASFV-ELISA kit, 68 parts of clinical test pig serum (36 parts of which are serum in one month after immunization) and 41 parts of clinical samples which are preserved in Harbin veterinary research institute are selected to be 109 parts of pig serum in total. Selecting an ASFV-ELISA antibody detection kit (aiming at P30 protein), an ASFV-ELISA detection kit (aiming at P54 protein) of Biochek company and an ASFV blocking ELISA antibody detection kit (aiming at VP72 protein) of INGENASE company, and carrying out serological detection with the ASF-IPMA method. And (3) analyzing the test results of the ASF-IPMA and the three ASFV-ELISA kits, and comparing the coincidence rate results of the ASF-IPMA and each kit.

Example 2: results of the experiment

1.12 screening of strains and cells PK15 cells after AEC staining, cells infected with rASFV delta 360 and HLJ/18-7GD viruses are uniformly stained, the shapes are uniform, the cells are small and regular dark rose red round stains, the negative control cell holes have clean backgrounds, and no non-specific stains are generated. Cells infected with rASFV delta 360 and HLJ/18-7GD viruses are not uniformly colored after AEC staining of Vero cells, the shapes are different in size, and the cells infected for a long time are deeply colored after rounding; the post-infected cells have good shapes, are irregular spindle-shaped and light in color due to low virus particle content and small damage to the cells, interfere observation and judgment, and the cells with light color have the possibility of being mistakenly judged as background color; the background of the negative control cell after AEC color development is clean and has no non-specific color, so that the HLJ/18-7GD virus with stronger safety is selected as the final inoculation antigen on the premise of ensuring that the cell infection rate and the cell color condition after AEC color development are not influenced; meanwhile, PK15 cells with more consistent cell morphology and coloring are selected as antigen to inoculate the cells. As shown in fig. 1.

1.13 infection dose and replication time optimization selection of virus content 3X 10⁷、1×10⁷、3×10⁶TCID₅₀The cells were infected by three infection gradients/mL and observed at three infection times of 24h, 48h and 72h, and the culture was not continued for 96h because the cell state of PK15 cells at three infection doses began to deteriorate and easily shed after 72h of infection. The results showed that the dose of virus infection was 1X 10⁷TCID₅₀When the virus replication time is 72h, the content of the fluorescent foci is moderate, and the virus begins to spread, so that obvious clone foci appear. In conclusion, 72h was chosen for the next IPMA test. As shown in fig. 2.

1.14 determination of dilution of secondary antibody determination test of ASF-IPMA was carried out by diluting ASFV standard positive and standard negative serum with serum diluent at four concentrations of 0, 1:100, 1:300, and 1:1000, and simultaneously diluting anti-porcine secondary antibody with secondary antibody preservative solution at five concentrations of 0, 1:1000, 1:2000, 1:4000, and 1:8000, and the results showed that ASFV standard positive serum was diluted at 1:100 times, and anti-porcine secondary antibody was slightly lighter in cell coloration at the dilution of 4000 times or more, compared to this, the cells were more deeply colored at the dilution of 1000 times and 2000 times, and the observation effect was better. However, the background of the cell reaction hole is colored when the anti-porcine secondary antibody is diluted by 1:1000 times, which affects the observation and judgment, so that the dilution range of the anti-porcine secondary antibody is set to be 1:2000 times. As shown in table 1.

Table 1: checkerboard test for serum and secondary antibody dilution

Note: "-" indicates no color reaction of the cells;

"+ ++" indicates that the cells have color reaction and are colored to dark rose red and dark brown;

"+ + + +" indicates that the cells had a color reaction and were more rose-colored;

"+ +" indicates that the cells had a color reaction and were rose-red; "+" indicates that the cells had a color reaction and were colored in light rose;

"+" indicates that the dilution has background color, which affects the observation

Determination of 1.15 serum dilution gradient dilution was performed by selecting 26 negative sera (SPF non-immune pig sera) and 4 weak positive sera (OD values of 0.56, 0.81, 0.97, 0.73) according to 1:10, 1:20, 1:40, 1:80, 1:160, respectively. When the serum is diluted by 1:80 times, the detection of the negative serum is negative, 4 weak positive sera can be detected as positive, and 1 of the four sera has background coloration influencing the observation; of the 30 sera, 3 sera had a background color that affected the determination. When the serum is diluted by 1:160 times, the detection of the negative serum is negative, 4 parts of weak positive serum can be detected to be positive, 30 parts of serum has no background color which can influence the judgment of the result, and 1:100 times between 1:80 and 1:160 is finally selected as the final dilution times of the serum, as shown in table 2. The serum dilution in the ASF-IPMA test was finally 1:100 fold and the anti-porcine secondary antibody dilution was finally 1:2000, as shown in FIG. 3.

TABLE 2ASF-IPMA serum dilution test

Note: a: "-" indicates no cell staining at this dilution and a negative reaction;

and b, "√ indicates that the cell well at that dilution is not background;

c: "+" indicates that there was staining of cells at this dilution and a positive reaction;

d: "X" indicates that the cell well at this dilution has a background color and affects the judgment of the result

1.16 specificity assay IPMA plate specificity was determined by reacting the prepared cell plate with positive sera from common porcine viruses (including PPRSV, PrV, SIV, PCV2, FMDV, TGEV, CSFV, BVDV, PDCoV, PPV) stored in Harbin veterinary institute, national academy of agricultural sciences, and by using ASF porcine standard positive and porcine negative sera for control. The result shows that the virus positive serum detection is negative, and the ASF-IPMA specificity is good. As shown in table 3.

TABLE 3ASF-IPMA specificity assay

Note: "+" indicates positive, and "-" indicates negative

1.17 coincidence rate comparison tests were conducted on 109 swine-derived sera obtained in the laboratory using ASF-P30-ELISA antibody detection kit, Biochek corporation ASF-P54-ELISA detection kit, INGENASE corporation ASF-VP 72-blocking ELISA antibody detection kit, and ASF-IPMA cell reaction plate prepared in this study, respectively, and the experimental results showed that ASF-IPMA had a coincidence rate of 99.08% as compared with the P30 protein ELISA detection kit, 98.17% as compared with the P54 protein ELISA detection kit, and 100% as compared with the blocking ELISA detection kit (VP72 protein). The results of the ASF-IPMA kit and the three ELISA kits are high in coincidence rate, the coincidence rate is more than 98%, and the results are shown in Table 4.

TABLE 4 comparison of ASF-IPMA with 3 ELISA detection kits

Note: the coincidence rate is (number of positive samples detected by two detection methods simultaneously + number of negative samples detected by two detection methods simultaneously)/total number of samples

The experiments show that the African swine fever attenuated virus which is popular in China and has gene deletion is suitable for the epidemic situation in China, and has good safety and natural immunogenicity. The invention compares two gene deletion attenuated strains of rASFV delta 360 and HLJ/18-7GD, although the capability of rASFV delta 360 to infect Vero and PK15 is about 10 times higher than that of HLJ/18-7GD, the rASFV delta 360 and the HLJ/18-7GD have no obvious difference after color development in IPMA detection. The HLJ/18-7GD strain is higher in safety because rASFV delta 360 still has strong virulence reversion after continuous generation in pigs.

For infected cells, Vero cells are a common cell line for domestication and adaptation of ASFV, but we find in the research that PK15 can well infect ASFV virus, the cells have strong adherence and are not easy to fall off, the cells grow faster, the serum content required by culture is lower than that of Vero, the cost can be reduced, and the research compares the two cells. The result shows that Vero cell staining has uneven staining depth and different sizes of infected cells compared with PK15 cells, and cells which are lighter in staining and larger in staining are easily connected with the background staining into a whole and are not easy to distinguish; and the PK15 cells have more uniform color and uniform size due to small cell volume, are easier to observe and can effectively reduce the misjudgment.

On the basis, the research carries out further optimization experiments of pathogen infection dosage, infection time, serum dilution and secondary antibody dilution, and determines parameters of the detection reagent which is good in coloring and sensitive to the detection method.

The study was performed by comparing the IPMA method with three ELISA kits (ELISA method for P54 protein, ELISA for P30 protein and blocking ELISA for VP 72). The results show that the coincidence rate of the ASF-IPMA and the P54-iELISA method, the P30-iELISA method (aiming at P30 protein) and the VP72 blocking ELISA method (aiming at VP72 protein) is more than 98%, and the ASF-IPMA method shows good detection performance (specificity and sensitivity) and stable and reliable results.