Quantum dot microsphere fluorescent immunochromatographic test paper for combined detection of asfv antibody and nucleic acid and application thereof

Abstract

The application provides a quantum dot microsphere fluorescent immunochromatography test paper for combined detection of ASFV antibodies and nucleic acids and an application thereof, and belongs to the technical field of veterinary biological diagnosis products. The quantum dot microsphere fluorescent immunochromatography test paper for combined detection of ASFV antibodies and nucleic acids comprises a base plate, on the base plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially connected in a head-to-tail mode, from the combination pad to the water absorption pad, the nitrocellulose membrane is sequentially provided with an antibody detection line, a nucleic acid detection line and a quality control line, the antibody detection line is coated with a goat anti-pig IgG-Fc antibody, the nucleic acid detection line is coated with streptavidin, and the quality control line is coated with a rabbit anti-sheep IgG antibody; the combination pad is coated with a quantum dot microsphere labeled recombinant protein P22 and a quantum dot microsphere labeled sheep anti-digoxin antibody. The test paper can rapidly, highly specifically and highly sensitively detect ASFV antibodies and nucleic acids.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological diagnostic products technology, specifically relating to a quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids and its application. Background Technology

[0002] African swine fever (ASF) is a devastating infectious disease caused by the African swine fever virus (ASFV), affecting pigs of all breeds and ages. It is listed as a disease requiring immediate reporting by the World Organisation for Animal Health (WOAH), and is classified as a Class A animal disease in my country.

[0003] Direct contact between healthy and infected pigs with ASFV contaminants or bites from infected ticks and other vector insects are the main routes of ASFV transmission. Currently, there are no specific drugs or vaccines for ASFV. The main control strategies rely on strict biosecurity measures and the culling of infected or exposed animals. Therefore, ASFV detection and early diagnosis are crucial for the confirmation and control of outbreaks.

[0004] Currently, ASF field testing still mainly relies on clinical manifestations and pathological lesions. In field diagnosis, veterinarians make differential diagnoses based on certain characteristic symptoms or lesions in the pig herd, providing a relatively rapid direction for disease control. However, African swine fever has multiple manifestations, including peracute, acute, subacute, and chronic, with diverse clinical symptoms and a lack of specific symptoms. Therefore, laboratory testing is necessary for the differential diagnosis of African swine fever. Various effective detection methods are used to detect the African swine fever virus and antibodies.

[0005] In clinical applications, the sensitivity of ASFV antibody detection is significantly lower than that of nucleic acid detection. Sometimes, there are cases where the antibody test is negative but ASFV infection occurs. This is because pigs with ASF may die before developing antibodies due to acute onset of the disease. Live diagnosis requires routine tissue polymerase chain reaction (PCR) and serological testing to detect antibodies.

[0006] Currently, the laboratory testing methods for African swine fever virus (ASFV) recommended by the World Organisation for Animal Health (WOAH) include virus isolation, fluorescent antibody detection, and conventional polymerase chain reaction (PCR). Virus isolation requires stringent environmental conditions and is time-consuming, while fluorescent antibody detection and PCR offer high specificity and sensitivity, but both demand high-level experimental conditions and equipment, making rapid on-site detection difficult. In addition, detection methods include enzyme-linked immunosorbent assay (ELISA) kits and colloidal gold immunochromatographic strips. ELISA kits offer high specificity and sensitivity and are relatively easy to operate, but they are highly sensitive to environmental factors such as temperature and pH, and are prone to false positives due to interference from autoantibodies and heterophile antibodies, making them unsuitable for rapid on-site detection. Colloidal gold immunochromatographic strips for rapid ASFV antibody detection have low sensitivity. Therefore, the market currently lacks on-site detection technologies that simultaneously achieve rapid, highly specific, and highly sensitive ASFV nucleic acid and antibody testing. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the first objective of this invention is to provide a quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids, enabling rapid, highly specific, and highly sensitive detection of ASFV antibodies and nucleic acids.

[0008] A second objective of the present invention is to provide a method for detecting blood samples using the test strip for non-diagnostic purposes.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids includes a base plate. On the base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially overlapped end-to-end. From the conjugate pad to the absorbent pad, the nitrocellulose membrane is sequentially provided with an antibody detection line, a nucleic acid detection line, and a control line. The antibody detection line is coated with goat anti-pig IgG-Fc antibody, the nucleic acid detection line is coated with streptavidin, and the control line is coated with rabbit anti-sheep IgG antibody. The conjugate pad is coated with recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres.

[0011] In this invention, the amino acid sequence of the recombinant protein P22 is shown in SEQ ID NO:5. In this invention, the sheep anti-digoxin antibody was purchased from Beijing Novogene Biotechnology Co., Ltd., product number 3210-0488.

[0012] In this invention, the goat anti-pig IgG-Fc antibody was purchased from Abmart, product number ab112748, and the rabbit anti-sheep IgG antibody was purchased from Abcam, product number 31480.

[0013] In this invention, a glass cellulose membrane is soaked in a borate buffer containing Triton, BSA and glucose, and then dried to obtain a sample pad.

[0014] In this invention, a glass cellulose membrane is soaked in a Tris-HCl buffer containing BSA, sucrose, PEG1500, and Tween20, and then sequentially sprayed with recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres, and dried to obtain a conjugation pad.

[0015] In this invention, quantum dot microspheres are conjugated with recombinant protein P22 and sheep anti-digoxin antibody, respectively, to obtain recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres, respectively.

[0016] In this invention, the mass ratio of the quantum dot microspheres to recombinant protein P22 is 5-7:1, and the mass ratio of the quantum dot microspheres to sheep anti-digoxin antibody is 6-10:1.

[0017] This invention also provides a method for detecting blood samples using the test strip for non-diagnostic purposes, characterized by comprising the following steps:

[0018] (1) Using genomic DNA from blood samples as a template, isothermal nucleic acid amplification was performed using primers F, primers R, and probes;

[0019] (2) The nucleic acid amplification product is diluted and mixed with the serum of the blood sample to obtain a mixed product;

[0020] (3) Add quantum dot microsphere-labeled recombinant protein P22 and quantum dot microsphere-labeled sheep anti-digoxin antibody to the mixture, shake to mix, then dilute with buffer containing Tween-20, add to the sample pad of the test strip, and after chromatography, use a fluorescence analyzer or ultraviolet laser lamp to interpret the results.

[0021] In this invention, the nucleotide sequence of primer F is shown in SEQ ID NO:1; the nucleotide sequence of primer R is shown in SEQ ID NO:2, and the 5' end of primer R is labeled with biotin; the nucleotide sequence of probe is shown in SEQ ID NO:3, the 5' end of probe is connected with digoxigenin, the 3' end of probe is connected with C3-spacer, and a tetrahydrofuran is labeled between the 30th and 31st nucleotides of SEQ ID NO:3.

[0022] The experimental principle of this invention is an indirect method. The 5' end of the downstream primer of the RPA is labeled with a modifying group (biotin); the 5' end of the fluorescent probe is modified with an antigen label (digoxigenin); a dSpacer (tetrahydrofuran, THF) is labeled at the midpoint between the 5' and 3' ends as the recognition site for the NFO; and a C3-Spacer modifying group is labeled at the 3' end. When African swine fever virus target DNA is present in the sample, a digoxigenin-labeled amplification product is generated via the RPA reaction. After the probe hybridizes with this product, the endonuclease in the system cleaves the THF residues, allowing the probe to act as a new primer to generate a digoxigenin- and biotin-labeled double-labeled target DNA amplicon. Sheep anti-digoxigenin antibody labeled with quantum dot microspheres captures double-labeled target DNA amplicones. The captured target DNA amplicones bind and are immobilized by streptavidin coated on the DNA test line (T2). Excess sheep anti-digoxigenin antibody labeled with quantum dot microspheres binds and is immobilized by rabbit anti-sheep IgG antibody coated on the control line (C), and the results are visualized by the labeled quantum dot microspheres. At this point, the nucleic acid detection line T2 and the control line C are both visible. When African swine fever virus antibodies are present in the sample, recombinant protein P22 labeled with quantum dot microspheres captures African swine fever virus antibodies in the serum. The captured antibodies bind and are immobilized by goat anti-pig IgG-Fc antibody coated on the test line, and the results are visualized by the labeled quantum dot microspheres. Excess sheep anti-digoxigenin antibody labeled with quantum dot microspheres binds and is immobilized by rabbit anti-sheep IgG antibody coated on the control line (C), and the results are visualized by the labeled quantum dot microspheres. At this point, the antibody detection line T1 and the control line C are both visible.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The quantum dot microsphere fluorescent immunochromatographic test strip of the present invention for the combined detection of ASFV antibodies and nucleic acids has advantages such as high uniformity, good monodispersity and strong stability. Therefore, the test strips made using quantum dot microspheres as markers have small batch-to-batch differences.

[0025] (2) The quantum dot microsphere fluorescent immunochromatographic test strip of the present invention for the combined detection of ASFV antibodies and nucleic acids is easy to prepare and operate, requires simple raw materials, can be mastered in a short time, and has broad market prospects and great economic benefits.

[0026] (3) The test strip of the present invention, combined with the isothermal reaction system of recombinant enzyme polymerase amplification, can rapidly and simultaneously detect African swine fever virus antibodies and nucleic acids. It has high specificity, high sensitivity, stability and good repeatability.

[0027] (4) The method of using the test strip of the present invention to detect blood samples for non-diagnostic purposes is simple to operate, requires no professional training, and can be well used in various field and grassroots laboratory environments. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of a quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids; wherein: 1-fluorescent immunochromatographic test strip; 2-PVC base plate; 3-sample pad; 4-conjugation pad; 5-nitrocellulose membrane; 6-antibody detection line; 7-nucleic acid detection line; 8-control line; 9-absorbent pad.

[0029] Figure 2 This is a schematic diagram showing the results of a quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids in samples with double positive nucleic acid antibody test, samples with negative nucleic acid antibody test, samples with positive nucleic acid antibody test, and samples with double negative nucleic acid antibody test. "+" indicates positive and "-" indicates negative.

[0030] Figure 3 The results are obtained by 2% agarose gel electrophoresis of the isothermal rapid amplification products of the positive samples, where M is the DNA Marker, 1 is the ddH2O negative control, and 2 is the isothermal rapid amplification product of the positive samples.

[0031] Figure 4 This is a Western blotting image of recombinant protein P22, where M is the protein marker and lane 1 is the purified recombinant protein P22. Detailed Implementation

[0032] Example 1: Preparation of recombinant protein P22

[0033] 1. Construction of the P22 gene recombinant vector

[0034] Referring to the gene sequence of CP204L (MH766894, KP177R, 3251-3784) of my country's first case of African swine fever published in the NCBI database, codons were optimized for E. coli tropism, resulting in the gene sequence of recombinant protein P22 (as shown in SEQ ID NO: 4). The amino acid sequence of recombinant protein P22 is shown in SEQ ID NO: 5. The gene for recombinant protein P22 was prepared using gene synthesis (Nanjing Genscript Biotech Co., Ltd.). This gene sequence was inserted between the NdeⅠ and XholⅠ restriction enzyme sites of the vector pET-21a(+), yielding the recombinant plasmid pET-21a-P22. The recombinant plasmid pET-21a-P22 was transformed into E. coli BL21(DE3) competent cells using the heat shock method, obtaining the recombinant bacterium pET-21a-P22(BL21). Colony PCR identification showed a specific band at 768 bp, indicating successful construction of the recombinant vector.

[0035] 3. Expression and purification of recombinant proteins

[0036] pET-21a-P22 (BL21) was cultured in LB liquid medium containing 100 μg / mL ampicillin under the following conditions: 37°C, shaking speed 180 rpm. When the OD600 reached 0.6–0.8, the temperature was lowered to 20°C. After 30 min, IPTG at a final concentration of 1.0 mmol / L was added to induce expression. The culture was then continued at 20°C for 17 hours, and the cells were collected. The cells were washed with buffer, resuspended, and subjected to ultrasonic lysis with PMSF (phenylmethylsulfonyl fluoride). The cells were then centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and the recombinant protein P22 was purified according to the instructions of Ni-NTA affinity chromatography medium (GenScript Biotechnology Co., Ltd., Cat. No: L00250). The purified recombinant protein P22 showed a specific band at approximately 20 kDa, consistent with expectations. Therefore, the recombinant protein P22 was successfully expressed and stored at -70°C for later use.

[0037] 3. Antigenicity detection of recombinant proteins

[0038] The purified recombinant protein P22 was transferred to an NC membrane after SDS-PAGE electrophoresis and then detected by Western blotting. After blocking with 5% skim milk overnight, the membrane was incubated at 37°C for 2 hours with a 1:200 dilution of African swine fever antibody-positive reference serum (Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) as the primary antibody. The membrane was washed 5 times with TBST for 5 minutes each time. A 1:20,000 dilution of HRP-goat anti-pig IgG enzyme-labeled antibody (purchased from Bethyl, catalog number a100-105p) was used as the secondary antibody, and the membrane was incubated at 37°C for 1 hour. The membrane was washed 5 times with TBST for 5 minutes each time. ECL was applied for staining in the dark for 5 minutes, followed by exposure for 10 seconds. Results: A specific reaction band for recombinant protein P22 appeared at approximately 20 kDa. Figure 4 The above results indicate that recombinant protein P22 can specifically react with African swine fever antibody-positive reference serum, demonstrating good antigenicity.

[0039] Example 2: Quantum dot microsphere fluorescent immunochromatographic test strip for combined detection of ASFV antibodies and nucleic acids

[0040] 1. Quantum dot microspheres labeled with sheep anti-digoxin antibodies

[0041] (1) Activation of quantum dot microspheres: 50 μL of a quantum dot microsphere suspension with a concentration of 1.2 μg / μL (purchased from Beijing Najing Biotechnology Co., Ltd., product number FM610C) was added to 100 μL of MES buffer with a concentration of 0.01 M and pH 6.0 containing 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mg / mL of N-hydroxysuccinimide. After continuous shaking and incubation at 37 °C for 30 min, the precipitate was collected by centrifugation. The precipitate was dispersed in 100 μL of MES buffer with a concentration of 0.02 M and pH 7.2 and resuspended to obtain activated quantum dot microspheres.

[0042] (2) Coupling of quantum dot microspheres: At room temperature, 7 μg of sheep anti-digoxin antibody (purchased from Beijing Nuowei Biotechnology Co., Ltd., product number 3210-0488) was added to activated quantum dot microspheres and reacted for 4 h to couple the sheep anti-digoxin antibody with the quantum dot microspheres to obtain a conjugate of anti-digoxin antibody and quantum dot microspheres.

[0043] (3) Blocking of quantum dot microspheres: Add 2 μL of PBS buffer solution containing 10% (w / w) BSA at a concentration of 0.01 M and pH 7.0 to the conjugate of anti-digoxin antibody and quantum dot microspheres, and stir gently at room temperature for 20 minutes to block the conjugate of anti-digoxin antibody and quantum dot microspheres.

[0044] (4) Resuspension of quantum dot microspheres: The conjugate of blocked anti-digoxigenin antibody and quantum dot microspheres was centrifuged at 10,000 rpm for 15 min. The precipitate was collected and resuspended in 100 μL of 0.02 M MES buffer at pH 7.0 to obtain sheep anti-digoxigenin antibody labeled with quantum dot microspheres. The antibody was stored in a refrigerator at 4 °C.

[0045] 2. Quantum dot microspheres labeling recombinant protein P22

[0046] (1) Activation of quantum dot microspheres: 50 μL of a quantum dot microsphere suspension with a concentration of 1.2 μg / μL (purchased from Beijing Najing Biotechnology Co., Ltd., product number FM610C) was added to 100 μL of MES buffer with a concentration of 0.01 M and pH 6.0 containing 10 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mg / mL of N-hydroxysuccinimide. After continuous shaking and incubation at 37 °C for 30 min, the precipitate was collected by centrifugation. The precipitate was dispersed in 100 μL of MES buffer with a concentration of 0.02 M and pH 7.2 and resuspended to obtain activated quantum dot microspheres.

[0047] (2) Coupling of quantum dot microspheres: 10 μg of recombinant protein P22 was added to activated quantum dot microspheres and incubated at 37°C in a shaker for 2 h to couple recombinant protein P22 with quantum dot microspheres to obtain a conjugate of recombinant protein P22 and quantum dot microspheres.

[0048] (3) Blocking of quantum dot microspheres: 2 μL of 10% BSA solution was added to the conjugate of recombinant protein P22 and quantum dot microspheres, and the conjugate was incubated at 37°C for 15 min to block the conjugate of recombinant protein P22 and quantum dot microspheres.

[0049] (4) Resuspension of quantum dot microspheres: The conjugate of the blocked recombinant protein P22 and quantum dot microspheres was centrifuged at 10,000 rpm for 10 minutes. The precipitate was collected and resuspended in 0.02 M, pH 7.0 MES buffer to a total volume of 500 μL to obtain quantum dot microsphere-labeled recombinant protein P22. It was then stored in a 4°C refrigerator.

[0050] 3. Preparation of quantum dot microsphere fluorescent immunochromatographic test strip for combined detection of ASFV antibodies and nucleic acids

[0051] The structure of quantum dot microsphere fluorescent immunochromatographic test strip 1 for combined detection of ASFV antibodies and nucleic acids is as follows: Figure 1On the PVC base plate 2, the sample pad 3, conjugate pad 4, nitrocellulose membrane 5, and absorbent pad 9 are sequentially overlapped end to end; the nitrocellulose membrane 5 is adhered to the middle of the base plate 2, with the conjugate pad 4 pasted and overlapped at one end of the nitrocellulose membrane 5, and the absorbent pad 9 (absorbent paper) pasted and overlapped at the other end. From the conjugate pad to the absorbent pad, the nitrocellulose membrane 5 is sequentially provided with antibody detection line 6, nucleic acid detection line 7, and control line 8. The antibody detection line 6 is coated with goat anti-pig IgG-Fc antibody, the nucleic acid detection line 7 is coated with streptavidin, and the control line 8 is coated with rabbit anti-sheep IgG antibody.

[0052] The specific preparation steps for the above-mentioned fluorescent immunochromatographic test strips are as follows:

[0053] (1) Preparation of sample pad: Cut a glass cellulose membrane 16 mm wide, soak it in a 50 mm borate buffer solution at pH 8.0 containing 1% (volume percentage) Triton, 1% (mass percentage) BSA (bovine serum albumin) and 2% (mass percentage) glucose, and dry it to obtain the sample pad.

[0054] (2) Preparation of conjugate pads: Glass cellulose membranes were soaked in 50mM Tris-HCl buffer (pH 8.0) containing 5% (w / v) BSA, 8% (w / v) sucrose, 0.5% (w / v) PEG1500, and 0.5% (v / v) Tween20. Recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres were then uniformly sprayed onto the treated glass cellulose membranes at a spraying rate of 0.5 μg / cm. The spraying rate refers to the amount of protein sprayed per centimeter of a 0.9 cm wide conjugate pad. The membranes were dried at 37℃ for 5 h to obtain the conjugate pads.

[0055] (3) Streptavidin was diluted to 0.6 mg / mL with 0.01 M PBS buffer containing 3% (w / v) sucrose at pH 7.4 to prepare the nucleic acid detection line (T2) working solution. Goat anti-pig IgG-Fc antibody (purchased from Abmart, product number ab112748) was diluted to 0.6 mg / mL with 0.01 M PBS buffer containing 3% (w / v) sucrose at pH 7.4 to prepare the antibody detection line (T1) working solution. Rabbit anti-sheep IgG antibody (purchased from Abcam, product number 31480) was diluted to 0.8 mg / mL with 0.01 M PBS buffer containing 3% (w / v) sucrose at pH 7.4 to prepare the control line (C) working solution. The nucleic acid detection line, antibody detection line and control line solutions were sprayed onto the nitrocellulose membrane using a three-dimensional planar gold spraying instrument. The run-through speed for the control line, nucleic acid detection line, and antibody detection line was 1 μL / cm, and the spacing between adjacent detection lines and control lines was controlled at about 6 mm. The run-through nitrocellulose membrane was dried at 37°C for 12 hours to obtain the treated nitrocellulose membrane, which was then stored in a drying cabinet for later use.

[0056] (4) Assembly: On a clean workbench under normal humidity and temperature, according to... Figure 1 The prepared sample pad 3, binding pad 4, nitrocellulose membrane 5 coated with detection and control lines, and absorbent pad 9 (made of absorbent filter paper) are sequentially overlapped in pairs (2-4 mm) and adhered to the base plate 2. Then, they are fed into a strip cutter to obtain test strips with a width of 4 ± 0.5 mm. Well-formed and neat test strips are selected, placed in a cartridge, and sealed along with one desiccant capsule in an aluminum foil bag. The base plate 2 is made of PVC board.

[0057] 4. Instructions for using the quantum dot microsphere fluorescent immunochromatographic test strip for combined detection of ASFV antibodies and nucleic acids.

[0058] The sample was tested using a quantum dot microsphere fluorescent immunochromatographic test strip that combines the detection of ASFV antibodies and nucleic acids, including the following steps:

[0059] (1) Serum was prepared using blood from healthy pigs to obtain antibody-negative samples. The SimpleViral DNA / RNA Kit (purchased from Beijing TransGen Biotech Co., Ltd., catalog number EC311-11) was used to extract nucleic acids from the blood of healthy pigs, yielding a nucleic acid extract, which served as a nucleic acid-negative sample. Serum was prepared from the blood of ASFV-positive pigs to obtain an antibody-positive sample. Nucleic acid was then extracted from the blood of ASFV-positive pigs to obtain a nucleic acid extract, which served as a nucleic acid-positive sample.

[0060] (2) RPA amplification was performed using a DNA isothermal rapid amplification kit (purchased from Weifang Anpu Future Biotechnology Co., Ltd., product number WLN8203KIT). RPA system: In a dry powder reaction tube (containing RPA reaction particles), add 2 μL of the sample to be tested (nucleic acid positive or negative), 2.5 μL of B buffer, 29.4 μL of A buffer, 2 μL of 10 μM upstream primer F, 2 μL of 10 μM downstream primer R, 0.6 μL of 10 μM RPA probe, and 11.15 μL of ddH2O. Mix the RPA system thoroughly and place on ice for later use.

[0061] The sequences of the upstream primer F, downstream primer R, and RPA primer-probe are shown below:

[0062] The nucleotide sequence of primer F (SEQ ID NO:1) is as follows: 5'-TGATAGACCCCACGTAATCCGTGTCCCAAC-3'.

[0063] The structure of primer R is as follows: 5'-biotin–GTTTCCATCAAAGTTCTGCAGCTCTTACATA-3'. The nucleotide sequence of primer R is shown in SEQ ID NO:2, with biotin attached to the 5' end.

[0064] The structure of the RPA probe is as follows:

[0065] 5'-DIG-CTCCCGTGGCTTCAAAGCAAAGGTAATCAT–THF–

[0066] ATCGCACCCGGATCATCG–C3–spacer-3’.

[0067] The nucleotide sequence of the RPA probe is shown in SEQ ID NO:3. The 5' end is connected to digoxigenin (DIG), and the 3' end is connected to a modification group C3–spacer. A dSpacer (tetrahydrofuran, THF) is marked between the 30th and 31st nucleotides of the RPA probe as a recognition site for exonuclease IV (nfo).

[0068] The upstream primer F, downstream primer R, and RPA primer probe were synthesized by General Biotech.

[0069] (3) After allowing the RPA system to stand at 37°C for 20 minutes, immediately place it on ice for later use; Figure 3 The amplification products of the positive sample are shown.

[0070] (4) Take 1 μL of the amplification product obtained in step (3) and add it to 1000 μL of sample diluent for dilution. The sample diluent is a 0.1 M Tris-HCl buffer with a pH of 8.0 containing 0.5% (w / w) PVP-40 (polyvinylpyrrolidone 40), 0.1% (w / w) Proclin 300, and 1% (v / v) Tween-20. Mix the diluted amplification product with an equal volume of serum sample (antibody-positive or antibody-negative sample) to obtain a mixed product. Add 100 μL of the mixed product to a PCR tube, add 0.5 μL of quantum dot microsphere-labeled recombinant protein P22 and 2 μL of quantum dot microsphere-labeled sheep anti-digoxigenin antibody, and vortex to mix (vortex before each addition of microspheres). Then dilute 10-fold with 0.1 M Tris-HCl buffer (pH 8.0) containing 1% (v / v) Tween-20, and add the solution to the sample pad of the quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids. Spectrometry is performed for 10 minutes. The mixture of amplification products from healthy swine serum and healthy swine blood nucleic acid extracts is a double-negative sample for nucleic acid and antibodies; the mixture of amplification products from ASFV-positive swine serum and ASFV-positive swine blood nucleic acid extracts is a double-positive sample for nucleic acid and antibodies; the mixture of amplification products from healthy swine serum and ASFV-positive swine blood nucleic acid extracts is a nucleic acid-negative and antibody-positive sample; and the mixture of amplification products from ASFV-positive swine serum and healthy swine blood nucleic acid extracts is a nucleic acid-positive and antibody-negative sample.

[0071] (5) Irradiate the nucleic acid detection line, antibody detection line, and control line with ultraviolet excitation light with a wavelength of 300–450 nm and observe the color development (note that you should wear the matching protective goggles); the results are as follows Figure 2 As shown.

[0072] from Figure 2 As can be seen, if the control line, antibody test line, and nucleic acid test line all show color, it indicates the presence of ASFV antibodies and nucleic acid in the sample. If the control line shows color but neither the antibody nor nucleic acid test lines show color, it indicates the absence of ASFV antibodies and ASFV nucleic acid in the sample. If the control line shows color and the antibody test line shows color but the nucleic acid test line does not, it indicates the presence of ASFV antibodies but not ASFV nucleic acid in the sample. If the control line does not show color, the test result is invalid.

[0073] Example 3: Sensitivity of the quantum dot microsphere fluorescent immunoassay strip for combined detection of ASFV antibodies and nucleic acids

[0074] The sensitivity analysis of the quantum dot microsphere fluorescent immunoassay strip for the combined detection of ASFV antibodies and nucleic acids is performed using the following steps:

[0075] (1) Sensitivity for positive serum detection

[0076] Preparation of negative serum: Select piglets aged 4-5 weeks with normal body temperature, appetite, and excretion, and which tested negative for African swine fever virus using a real-time quantitative PCR detection kit (purchased from Qingdao Lijian Biotechnology Co., Ltd.). Collect blood and separate serum, add thimerosal to a final concentration of 0.01% (w / w), and store at -20℃ to obtain negative serum.

[0077] Preparation of positive serum: Recombinant protein P22 was adjusted to 1 mg / mL using PBS buffer (0.01 M, pH 7.4) to obtain P22 antigen. Piglets were immunized with P22 antigen as follows: 1 mg / mL antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified. This mixture was then injected intramuscularly into one healthy 2-month-old piglet at a dose of 2 mL (containing 1 mg of protein) per piglet. Ten days after the first immunization, 1 mg / mL antigen was mixed with Freund's incomplete adjuvant at a 1:1 volume ratio and emulsified. A booster immunization was performed using the same dose as the initial immunization. Ten days later, a second booster immunization was performed using the same method and dose as the second immunization. Seven days after the third immunization, swine blood was aseptically collected to separate serum, yielding positive serum.

[0078] Positive serum was diluted with negative serum at dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, and 1:1024000. Quantum dot microsphere fluorescent immunoassay strips for the combined detection of ASFV antibodies and nucleic acids were used to test both the different dilutions of positive serum and the negative serum.

[0079] Test results: The quantum dot microsphere fluorescent immunoassay strip, which combines the detection of ASFV antibodies and nucleic acids, showed a positive result for a 1:1024000 dilution of positive serum, indicating that the detection limit of the strip for African swine fever virus antibodies in positive serum can be as low as a 1:1024000 dilution.

[0080] (2) Sensitivity to nucleic acid detection

[0081] Preparation of negative nucleic acid samples: Nucleic acid extracts from whole blood samples of healthy pigs were used as negative nucleic acid samples.

[0082] Preparation of positive nucleic acid samples: The full-length ASFV P72 plasmid was used as the positive nucleic acid sample. After the content was determined by spectrophotometry, it was diluted with nucleic acid extracts from healthy pig whole blood samples to 1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copies / μL, and 0.1 copies / μL, respectively. The full-length ASFV P72 plasmid was synthesized by Mingyan Biotechnology Co., Ltd., and the specific gene sequence is shown in SEQ ID NO:6.

[0083] A quantum dot microsphere fluorescent immunoassay strip for the combined detection of ASFV antibodies and nucleic acids was used to detect positive and negative nucleic acid samples of different concentrations. Results: The strip achieved a detection limit as low as 1 copies / μL for positive nucleic acid samples, but showed no color development for negative nucleic acid samples.

[0084] Example 3: Specificity of quantum dot microsphere fluorescent immunoassay strip for combined detection of ASFV antibodies and nucleic acids

[0085] A specific detection method for quantum dot microsphere fluorescent immunoassay strips for combined detection of ASFV antibodies and nucleic acids: Quantum dot microsphere fluorescent immunoassay strips for combined detection of ASFV antibodies and nucleic acids were used to detect standard positive serum and nucleic acid extracts of porcine circovirus antibody, foot-and-mouth disease virus antibody, porcine reproductive and respiratory syndrome virus antibody, classical swine fever virus antibody, and porcine pseudorabies virus antibody.

[0086] Test results: All viral nucleic acid and antibody test results were negative, indicating that the test strip of this invention has no cross-reaction with other swine viruses and has good specificity.

Claims

1. A quantum dot microsphere fluorescent immunochromatographic test strip for the combined detection of ASFV antibodies and nucleic acids, characterized in that... The device includes a base plate on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially overlapped end-to-end. From the conjugate pad to the absorbent pad, the nitrocellulose membrane is sequentially provided with an antibody detection line, a nucleic acid detection line, and a control line. The antibody detection line is coated with goat anti-pig IgG-Fc antibody, the nucleic acid detection line is coated with streptavidin, and the control line is coated with rabbit anti-sheep IgG antibody. The conjugate pad is coated with recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxigenin antibody labeled with quantum dot microspheres. The amino acid sequence of the recombinant protein P22 is shown in SEQ ID NO:

5. The sheep anti-digoxigenin antibody was purchased from Beijing Nuowei Biotechnology Co., Ltd., product number 3210-0488; the goat anti-pig IgG-Fc antibody was purchased from Abmart, product number ab112748; and the rabbit anti-sheep IgG antibody was purchased from Abcam, product number 31480.

2. The test strip according to claim 1, characterized in that... The glass cellulose membrane was immersed in a borate buffer containing Triton, BSA and glucose, and then dried to obtain the sample pad.

3. The test strip according to claim 2, characterized in that... Glass cellulose membranes were soaked in Tris-HCl buffer containing BSA, sucrose, PEG1500, and Tween20, and then sequentially sprayed with recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres. After drying, the binding pads were obtained.

4. The test strip according to claim 3, characterized in that... Quantum dot microspheres were conjugated with recombinant protein P22 and sheep anti-digoxin antibody, respectively, to obtain recombinant protein P22 labeled with quantum dot microspheres and sheep anti-digoxin antibody labeled with quantum dot microspheres, respectively.

5. The test strip according to claim 4, characterized in that... The mass ratio of the quantum dot microspheres to recombinant protein P22 is 5-7:1, and the mass ratio of the quantum dot microspheres to sheep anti-digoxin antibody is 6-10:

1.

6. A method for detecting blood samples using the test strip of claim 1 for non-diagnostic purposes, characterized in that... Includes the following steps: (1) Using genomic DNA from blood samples as a template, isothermal nucleic acid amplification was performed using primers F, primers R, and probes; (2) The nucleic acid amplification product is diluted and mixed with the serum of the blood sample to obtain a mixed product; (3) Add quantum dot microsphere-labeled recombinant protein P22 and quantum dot microsphere-labeled sheep anti-digoxin antibody to the mixed product, shake to mix, then dilute with buffer containing Tween-20, add to the sample pad of the test strip, and after chromatography, use a fluorescence analyzer or ultraviolet laser lamp to interpret the results.

7. The method according to claim 6, characterized in that... The nucleotide sequence of primer F is shown in SEQ ID NO:1; the nucleotide sequence of primer R is shown in SEQ ID NO:2, and the 5' end of primer R is labeled with biotin; the nucleotide sequence of probe is shown in SEQ ID NO:3, the 5' end of probe is connected with digoxigenin, the 3' end is connected with a C3-spacer, and a tetrahydrofuran is labeled between the 30th and 31st nucleotides of SEQ ID NO:3.

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