CN111912991A - Test strip for detecting serum antibody of African swine fever virus and application thereof - Google Patents
Test strip for detecting serum antibody of African swine fever virus and application thereof Download PDFInfo
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- CN111912991A CN111912991A CN202010806186.5A CN202010806186A CN111912991A CN 111912991 A CN111912991 A CN 111912991A CN 202010806186 A CN202010806186 A CN 202010806186A CN 111912991 A CN111912991 A CN 111912991A
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- test strip
- recombinant protein
- pad
- spa
- swine fever
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Abstract
The invention provides a test strip for detecting an African swine fever virus serum antibody and application thereof, belonging to the production and technical field of veterinary biological diagnostic products. The test strip comprises a bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate; the bonding pad is coated with SPA marked by quantum dot microspheres; the nitrocellulose membrane is provided with a first detection line and a quality control line, the first detection line is coated with a recombinant protein CD2v and a recombinant protein MGF360, and the first detection line is arranged close to one side of the combination pad; the quality control line is coated with chicken anti-SPA polyclonal antibody and is arranged close to one side of the water absorption pad. The sample diluent used in the test strip was a borate buffered saline containing BSA and Tween 20. The test strip can be used for rapidly detecting the serum antibody of the African swine fever virus with high specificity and high sensitivity.
Description
Technical Field
The invention belongs to the field of production and technology of veterinary biological diagnostic products, and particularly relates to a test strip for detecting an African swine fever virus serum antibody and application thereof.
Background
African Swine Fever (ASF) is an acute, febrile and highly contagious disease of domestic and wild pigs caused by African Swine Fever Virus (ASFV). ASFV is a large double-stranded DNA virus, about 180kb in length, encoding up to 180 proteins. ASFV is mainly transmitted by contact and also transmitted by air in a short distance, and infects porcine alveolar macrophages to cause acute hyperpyrexia of domestic pigs and wild pigs.
Although there is no commercial ASFV vaccine so far, researchers have disclosed the possibility of genetically modifying African swine fever virus as a potential vaccine strain.
At present, the traditional antibody detection method for African swine fever, such as indirect Immunofluorescence (IFA), enzyme-linked immunosorbent assay (ELISA) and the like, has low sensitivity and expensive instrument and equipment; or the operation is complicated, the cost is high, and the like, and the application and the development in basic-level farms are difficult.
Disclosure of Invention
Aiming at the defects of the existing problems, the first object of the invention is to provide a test strip for detecting the serum antibody of the African swine fever virus, which can detect the serum antibody of the African swine fever virus rapidly, with high specificity and high sensitivity.
The second purpose of the invention is a method for identifying and detecting antibodies generated by African swine fever virus infection and vaccine immunization for non-diagnosis purpose, the method is simple and convenient to operate, needs short time and is low in cost, and the method is suitable for detection under various conditions.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a test strip for detecting serum antibodies of African swine fever viruses comprises a bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate; the bonding pad is coated with SPA marked by quantum dot microspheres; the nitrocellulose membrane is provided with a first detection line and a quality control line, the first detection line is coated with a recombinant protein CD2v and a recombinant protein MGF360, and the first detection line is arranged close to one side of the binding pad; the quality control line is coated with chicken anti-SPA polyclonal antibody and is arranged close to one side of the water absorption pad.
In the present invention, the chicken anti-SPA polyclonal antibody was purchased from abcam, cat # ab 19483.
In the invention, the sample pad is obtained by soaking glass fiber in a boric acid buffer solution containing bovine serum albumin and Tween 20.
In the invention, the bonding pad is obtained by soaking glass fiber in boric acid buffer solution containing bovine serum albumin, sucrose, Tween20 and PEG1500, and spraying SPA solution marked by quantum dot microspheres.
In the invention, the sequences of the recombinant protein CD2v and the recombinant protein MGF360 are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 4.
In the invention, quantum dot microspheres are coupled with SPA to obtain a coupling compound of SPA and quantum dot microspheres; and then blocking the SPA and quantum dot microsphere conjugate by BSA, and suspending the SPA and quantum dot microsphere conjugate in a boric acid buffer solution containing BSA, sucrose and Tween20 to obtain a quantum dot microsphere labeled SPA solution.
In the invention, the mass ratio of the coated recombinant protein CD2v to the recombinant protein MGF360 at the first detection line is 1: 0.8-1.2.
In the invention, the nitrocellulose membrane is also provided with a second detection line coated with a recombinant protein P30V, and the amino acid sequence of the recombinant protein P30V is shown as SEQ ID NO. 6.
The invention also provides a sample diluent matched with the test strip, which is a boric acid buffer solution containing BSA and Tween 20.
The invention also provides a method for detecting African swine fever virus antibodies by using the test strip and the sample diluent, which aims at non-diagnosis: and (3) adding a serum sample into the sample diluent for dilution, then dropwise adding the diluted serum sample onto the sample pad of the test strip, and after reaction, detecting the line C and the detection line by adopting an ultraviolet light emitter.
SPA marked by quantum dot microspheres is combined with IgG antibody of pig in serum sample, corresponding African swine fever antibody is captured by the recombinant protein CD2v and the recombinant protein MGF360 coated at the first detection line, corresponding African swine fever antibody is captured by the recombinant protein P30V coated at the second detection line, chicken anti-SPA multi-antibody capturing SPA coated at the quality control line, and light emission and color development are carried out by the quantum dots. The African swine fever wild virus infection antibody can be combined with the recombinant protein CD2v, the recombinant protein MGF360 and the recombinant protein P30V, so that the first detection line and the second detection line are colored. The African swine fever vaccine immune antibody can only be combined with the recombinant protein P30V, so the first detection line does not develop color, and the second detection line develops color.
Compared with the prior art, the invention has the following beneficial effects:
1. the test strip for detecting the serum antibody of the African swine fever virus provided by the invention can be used for quickly detecting the serum antibody of the African swine fever virus with high specificity and high sensitivity, and has stable effect and repeatability.
2. The test strip for detecting the serum antibody of the African swine fever virus provided by the invention adopts the quantum dot microspheres as the markers, the uniformity is high, the stability is strong, and the difference between batches of the prepared test strip is small.
3. The test strip for detecting the serum antibody of the African swine fever virus provided by the invention is simple in preparation method, easily available in raw materials and wide in market prospect.
4. The test strip for detecting the serum antibody of the African swine fever virus is simple and convenient to operate, low in cost and low in condition requirement, does not need expensive instruments and equipment, and is suitable for carrying out detection work in various basic laboratories and on the site. The test strip for detecting the African swine fever virus serum antibody provided by the invention can be used for identifying whether the African swine fever antibody generated by an animal is derived from an antibody generated by the immunization of an African swine fever gene deletion strain vaccine or an antibody generated after wild virus infection, and makes up for the vacancy of the market.
Drawings
FIG. 1 is an expression identification diagram of a recombinant protein CD2v in example 1 of the present invention, wherein lane 1 is a purified recombinant protein CD2v, lane 2 is a Western-blotting identification of a purified recombinant protein CD2v and a positive reference serum of African swine fever antibody, and M is a protein marker;
FIG. 2 is an expression identification diagram of a recombinant protein MGF360 in example 1 of the present invention, wherein lane 1 is a purified recombinant protein MGF360, lane 2 is a Western-blotting identification of the purified recombinant protein MGF360 and a positive reference serum of African swine fever antibody, and M is a protein marker;
FIG. 3 is an expression identification diagram of the recombinant protein P30V in example 2 of the present invention, wherein lane 10 is a purified recombinant protein P30V, lane 11 is a Western-blotting identification of the purified recombinant protein P30V and ASFV antibody positive reference serum, and M is a protein marker.
FIG. 4 is a schematic structural diagram of the test strip for detecting serum antibodies of African swine fever virus according to the present invention;
FIG. 5 is a cross-sectional view of a test strip for detecting serum antibodies against African swine fever virus according to the present invention;
FIG. 6 is a top view of the test strip for detecting serum antibodies against African swine fever virus according to the present invention;
wherein: 1-test paper strip for detecting serum antibody of African swine fever virus; 2-a bottom plate; 3-sample pad; 4-a conjugate pad; 5-nitrocellulose membrane; 6-a first detection line; 7-a second detection line; 8-quality control line; 9-absorbent pad.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples of the present invention. Furthermore, it should be understood that the following examples are provided for better understanding of the present invention, are not limited to the best mode and are not intended to limit the scope of the present invention, and any methods and products similar or equivalent to the present invention, which can be obtained by combining the present invention with other features of the prior art, while the present invention is taught by the present application, are within the scope of the present invention.
In the invention: MES, EDC, NHS, Tween-20, PEG1500, sucrose were purchased from Sigma; Tris-HCl, BSA from Thermo Fisher; SPA and chicken anti-SPA polyclonal antibodies were purchased from abcam; glass fiber membranes purchased from Ahlstrom; nitrocellulose membranes were purchased from Sartorius; the water absorption filter paper and the bottom plate are purchased from Shanghai gold-labeled Biotech limited; quantum dot microspheres (product number FM610C, hydrated particle size 120nm) were purchased from Beijing Najing Biotech Ltd; the African swine fever antibody positive reference serum and the African swine fever antibody negative reference serum come from Harbin veterinary research institute of Chinese agricultural science institute.
MES buffer 0.02M, pH 6.0.6.0: 4.265g MES were dissolved in 1L double distilled water and the pH was adjusted to 6.0 with 1M sodium hydroxide solution or hydrochloric acid.
Boric acid buffer (concentration 0.02M, ph 8.0): 1.236H3BO3Dissolved in 1L of double distilled water, and the pH was adjusted to 8.0 with 1M hydrochloric acid.
Tris-HCl buffer (50 mM, pH 8.0): 7.88g Tris was dissolved in 1L double distilled water and the pH was adjusted to 8.0 with 1M hydrochloric acid.
PBS buffer (concentration 0.01M, pH7.4): 3g of Na are taken2HPO4.12H2O, 0.2g KH2PO48g of NaCl and 0.2g of KCl were dissolved in 1L of double distilled water, and the pH was adjusted to 7.4 with 1M sodium hydroxide solution or hydrochloric acid.
Example 1 preparation of recombinant protein CD2v and recombinant protein MGF360
1. Construction of recombinant bacterium for preparing recombinant protein CD2v
Referring to a first example of an African swine fever SY18 full gene sequence (MH766894, EP402R, 73369-74451) in China published by an NCBI database, a gene of a recombinant protein CD2v is designed, wherein the sequence is shown as SEQ ID NO:1, and the amino acid sequence of the recombinant protein CD2v is shown as SEQ ID NO: 2. The gene of the recombinant protein CD2v is obtained by adopting a gene synthesis mode (from Nanjing Kingsrey Biotech Co., Ltd.), and the gene of the recombinant protein CD2v is inserted between the multiple cloning enzyme cutting sites EcoRI and HindIII of a vector pET-32a (+), so as to obtain a recombinant plasmid pET-32a-CD2 v.
The recombinant plasmid pET-32a-CD2v is transformed into E.coli BL21(DE3) competent cells by a heat shock method to obtain a recombinant bacterium pET-32a-CD2v (BL 21).
2. Construction of recombinant bacterium for preparing recombinant protein MGF360
Referring to a first African swine fever SY18 full gene sequence (MH766894, MGF360-12L, 29382-30434) published by NCBI database, a gene of a recombinant protein MGF360 is designed, the sequence is shown as SEQ ID NO. 3, and the amino acid sequence of the recombinant protein MGF360 is shown as SEQ ID NO. 4. The gene of the recombinant protein MGF360 is obtained by adopting a gene synthesis mode (from Nanjing Kingsry Biotech Co., Ltd.), and the gene of the recombinant protein MGF360 is inserted between the cloning enzyme cutting sites EcoRI and HindIII of a vector pET-32a (+) to obtain a recombinant plasmid pET-32a-MGF 360.
E.coli BL21(DE3) competent cells were transformed with the recombinant plasmid pET-32a-MGF360 by "heat shock" method to obtain recombinant bacterium pET-32a-MGF360(BL 21).
3. Expression and purification of recombinant proteins
The recombinant bacterium pET-32a-CD2v (BL21) and the recombinant bacterium pET-32a-MGF360(BL21) were cultured in LB liquid medium containing 100. mu.g/mL ampicillin, respectively, under the following conditions: culturing at 37 deg.C and shaking table rotation speed of 180r/min until OD is reached600Cooling to 20 deg.C when the concentration is 0.6-0.8 deg.C, adding IPTG with final concentration of 0.2mmol/L for inducing expression after 30min, culturing at 20 deg.C for 12-18 hr, and collecting thallus. The cells were washed with a buffer solution, resuspended, and then subjected to ultrasonic lysis by adding PMSF (phenylmethylsulfonyl fluoride), and centrifuged at 10000r/min at 4 ℃ for 30 minutes. The supernatant was collected and purified according to the instructions of Ni-NTA affinity chromatography medium (product of King Bio-technology Co., Ltd., Cat. No: L00250). As can be seen from lane 1 of FIG. 1, the purified recombinant protein CD2v showed a specific band around 30kDa, consistent with the expectation. As can be seen from lane 1 of FIG. 2, the purified recombinant protein MGF360 showed a specific band around 50kDa, consistent with the expectation. Therefore, the recombinant protein CD2v and the recombinant protein MGF360 are successfully expressed and stored below-70 ℃ for later use.
4. Antigenicity testing of recombinant proteins
And (3) transferring the purified recombinant protein CD2v and recombinant protein MGF360 to an NC membrane after SDS-PAGE electrophoresis, and performing Western-blotting detection respectively. After being sealed with 5% skimmed milk overnight, a mixture of African swine fever antibody positive reference serum and 5% skimmed milk solution in a volume ratio of 1:1 is used as a primary antibody, and the mixture is incubated at 2-8 ℃ for 12-18 hours. TBST washes were 5 times, 5 min/time. Using a HRP-goat anti-porcine enzyme-labeled antibody (available from Bethyyl, cat. No. A100-102p) diluted 1:20000 as a secondary antibody, the incubation was carried out at 37 ℃ for 1.0 hour. TBST washes were 5 times, 5 min/time. ECL was developed in the dark for 5 min. As a result: the recombinant protein CD2v shows a specific reaction band at about 30kDa (shown in lane 2 of FIG. 1). The recombinant protein MGF360 shows a specific reaction band at about 50kDa (as shown in lane 2 of FIG. 2). The results show that both the recombinant protein CD2v and the recombinant protein MGF360 can specifically react with the African swine fever antibody positive reference serum, and have good antigenicity.
EXAMPLE 2 preparation of recombinant protein P30V
Construction of P30V Gene recombination vector
Referring to a first African swine fever CP204L gene sequence (MH766894, CP204L, 124770-125375) in China published by NCBI database, codon preference optimization aiming at Escherichia coli is carried out, and a gene sequence of a recombinant protein P30V is obtained, such as SEQ ID NO: 5, and the corresponding amino acid sequence is shown as SEQ ID NO: and 6. The gene of the recombinant protein P30V is obtained by adopting a gene synthesis mode (Nanjing Kingsrei Biotech Co., Ltd.), and the gene sequence is inserted between the polyclonal enzyme cutting site Nde I and Xhol I of a vector pET-21a (+) to obtain a recombinant plasmid pET-21 a-P30V.
The recombinant plasmid pET-21a-P30V is transformed into E.coli BL21(DE3) competent cells by a heat shock method to obtain a recombinant bacterium pET-21a-P30V (BL 21).
2. Expression and purification of recombinant proteins
pET-21a-P30V (BL21) was cultured in LB liquid medium containing 100. mu.g/mL ampicillin under the following conditions: culturing at 37 deg.C and shaking table rotation speed of 180r/min until OD is reached600Cooling to 20 ℃ when the concentration is 0.6-0.8, adding IPTG with the final concentration of 1.0mmol/L for induction expression after 30min, then culturing for 17 hours at 20 ℃, and collecting thalli. The cells were washed with a buffer solution, resuspended, and then subjected to ultrasonic lysis by adding PMSF (phenylmethylsulfonyl fluoride), and centrifuged at 10000r/min at 4 ℃ for 30 minutes. The supernatant was collected and each recombinant protein was purified according to the instructions of Ni-NTA affinity chromatography medium (product of King Bio-technology Co., Ltd., Cat. No: L00250). FromAs can be seen in lane 10 of FIG. 3, a specific band appeared around 43kDa for the recombinant protein P30V, consistent with the expectation. Therefore, the recombinant protein P30V is successfully expressed and stored below 70 ℃ for later use.
3. And (3) detecting the antigenicity of the recombinant protein, namely transferring the purified recombinant protein P30V to an NC membrane after SDS-PAGE electrophoresis, and carrying out Western-blotting detection. After blocking overnight with 5% skim milk, add 1:200 dilutions of African swine fever antibody positive reference serum (Harbin veterinary institute of Chinese academy of agricultural sciences) were used as primary antibody and incubated at 37 ℃ for 2 hours. TBST washes were 5 times, 5 min/time. The secondary antibody was incubated with HRP-goat anti-porcine IgG enzyme-labeled antibody (available from Bethyyl under the cat designation a100-105p) diluted 1:20000 times at 37 ℃ for 1.0 hour. TBST washes were 5 times, 5 min/time. ECL was developed in the dark for 5min and exposed for 10 sec. As a result: the recombinant protein P30V shows a specific reaction band around 43kDa (see lane 11 in FIG. 3). The results show that the recombinant protein P30V can specifically react with the African swine fever antibody positive reference serum, and has good antigenicity.
Example 3 preparation of the test strip of the present invention
Referring to fig. 4-6, the test strip for detecting serum antibody of african swine fever virus comprises a base plate 2, and a sample pad 3, a binding pad 4, a nitrocellulose membrane 5 and a water absorption pad 9 are sequentially arranged on the base plate. The sample pad 3, the combination pad 4, the nitrocellulose membrane 5 and the water absorption pad 9 are sequentially connected end to end and are adhered to the bottom plate 2, and the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are overlapped by 2mm respectively. From the combination pad to the absorbent pad, a first detection line 6, a second detection line 7 and a quality control line 8 are sequentially arranged on the nitrocellulose membrane 5. The first detection line 6 is coated with a recombinant protein CD2v and a recombinant protein MGF 360; the second detection line 7 is coated with recombinant protein P30V. The control line is coated with chicken anti-SPA polyclonal antibody.
The preparation method of the test strip for detecting the serum antibody of the African swine fever virus comprises the following steps:
1. preparation of quantum dot microsphere labeled SPA
Activation of quantum dot microspheres: 50 μ L of the quantum dot microsphere suspension with a concentration of 1.2 μ g/μ L was added to 50 μ L of MES buffer with a concentration of 0.02M, pH 6.0.0, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) with a final concentration of 10mg/mL and NHS (N-hydroxysuccinimide) with a final concentration of 10mg/mL were added, and the mixture was incubated in a shaker at 37 ℃ for 20 minutes. Centrifuging for 20 minutes at 10000g in a centrifuge, discarding the supernatant, adding 50 mu L MES buffer solution with the concentration of 0.02M, pH 6.0.0 for heavy suspension, and obtaining the activated quantum dot microspheres.
Coupling of quantum dot microspheres: adding 13 μ g SPA (staphylococcal protein A, purchased from abcam, cat # ab52953) into the activated quantum dot microspheres, quickly mixing uniformly, incubating in a shaker at 37 ℃ for 3 hours, and coupling the SPA and the quantum dot microspheres to obtain a coupling agent of the SPA and the quantum dot microspheres.
Washing and sealing the quantum dot microspheres: adding 20 mu L of BSA (bovine serum albumin) aqueous solution with the mass percentage concentration of 1% into the obtained SPA and quantum dot microsphere conjugate, quickly mixing uniformly, reacting for 0.5h at 37 ℃, centrifuging for 15 minutes at 10000g, removing supernatant, adding 600 mu L of 0.02M boric acid buffer solution with the pH of 8.0 and containing 0.5% (mass percentage concentration) BSA, 5% (mass percentage concentration) sucrose and 0.2% (volume percentage concentration) Tween20 for re-suspension to obtain the SPA solution marked by the quantum dot microsphere, and placing the SPA solution in a refrigerator with the temperature of 4 ℃ for dark storage.
2. Preparation of sample pad
The glass fiber was soaked in 0.02M, pH8.0 boric acid buffer containing 2% (mass percent) BSA, 1% (volume percent) Tween20 for 20min, and then dried to obtain a sample pad.
3. Preparation of the conjugate pad
(1) The glass fibers were soaked for 20min in 0.02M pH8.0 boric acid buffer containing 0.5% (mass percent) BSA, 6% (mass percent) sucrose, 0.5% (volume percent) PEG1500, 0.5% (volume percent) Tween20, and then dried for use.
(2) The quantum dot microsphere-labeled SPA solution prepared in the title 1 of this example was sprayed onto the glass fibers treated as described above in an amount of 0.7. mu.g/cm using a gold spraying apparatus. The amount of spray applied refers to the amount of protein sprayed per cm of long conjugate pad (width 0.9 cm).
(3) Drying at 37 ℃ for 5h to obtain the SPA bonding pad sprayed with the quantum dot microsphere label.
4. Preparation of nitrocellulose membranes
(1) After mixing the recombinant protein CD2v and the recombinant protein MGF360 (prepared in example 1) at a mass ratio of 1:1, the mixture was diluted to a total protein concentration of 0.9mg/mL with a 0.01M PBS buffer solution containing 3% (mass percent) sucrose at pH7.4 to obtain a first detection line solution. The 0.01M, pH7.4 PBS buffer containing 3% (mass percentage concentration) of sucrose was obtained by dissolving sucrose in 0.01M, pH7.4 PBS buffer.
(2) The recombinant protein P30V (prepared in example 2) was diluted to 0.5mg/mL with 3% (mass percent concentration) sucrose in 0.01M PBS buffer (pH 7.4) to obtain a second test line solution.
(3) Chicken anti-SPA polyclonal antibody (purchased from abcam, cat # ab19483, where SPA refers to staphylococcal protein A) was diluted to 0.8mg/mL with 0.01M, pH7.4 PBS buffer containing 3% (mass percent) sucrose to obtain a quality control line solution.
(4) Spraying a first detection line solution on a nitrocellulose membrane by using a membrane scribing instrument to form a first detection line, wherein the spraying amount is 0.9 mu g/cm; spraying a second detection line solution to form a second detection line, wherein the spraying amount is 0.5 mu g/cm; spraying the quality control line solution to form a quality control line (C line), wherein the spraying amount is 0.8 mu g/cm. The spraying amount refers to the amount of protein sprayed on a detection line or a quality control line with the length of each centimeter.
(5) And (5) drying overnight to obtain the nitrocellulose membrane sprayed with the detection line and the quality control line.
5. Preparation of sample dilutions
A0.02M, pH8.0 boric acid buffer containing 2% (mass percent) BSA and 0.5% (volume percent) Tween20 was prepared as a sample diluent according to the present invention.
6. Assembly of test strips
According to the structure of the test strip in fig. 4-6, the test strip is assembled by the following method: on a clean operation table at normal humidity and temperature, the sample pad 3 prepared in the embodiment, the binding pad 4 sprayed with the SPA marked with quantum dot microspheres, the nitrocellulose membrane 5 sprayed with the detection line and the quality control line, and the water absorption pad 9 (made of water absorption filter paper) are sequentially overlapped and adhered to the bottom plate 2 by 2-4mm, and then are sent to a slitter to obtain a test strip with a width of 4 +/-0.5 mm, which is marked as the test strip of the invention. Picking up the intact and tidy test paper strips, putting the test paper strips into a card shell, putting the test paper strips and 1 desiccant into an aluminum foil bag after the capping is finished, and sealing and storing the test paper strips and the desiccant. Wherein the bottom plate 2 is a PVC plate.
1. The method for detecting the African swine fever virus serum antibody by adopting the test strip and the sample diluent comprises the following steps:
(1) sample treatment: and (3) centrifuging the collected whole blood of the pig at 4000g for 15min or overnight at 4 ℃, and naturally precipitating, wherein serum or a diluent obtained by diluting the serum by adopting standard negative serum is taken as a detection sample.
(2) Tearing the aluminum foil bag of the test strip, taking out the test strip and placing the test strip on a clean operation table.
(3) The test sample is aspirated by a 1mL pasteur pipette, 1 drop of test sample (about 30. mu.L per drop) is added to 2 mL of sample dilution, and the mixture is mixed well.
(4) 3 drops (about 30. mu.L per drop) of the mixture from step (3) were pipetted with a 1mL Pasteur pipette and slowly added vertically above the sample pad.
(5) And (5) after the dripping is finished, waiting for 10-15min, and judging the result.
2. Determination of results
Irradiating the reacted test strip by using a 365nm ultraviolet light emitter, judging that the test strip is infected by the African swine fever virus if the C line of the test strip is colored, the first detection line is colored and the second detection line is also colored, and judging that the result is the wild virus infection, wherein the darker the colors of the first and second detection lines are, the higher the antibody titer generated by the African swine fever virus wild virus infection in the sample is. When the C line of the test strip is colored, the first detection line is not colored, and the second detection line is colored, the result is judged that the antibody produced after the gene deletion vaccine is immunized is positive, and the darker the color of the second detection line is, the higher the antibody titer produced by the vaccine immunization in the sample is. When the C line of the test strip is colored, the first detection line is not colored, and the second detection line is not colored, the test strip is judged to be negative, and the test strip is not infected by wild viruses and fails in vaccine immunization. And if the C line of the test strip does not develop color, the test result of the test strip is invalid.
Example 5 evaluation of the test strip of the present invention
1. Specificity detection
By using the sample diluent of the invention in combination with the test strip of the invention, according to the method of the embodiment 4, African swine fever antibody positive reference serum (Harbin veterinary institute of Chinese academy of agricultural sciences), porcine circovirus antibody standard positive serum, foot-and-mouth disease virus antibody standard positive serum, porcine reproductive and respiratory syndrome virus antibody standard positive serum, classical swine fever virus antibody standard positive serum and porcine pseudorabies virus antibody standard positive serum are detected.
The African swine fever virus positive serum is serum generated after the African swine fever virus is attacked by virus after being identified by a nucleic acid sequence in a laboratory. The African swine fever virus refers to an African swine fever virus from nature, and the African swine fever virus is not artificially subjected to any gene deletion or modification.
And (3) detection results: detection result of positive reference serum of African swine fever antibody: the first detection line, the second detection line and the quality control line (C line) are all colored. Results of the other 5 specific sera: only C line develops color. The experimental results show that: the test strip has good specificity and has no cross reaction with other virus positive serum.
2. Sensitivity detection
By matching the sample diluent with the test strip of the invention and according to the method in the embodiment 4, the African swine fever antibody positive reference serum diluted by 2 times is detected by using the African swine fever negative reference serum (Harbin veterinary institute of Chinese academy of agricultural sciences), and the result shows that: the test strip still has the detection result that the first detection line, the second detection line and the quality control line (C line) are all colored for the diluent with the dilution of 1: 128000. For the diluent with the dilution degree of 1:256000, the detection result shows that the first detection line and the second detection line do not develop color, and the quality control line (C line) develops color. Therefore, the lowest detection limit of the sample diluent and the test strip for detecting the African swine fever antibody positive reference serum is 1:128000 dilution.
3. Stability detection
The test paper strip is put into an aluminum foil bag containing 1 desiccant and is sealed for storage, and then is respectively stored for 7 days, 14 days, 21 days and 28 days at 20 ℃ and 37 ℃, and the test paper strip is subjected to sensitivity and specificity detection. The result shows that the test strip has good specificity and no cross reaction, and the test result of the diluent of the African swine fever antibody positive reference serum is 1: 128000: the first detection line, the second detection line and the quality control line (C line) are all colored.
Example 6 comparison of the sensitivity of the test strip of the invention with that of the colloidal gold test strip
1. Preparing colloidal gold particles: reducing chloroauric acid by using a trisodium citrate reducing agent to prepare 20-40nm colloidal gold particles, wherein the specific method comprises the following steps: heating 800mL of chloroauric acid aqueous solution with the mass percentage concentration of 1% to boiling by using a constant-temperature electromagnetic stirrer, adding 1mL of trisodium citrate aqueous solution with the mass percentage concentration of 16% under the condition of continuous stirring, and continuously stirring and heating for 5-10min to obtain a bright red solution. Cooling at room temperature, supplementing the volume to 800mL with deionized water to obtain 20-40nm colloidal gold particles, and storing at 4 ℃.
Preparation of SPA-colloidal gold marker: taking 1mL of the colloidal gold particles prepared in the step 1, and adding 4.2 muL of K with the concentration of 0.1mol/L2CO3Adjusting the pH value of the solution, adding 1.8mg of SPA, uniformly mixing, standing for 5min, adding 10 mu L of polyethylene glycol 2000 solution with the mass percentage concentration of 10%, centrifuging at 12000 rpm for 9min, removing the supernatant, adding 100 mu L of complex solution (aqueous solution containing 0.05M of tris (hydroxymethyl) aminomethane and 5% of sucrose), and uniformly mixing to obtain the SPA marked by the colloidal gold.
3. Preparing a colloidal gold pad: the colloidal gold labeled SPA and 50mM Tris-HCl buffer solution with pH8.0 and containing 0.4% (mass percentage concentration) BSA, 0.3% (volume percentage concentration) Tween20 and 2.5% (mass percentage concentration) sucrose were mixed uniformly according to the volume ratio of 1:90, and then used for soaking glass fiber for 30 minutes, and dried at 37 ℃ for 30 minutes, thus obtaining the colloidal gold pad. In the preparation of the colloidal gold pad, 50mM, pH8.0 Tris-HCl buffer containing 0.4% BSA, 0.3% Tween-20 and 2.5% sucrose and its volume ratio to the colloidal gold labeled SPA were used under optimized conditions.
4. Assembling the test strip: the colloidal gold test strip is assembled according to the method for assembling the test strip of the present invention, except that the binding pad is replaced with the colloidal gold pad (prepared in title 3 of this example) to obtain the colloidal gold test strip. The detection method of the colloidal gold test strip comprises the following steps: diluting the African swine fever antibody positive reference serum by 2 times of the African swine fever negative reference serum (Harbin veterinary institute of Chinese academy of agricultural sciences), sucking 3 drops (about 30 microliters per drop) of each concentration of diluent by using a 1mL pasteur pipette, dripping the diluent on a sample pad, directly observing by naked eyes, and showing that a detection sample is African swine fever wild virus infection when a first detection line, a second detection line and a quality control line are all colored; when the C line of the test strip is colored, the first detection line is not colored, and the second detection line is colored, the result is judged that the antibody generated after the gene deletion vaccine is immunized is positive; when the C line of the test strip is colored, the first detection line is not colored, and the second detection line is not colored, the test strip is judged to be negative, and the test strip is not infected by wild viruses and fails in vaccine immunization.
5. The test paper strip of the invention and the colloidal gold test paper strip sensitivity contrast test
The African swine fever antibody positive reference serum is diluted by 2 times of the African swine fever negative reference serum (Harbin veterinary institute of Chinese academy of agricultural sciences), then the sample diluent is matched with the test strip (according to the method in the embodiment 4) for detection, and meanwhile, the diluent is detected by the colloidal gold test strip prepared in the embodiment, and the detection limits of the two methods are compared. As a result, the lowest detection limit of the colloidal gold test strip is 1:8000 dilution, which is far lower than the lowest detection limit of the sample diluent of the invention matched with the test strip of the invention for detection, of 1:128000 dilution.
Example 7 Effect of first detection line coating protein on detection sensitivity in the test strip of the present invention
The recombinant protein CD2v (prepared in example 1) was diluted to 0.9mg/mL with 3% (mass percent concentration) sucrose in 0.01M PBS buffer at pH7.4 to obtain a control first test line solution A. The recombinant protein MGF360 (prepared in example 1) was diluted to 0.9mg/mL with 3% (mass percent concentration) sucrose in 0.01M PBS buffer, pH7.4, to obtain a control first test line solution B. The control test strip 1 was prepared in the same manner as in example 3, except that the first test line solution therein was replaced with the control first test line solution a. The control test strip 2 was prepared in the same manner as in example 3, except that the first test line solution therein was replaced with the control first test line solution B.
The African swine fever antibody positive reference serum is diluted by 2 times of the African swine fever negative reference serum (Harbin veterinary institute of Chinese academy of agricultural science), and then the sample diluent is matched with the test strip of the invention respectively, the sample diluent is matched with the control test strip 1, and the sample diluent is matched with the control test strip 2 for detection, so as to carry out sensitivity comparison test. The result shows that the lowest detection limit of the control test strip 1 is dilution 1:32000, the lowest detection limit of the control test strip 2 is 1:16000, and the sensitivity is far lower than the lowest detection limit when the sample diluent is matched with the test strip of the invention for detection: dilution 1: 128000.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.
SEQUENCE LISTING
<110> Nanjing university of agriculture
Jiangsu province academy of agricultural science
<120> test strip for detecting serum antibody of African swine fever virus and application thereof
<130> 20200804
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 300
<212> DNA
<213> artificial
<220>
<223> recombinant protein CD2v
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gacctgcacg aagcttacga aaaagacaaa atccacatgg acatcgaaga aatgatgcgt 480
atcgcttgca tcaaagacca caacctgtct accatgtact actgctacgt tctgggtgct 540
aacatcaacc aggctatgct gtcttctatc cagtactaca acatcgaaaa catgttcttc 600
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gaactgatca aaaacatcct gtctctgaaa atctactctc cggctaccac cccgctgccg 720
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Claims (10)
1. The test strip for detecting the serum antibody of the African swine fever virus is characterized by comprising a bottom plate, wherein a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the bottom plate; the bonding pad is coated with SPA marked by quantum dot microspheres; the nitrocellulose membrane is provided with a first detection line and a quality control line, the first detection line is coated with a recombinant protein CD2v and a recombinant protein MGF360, and the first detection line is arranged close to one side of the binding pad; the quality control line is coated with chicken anti-SPA polyclonal antibody and is arranged close to one side of the water absorption pad.
2. The test strip of claim 1, wherein the chicken anti-SPA polyclonal antibody is purchased from abcam, cat # ab 19483.
3. The test strip of claim 1 or 2, wherein: the sample pad is obtained by soaking glass fibers in a boric acid buffer solution containing bovine serum albumin and Tween 20.
4. The test strip of claim 3, wherein: the combination pad is obtained by soaking glass fiber in boric acid buffer solution containing bovine serum albumin, sucrose, Tween20 and PEG1500, and spraying SPA solution marked by quantum dot microspheres.
5. The test strip of claim 4, wherein the sequences of the recombinant protein CD2v and the recombinant protein MGF360 are shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively.
6. The test strip of claim 5, wherein the quantum dot microspheres are coupled with SPA to obtain a conjugate of SPA and quantum dot microspheres; and then blocking the SPA and quantum dot microsphere conjugate by BSA, and suspending the SPA and quantum dot microsphere conjugate in a boric acid buffer solution containing BSA, sucrose and Tween20 to obtain a quantum dot microsphere labeled SPA solution.
7. The test strip of claim 6, wherein the mass ratio of the recombinant protein CD2v and the recombinant protein MGF360 coated on the first detection line is 1: 0.8-1.2.
8. The test strip of any one of claims 1-7, wherein the nitrocellulose membrane is further provided with a second detection line coated with a recombinant protein P30V, and the amino acid sequence of the recombinant protein P30V is shown in SEQ ID NO 6.
9. A sample diluent for use with the test strip of any one of claims 1 to 8, wherein the sample diluent is a borate buffered saline containing BSA and Tween 20.
10. A method for non-diagnostic purposes of detecting antibodies to african swine fever virus using the test strip of claim 1 and the sample dilution of claim 9: and (3) adding a serum sample into the sample diluent for dilution, then dropwise adding the diluted serum sample onto the sample pad of the test strip, and after reaction, detecting the line C and the detection line by adopting an ultraviolet light emitter.
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Inventor after: Liu Fei Inventor after: Liu Beibei Inventor after: Hua Lizhong Inventor after: Xie Qingyun Inventor after: Xiong Qiyan Inventor after: Shao Guoqing Inventor after: Shan Yanke Inventor after: Lu Yunan Inventor after: Wang Li Inventor after: Feng Zhixin Inventor after: Li Jiahao Inventor after: Bai Yun Inventor after: Chen Rong Inventor after: Zhang Lei Inventor after: Wei Yanna Inventor after: Zhang Yue Inventor after: Li Yue Inventor before: Liu Fei Inventor before: Liu Beibei Inventor before: Hua Lizhong Inventor before: Xie Qingyun Inventor before: Xiong Qiyan Inventor before: Shao Guoqing Inventor before: Shan Yanke Inventor before: Lu Yunan Inventor before: Wang Li Inventor before: Feng Zhixin Inventor before: Li Jiahao Inventor before: Bai Yun Inventor before: Chen Rong Inventor before: Zhang Lei Inventor before: Wei Yanna Inventor before: Zhang Yue Inventor before: Li Yue |
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Application publication date: 20201110 |