阅读说明：本技术 非洲猪瘟病毒的检测试纸、试剂盒及其制备方法 (Test paper and kit for detecting African swine fever virus and preparation method thereof ) 是由 杨春江 杨先富 赵荣茂 袁志波 杨晓霞 吴佳兴 于在江 马孝斌 朱琳 于 2019-12-02 设计创作，主要内容包括：本发明提供非洲猪瘟病毒的检测试纸、试剂盒及其制备方法。非洲猪瘟病毒检测试纸包括背板,背板上依次排列设置样品垫、乳胶微球垫、硝酸纤维素膜和吸水垫；乳胶微球垫上包被有乳胶微球标记的标记抗体；硝酸纤维素膜上设有检测线和质控线,检测线处包被有捕获抗体,检测线靠近乳胶微球垫一侧设置；质控线处包被有羊抗鼠抗抗体,质控线靠近吸水垫一侧设置；标记抗体为由保藏编号为CGMCC No.18540的杂交瘤细胞7A7分泌产生的单克隆抗体或由保藏编号为CGMCC No.18539的杂交瘤细胞3E5分泌产生的单克隆抗体,捕获抗体为非洲猪瘟病毒抗体。该非洲猪瘟病毒检测试纸可实现对非洲猪瘟病毒的快速、高特异性、高灵敏度检测。(The invention provides test paper and a kit for detecting African swine fever virus and a preparation method thereof. The African swine fever virus test paper comprises a back plate, wherein a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere cushion is coated with a labeled antibody labeled by latex microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one side of the latex microsphere pad; the quality control line is coated with goat anti-mouse anti-antibody and is arranged close to one side of the water absorption pad; the marking antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with the preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with the preservation number of CGMCC No.18539, and the capturing antibody is an African swine fever virus antibody. The African swine fever virus detection test paper can realize rapid, high-specificity and high-sensitivity detection on the African swine fever virus.)

1. The test paper for detecting the African swine fever virus is characterized by comprising a back plate, wherein a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere pad is coated with a labeled antibody labeled by latex microspheres, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one side of the latex microsphere pad; the quality control line is coated with goat anti-mouse anti-antibody and is arranged close to one side of the water absorption pad; the marker antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with the preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with the preservation number of CGMCC No. 18539; the capture antibody is an African swine fever virus antibody.

2. The test strip for detecting African swine fever virus according to claim 1, wherein: the marker antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with the preservation number of CGMCC No.18540, and the capture antibody is a monoclonal antibody secreted by a hybridoma cell 3E5 with the preservation number of CGMCC No. 18539.

3. The test strip for detecting African swine fever virus according to claim 1, wherein: the marker antibody is a monoclonal antibody secreted by a hybridoma cell 3E5 with the preservation number of CGMCC No.18539, and the capture antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with the preservation number of CGMCC No. 18540.

4. The test strip for detecting African swine fever virus according to claim 1, wherein: the sample pad includes first sample pad, second sample pad and third sample pad, first sample pad the second sample pad with different buffer solutions of infiltration respectively are gone up to the third sample, according to the third sample pad the second sample pad the latex microsphere pad the order of first sample pad is crisscross setting in proper order.

5. The test strip for detecting African swine fever virus according to claim 4, wherein: still include and strain the blood membrane, it locates to strain the blood membrane the second sample pad with between the latex microballon pad.

6. The test strip for detecting African swine fever virus according to claim 5, wherein: the distance between one end of the first sample pad close to the nitrocellulose membrane and the detection line is 4-10 mm.

7. The test strip for detecting African swine fever virus according to claim 6, wherein: the length of the overlapping part of the first sample pad and the nitrocellulose membrane is 1.5-2.0 mm.

8. A detection kit for African swine fever virus is characterized by comprising: a kit housing, a pipette, a sample diluent and the test paper for detecting African swine fever virus according to any one of claims 1 to 7.

9. A method for preparing a test strip for detecting African swine fever virus according to any one of claims 1 to 7, comprising the steps of:

s1 preparation of African swine fever virus p72 recombinant protein monoclonal antibody: immunizing animals by using the African swine fever virus p72 recombinant protein, and obtaining an African swine fever virus p72 recombinant protein monoclonal antibody by a hybridoma technology;

s2 preparation of the latex microsphere pad: labeling the African swine fever virus p72 recombinant protein monoclonal antibody obtained in the step S1 by using a latex microsphere, and then coating the obtained labeled antibody labeled by the latex microsphere in a glass fiber pad to obtain the latex microsphere pad;

s3 preparation of nitrocellulose membrane: spraying a capture antibody on a nitrocellulose membrane to obtain a detection line, and then spraying a goat anti-mouse anti-antibody to obtain a quality control line;

s4, assembling test paper: and sequentially and mutually staggered on the back plate, the sample pad, the latex microsphere pad, the nitrocellulose membrane and the water absorption pad are arranged, so that the test paper for detecting the African swine fever virus is obtained.

10. The method for preparing test paper for detecting African swine fever virus according to claim 9, wherein the method for preparing the labeled antibody labeled with latex microspheres in step S2 comprises the steps of:

s211, washing the latex microspheres: washing the latex microspheres at least twice by using a labeling buffer solution;

s212, activation of the latex microspheres: adding NHS and EDC into the labeling buffer solution, then adding the washed latex microspheres, and incubating and activating at room temperature to obtain a latex microsphere buffer solution;

s213. labeling of latex microspheres: adding the African swine fever virus p72 recombinant protein monoclonal antibody obtained in the step S1 into the latex microsphere buffer solution obtained in the step S212, and incubating at room temperature; then adding confining liquid, and incubating at room temperature; and centrifuging the obtained product, discarding the supernatant, and adding a labeling buffer solution to resuspend the latex microspheres to obtain the labeled antibody labeled by the latex microspheres.

Technical Field

The invention belongs to the technical field of animal epidemic disease detection, and particularly relates to test paper and a kit for detecting African swine fever virus and a preparation method thereof.

Background

The African Swine Fever (ASF) is a hemorrhagic, high-lethality and viral infectious disease of pigs caused by African Swine Fever Virus (ASFV). all day-old pigs are susceptible to the ASF, the incubation period of the ASF natural infection is long, the natural infection is 4 ~ 19 days, the average death time after infection is 2-10 days, the clinical expression forms comprise high fever, inappetence, skin and internal organ hemorrhage and the like, the clinical symptoms of the ASF natural infection are similar to swine fever and swine erysipelas virus, after the pigs or wild pigs are infected with the African swine fever virus, the incubation period is usually 3-15 days, the death rate of the infection of a virulent strain can reach 100 percent, and the world animal health Organization (OIE) considers that the research and the diagnosis are carried out in an ASF epidemic area and an area at the initial stage of low-toxicity ASFV infection, a serological method is a first-push diagnosis method, and the recombinant protein obtained by purification is used as a detection source, so that.

The ASFV genome is a double-strand linear DNA with the end covalently closed, the length is 170 kb ~ kb, and the ASFV genome has an end cross-linking and reversal repeat region, which codes 151-protein 167 proteins, and the mature virus particle contains about 50 structural proteins, wherein the nucleocapsid protein composed of p72 structural protein accounts for 1/3 of all virus proteins, and is the most abundant structural protein, p72 exists on the surface of the virus capsid, has better immunogenicity and antigenicity, can induce organism to generate neutralizing antibody, thus is the main antigen region for serological detection, and is also the main protein for ASFV diagnostic detection, and lays the foundation for establishing a non-infectious, rapid and sensitive serological detection method.

In addition, the traditional method for diagnosing and detecting African swine fever virus comprises a molecular biology method and an enzyme linked immunosorbent assay kit method, wherein the molecular biology method is represented by Polymerase Chain Reaction (PCR), whether animals are infected or not is confirmed by detecting virus molecules, the operation is complicated, reagents and equipment are expensive, and the operation is difficult for a pig farm; the enzyme-linked immunosorbent assay uses a 96-hole enzyme label plate as a carrier, detects whether an animal is infected with ASF virus by using an antigen-antibody specific reaction as a principle, and has the characteristics of high sensitivity, good specificity and the like, wherein the defects in the enzyme-linked immunosorbent assay are that the operation process is relatively complicated, the requirement on sample adding accuracy is high, experimental equipment such as an enzyme label instrument and a thermostat is needed to ensure the reaction environment, and the enzyme-linked immunosorbent assay is difficult to be carried out in basic-level farms, particularly in field detection; the above methods cannot achieve the purpose of rapid detection. At present, the most widely used test strip for detecting the African swine fever virus antigen is a colloidal gold test strip, the antigen-antibody reaction principle is also utilized to detect the African swine fever virus antigen, the operation is simple and convenient, the reaction time is short, but the defects are that the sensitivity is not high enough, false negative or false positive may exist, particularly, when the ASFV is rapidly detected, because samples are mostly whole blood, tissue samples and the like, the sample components are complex, long-time pretreatment cannot be carried out, the test strip may have the problems of interference, poor specificity and the like, and the requirement for rapidly detecting the ASFV is difficult to meet.

Disclosure of Invention

The invention aims to provide test paper and a kit for detecting African swine fever virus and a preparation method thereof, and the test paper can be used for the rapid, high-specificity and high-sensitivity detection of the African swine fever virus.

In order to solve the problems, the invention provides test paper for detecting African swine fever virus, which comprises a back plate, wherein a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere cushion is coated with a labeled antibody labeled by latex microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one side of the latex microsphere pad; the quality control line is coated with goat anti-mouse anti-antibody and is arranged close to one side of the water absorption pad; the marking antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with the preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with the preservation number of CGMCC No.18539, and the capturing antibody is an African swine fever virus antibody.

Wherein, the capture antibody can be one or more of African swine fever virus monoclonal antibody or polyclonal antibody; more specifically, the antibody can be one or a mixture of more of an African swine fever virus antibody, an African swine fever virus p72 protein antibody and an African swine fever virus p72 recombinant protein antibody; preferably, the capture antibody is the aforementioned african swine fever virus p72 recombinant protein antibody; further preferably, the capture antibody is a monoclonal antibody of recombinant protein p72 of African swine fever virus. Also, the capture antibody is different from the labeled antibody.

Preferably, the marker antibody is a monoclonal antibody secreted by the hybridoma cell 7A7 with the preservation number of CGMCC No.18540, and the capture antibody is a monoclonal antibody secreted by the hybridoma cell 3E5 with the preservation number of CGMCC No. 18539.

Preferably, the marker antibody is a monoclonal antibody secreted by the hybridoma cell 3E5 with the preservation number of CGMCC No.18539, and the capture antibody is a monoclonal antibody secreted by the hybridoma cell 7A7 with the preservation number of CGMCC No. 18540.

Preferably, the sample pad comprises a first sample pad, a second sample pad and a third sample pad, wherein the first sample pad, the second sample pad and the third sample pad are respectively soaked with different buffer solutions and are sequentially and alternately arranged according to the sequence of the third sample pad, the second sample pad, the latex microsphere pad and the first sample pad.

Preferably, the test paper for detecting African swine fever virus further comprises a blood filter membrane, and the blood filter membrane is arranged between the second sample pad and the latex microsphere pad.

Preferably, the end of the first sample pad close to the nitrocellulose membrane is at a distance of 4-10mm from the detection line.

Preferably, the length of the overlapping portion of the first sample pad and the nitrocellulose membrane is 1.5 to 2.0 mm.

Preferably, the length of the overlapping portion of the latex microsphere pad and the first sample pad is 1-1.5 mm.

Preferably, the length of the overlapping part of the blood filtering membrane and the latex microsphere pad is 2.5-3.0 mm.

Preferably, the length of the overlapping portion of the second sample pad and the blood filtration membrane is 9.0-9.5 mm.

In particular, the first, second and third sample pads may be glass fibre or cellulose pads or woven polymer pads.

Specifically, the absorbent pad is a glass fiber pad.

Specifically, the back sheet is a PVC back sheet.

The invention also provides a method for preparing the test paper for detecting African swine fever virus, which comprises the following steps:

s1 preparation of African swine fever virus p72 recombinant protein monoclonal antibody: immunizing animals by using the African swine fever virus p72 recombinant protein to obtain the African swine fever virus p72 recombinant protein monoclonal antibody by a hybridoma technology;

s2 preparation of the latex microsphere pad: labeling the African swine fever virus p72 recombinant protein monoclonal antibody obtained in the step S1 by using latex microspheres, and then coating the obtained labeled antibody labeled by the latex microspheres in a glass fiber pad to obtain a latex microsphere pad;

s3 preparation of nitrocellulose membrane: spraying a capture antibody on a nitrocellulose membrane to obtain a detection line, and then spraying a goat anti-mouse anti-antibody to obtain a quality control line;

s4, assembling test paper: and arranging a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad on the back plate in a mutually staggered manner in sequence to obtain the African swine fever virus latex microsphere detection test paper.

Specifically, the preparation of the African swine fever virus p72 recombinant protein monoclonal antibody in the step S1 specifically comprises the following steps:

s11 synthesis of African swine fever virus p72 recombinant gene: analyzing dominant epitopes of African swine fever virus p72 protein by DNAstar and IEDB databases, performing tandem expression on the dominant epitopes by B cell epitope prediction to obtain a sequence of SEQ ID No.4, performing gene synthesis, inserting the obtained recombinant gene of the African swine fever virus p72 into a pUC vector to obtain pUC-p 72;

s12, gene cloning and vector construction: designing primers according to a SEQ ID No.4 gene sequence, respectively adding BamH I and Sal I enzyme cutting sites at the near 5' ends of the upstream and downstream primers, amplifying a target fragment by using a pUC-p72 gene as a template, carrying out double enzyme cutting, identifying an amplification product through agarose gel electrophoresis, connecting the amplification product with an expression vector pFastBac HT-B to obtain a recombinant expression vector pFastBac HT-p72, and constructing sequencing identification after work;

s13, construction, induced expression and purification of recombinant gene engineering bacteria: transposing a positive recombinant plasmid pFastBac HT-p72 into a DH10Bac competent cell, screening positive clones through kanamycin-tetracycline-gentamicin and blue white spots, extracting baculovirus plasmids, transfecting SF9 insect cells, collecting recombinant baculovirus, infecting the SF9 insect cells with the recombinant baculovirus at MOI 0.1, centrifuging and collecting supernatant to obtain African swine fever virus p72 recombinant protein;

s14, immune animal and p72 monoclonal antibody preparation and screening: the African swine fever virus p72 recombinant protein is used for immunizing animals to obtain an African swine fever virus p72 recombinant protein polyclonal antibody, or the African swine fever virus p72 recombinant protein is used for immunizing animals, spleen cells and tumor cells are hybridized after the animals are immunized, hybridoma cells are obtained through screening, then the African swine fever virus p72 recombinant protein is used for screening specific antibodies, and swine fever virus live vaccines, swine pseudorabies live vaccines, swine reproduction and respiratory syndrome live vaccines, porcine parvovirus culture solutions, porcine circovirus type 2 cell culture solutions and other swine susceptible viruses are used for cross screening to obtain the African swine fever virus p72 recombinant protein monoclonal antibody.

Specifically, the preparation of the latex microsphere pad in the step S2 specifically comprises the following steps:

s21, preparing a labeled antibody labeled by latex microspheres;

and S22, infiltrating the glass fiber pad with the detection antibody marked by the latex microspheres to obtain the latex microsphere pad.

Specifically, the method for preparing the latex microsphere labeled antibody in step S2 includes the following steps:

s211, washing the latex microspheres: washing the latex microspheres at least twice by using a labeling buffer solution;

s212, activation of the latex microspheres: adding NHS and EDC into a labeling buffer solution, then adding the washed latex microspheres, and incubating and activating at room temperature to obtain a latex microsphere buffer solution;

s213. labeling of latex microspheres: adding the African swine fever virus p72 recombinant protein monoclonal antibody obtained in the step S1 into the latex microsphere buffer solution obtained in the step S212, and incubating at room temperature; then adding confining liquid, and incubating at room temperature; and centrifuging the obtained product, discarding the supernatant, and adding a labeling buffer solution to resuspend the latex microspheres to obtain the labeled antibody labeled by the latex microspheres.

Specifically, the preparation of the nitrocellulose membrane of step S3 specifically includes the following steps:

s31. preparation of capture antibody: the method comprises the steps of immunizing animals for multiple times by utilizing African swine fever virus or African swine fever virus p72 protein or African swine fever virus p72 recombinant protein, purifying to obtain an African swine fever virus polyclonal antibody or an African swine fever virus p72 protein polyclonal antibody or an African swine fever virus p72 recombinant protein polyclonal antibody, or preparing an African swine fever virus monoclonal antibody, an African swine fever virus p72 protein monoclonal antibody or an African swine fever virus p72 recombinant protein monoclonal antibody by adopting the method;

s32, scribing: and respectively spraying the capture antibody and the goat anti-mouse anti-antibody on a nitrocellulose membrane by using a membrane scratching instrument to form a detection line and a quality control line.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention analyzes the dominant epitope of the African swine fever virus p72 protein by DNAstar and IEDB databases, provides more choices for screening antibody libraries for exposing the N-terminal epitope and the C-terminal epitope of the protein more easily, the two main antigen epitope regions are expressed in series, the flexible amino acid fragment is used as a linker, retains more amino acids and simultaneously reduces the influence of steric hindrance as much as possible to obtain recombinant protein with strong immunogenicity, and the recombinant protein is used for immunizing animals, screening by a hybridoma cell technology to obtain monoclonal antibodies with strong specificity secreted by the hybridoma cell 7A7 with the preservation number of CGMCC No.18540 and the hybridoma cell 3E5 with the preservation number of CGMCC No.18539, the African swine fever virus detection test paper prepared by the monoclonal antibody has high specificity and high sensitivity to the African swine fever virus p72 protein;

2. the latex microspheres of the African swine fever virus detection test paper provided by the invention have the advantages of high uniformity, good monodispersity, good stability and the like, the latex microspheres are used as a marker, the prepared detection test paper has the advantages of small batch difference and high detection sensitivity, the latex microspheres of the African swine fever virus detection test paper prepared by the latex microspheres do not need organic reagents during detection, equipment such as a thermostat, an enzyme labeling instrument and the like are not needed, a special field is not needed, the detection can be completed only by collecting a good sample and an operation table board, the operation is simple, convenient and rapid, the whole time consumption is short, special training is not needed, the detection sensitivity is high, the specificity is good, the test paper is particularly suitable for detection in the fields such as fields of fields, basic laboratories and the like, the detection result can be qualitative and quantitative, and under the condition of a reading instrument, the color development value of a detection line and a quality control, quantitatively detecting the concentration of the antigen;

3. the preparation method of the latex microsphere test paper for detecting African swine fever virus provided by the invention is simple and convenient to operate, simple in required raw materials, capable of being successfully prepared in a short time and capable of generating great economic benefits.

Biological preservation information description

The hybridoma 7A7 was deposited in the general microbiological center of China Committee for culture Collection of microorganisms 24.10.2019, having the address of No.3 Siro-1, Beijing, Toyokuo, sunny region, the microbial research institute of Chinese academy of sciences, zip code 100101, and the deposition number CGMCC No. 18540.

The hybridoma cell 3E5 was deposited in the general microbiological center of China Committee for culture Collection of microorganisms 24.10.2019, having the address of No.3 Siro-1, Beijing, Toyokuo, sunny region, the microbial research institute of Chinese academy of sciences, the postal code is 100101, and the deposition number is CGMCC No. 18539.

Drawings

FIG. 1 is a schematic structural diagram of a test paper for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 2 is a cross-sectional view of a test strip for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 3 is a top view of a test strip for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 4 is an antigen identification diagram of the recombinant protein p72 of African swine fever virus obtained in the example of the invention;

FIG. 5 is a diagram showing the identification result of the cross reaction between the recombinant protein monoclonal antibody of African swine fever virus p72 and the common porcine virus obtained in the example of the present invention;

FIG. 6 is a standard curve of the African swine fever virus latex microsphere test paper obtained in the embodiment of the present invention for the quantification of the p72 protein concentration.

Wherein: 1-latex microsphere test paper for African swine fever virus; 2-a back plate; 3-nitrocellulose membrane; 4-quality control line; 5-detection line; 6-first sample pad; 7-latex microsphere pad; 8-a blood filtration membrane; 9-a second sample pad; 10-third sample pad; 11-absorbent pad.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.