Porcine epidemic diarrhea virus detection method based on magnetic nanoparticles

Abstract

The invention belongs to the technical field of animal medicine detection, and particularly relates to a porcine epidemic diarrhea virus detection method based on magnetic nanoparticles. The method belongs to a double-probe competitive ELISA (enzyme-linked immunosorbent assay) detection method, and comprises the steps of plate preparation, sample adding, detection, color development, judgment and the like. According to the invention, magnetic microspheres are used as a medium and are coupled with N protein of PEDV (Porcine Epidemic Diarrhea Virus) to prepare the magnetic nanoparticle MNPs-N probe. The preparation of an McAb-HRP probe is further combined, and a double-probe competitive enzyme-linked immunosorbent assay method aiming at the PEDV is established. Through optimization of related detection parameters and experiment conditions, rapid detection of PEDV can be realized. Related experimental results show that the detection method provided by the invention can obtain an accurate result within 50 minutes, and has excellent inter-batch and intra-batch reproducibility. The effect has very important technical significance on immediate detection and judgment and further prevention and control of PED.

Description

Technical Field

[0001] The present application belongs to the field of animal medical detection technology, and specifically relates to a porcine epidemic diarrhea virus detection method based on magnetic nanoparticles. Background Art

[0002] Porcine epidemic diarrhea (PED) is an acute, highly contagious disease caused by porcine epidemic diarrhea virus (PEDV). Its clinical manifestations include vomiting, diarrhea and dehydration. Statistics show that although pigs of all ages are infected with PEDV, piglets are particularly susceptible to infection, and the mortality rate can be as high as 100%.

[0003] Since PED was first reported in the UK in 1971, it has been reported all over the world and has become one of the most serious epidemics in the pig industry. Vaccines are the main way to prevent and control the disease, and the vaccine developed based on the classic vaccine strain CV777 is the most widely used prevention and control vaccine. However, with the emergence of new mutant virus strains, the piglet loss rate caused by PEDV variants can reach 20%-30%, seriously affecting the healthy development of the pig industry.

[0004] Studies on PEDV have shown that PEDV is a typical single-stranded RNA virus with a genome length of approximately 28Kb, encoding 4 structural proteins and at least 17 non-structural proteins. It is worth noting that the N protein, which is a nucleocapsid protein, accounts for a large part of the viral protein and shows a high degree of sequence conservation. This feature makes the N protein an ideal target for the development of highly sensitive and specific diagnostic tools, which will help in the early detection and accurate diagnosis of PED. Summary of the invention

[0005] The main purpose of this application is to provide an enzyme-linked immunosorbent assay method for porcine epidemic diarrhea virus (PEDV) based on magnetic nanoparticles, thereby laying a certain technical foundation for the diagnosis and prevention of PEDV.

[0006] The specific technical solutions adopted in this application are as follows.

[0007] A porcine epidemic diarrhea virus detection method based on magnetic nanoparticles, which belongs to a dual-probe competitive ELISA detection method, specifically comprising the following steps: (I) Prepare the plate. Use the ELISA plate as the test container, add 200 μL of 5% BSA solution to each well, and incubate at 37°C for 1 hour. After incubation, pat dry and proceed directly to subsequent tests, or store at 4°C for later use. (II) Sampling and Testing Add 50 μL of MNPs-N probe, 50 μL of McAb-HRP probe, and 50 μL of PEDV-positive serum (or sample to be tested) to each well; PBS was used as blank control; After adding the sample, shake well at room temperature (about 15-28°C) and let it stand for 30 minutes; (III) Color development and determination After the reaction, the ELISA plate was magnetically separated for 10 seconds, the supernatant was discarded, and the precipitate was washed 4 times with PBST buffer; after washing, 100 μL of TMB colorimetric solution was added, and the colorimetric reaction was carried out at room temperature in the dark. After the reaction was completed, 50 μL of reaction stop solution was added (to stop the color development); Finally, the OD value was measured at 450 nm with PBS buffer as the control, and the inhibition percentage (PI) was calculated. PI = [(1 - OD450 nm positive value (P)) / negative value (N)] x 100%.

[0008] The preparation method of the MNPs-N probe is as follows: Take 2 mg of MNPs, wash them twice with MES (0.015 M, pH = 5.5) solution, and then resuspend them in 0.015 ml of MES; After resuspension, add 0.2 mg of EDC and 0.2 mg of NHC (EDC and NHC are both 10 mg / ml solutions, take the corresponding volume), and shake the reaction at room temperature for 30 minutes; After the reaction, the mixture was separated by magnetic separation, the supernatant was discarded, the MNPs were washed once with MES solution, and resuspended with 0.015 ml MES. After resuspension, 40 μg of purified N protein of PEDV was added, and incubated at room temperature (about 15-25°C) with shaking for 4 h; After the incubation, the MNPs were separated by magnetic separation and washed with PBST buffer (pH = 7.4); Then, 1% BSA (solvent: PBST) was added and incubated at room temperature with shaking for about 1 h to block the unbound sites on the MNPs; After blocking the unbound sites on the MNPs, the coupled MNPs-N were washed with PBST buffer and resuspended with 0.2 mL of PBST buffer to obtain a solution containing the MNPs-N probe.

[0009] The McAb-HRP probe is prepared as follows: (I) Preparation of monoclonal antibodies against PEDV N protein The preparation method of the monoclonal antibody McAb against the N protein of PEDV is as follows: 30 μg (concentration 1 μg / μl) of purified N protein was mixed with immune adjuvant at a volume ratio of 1:1, and then intramuscular immunization was performed on BALB / c female mice (6 weeks old); After the injection, the antibody production in mice was detected by indirect enzyme-linked immunosorbent assay, and mice with higher antibody levels were euthanized, and their spleens were removed, ground, and fused with SP2 / 0 cells; The hybridoma cells are screened, the monoclonal cells that stably secrete N protein-specific antibodies are amplified and cultured, and ascites is prepared; finally, the ascites is purified using a protein G affinity chromatography column to obtain purified McAb; (II) Preparation of McAb-HRP probe Take 5 mg of HRP and dissolve it in 1 mL of deionized water, add 200 μL of 0.1 M NaIO4 solution, stir at room temperature in the dark for about 20 min, put it into a dialysis bag (dialysis molecular weight is 8000-14000D), and dialyze it in 1 mM sodium acetate buffer (pH=4.4) at 4°C for 12 h; After dialysis, add 200 μL of 0.2 M (pH = 9.6) CB solution to the dialysate, then quickly add 1 M NaOH (to enolize HRP), adjust the pH to about 9.5, and then quickly add 10 mg of purified antibody McAb (concentration of 4 mg / ml, PBS as solvent), and treat at 4°C with gentle stirring for about 12 hours; After the stirring treatment was completed, 235 μL of freshly prepared 4 mg / mL NaBH4 solution was added to the above mixture (47 μL of NaBH4 was added for every 1 mg of HRP), mixed well, and allowed to stand at 4°C for 2 h; After standing, add an equal volume of saturated ammonium sulfate solution, stand at 4°C for 30 min, centrifuge at 4°C and 12000 r / min for 30 min, discard the supernatant, resuspend the precipitate with PBS, and dialyze in PBS at 4°C for about 12 h; After the dialysis, the dialysate was centrifuged at 12000 r / min for 10 min, and the supernatant (the supernatant is the McAb-HRP probe solution) was taken for detection and use.

[0010] In the existing technology, the detection of PEDV includes methods such as immunofluorescence antibody technology (IFA), immunohistochemistry, fluorescence quantitative PCR and ELISA. Different detection methods have their own advantages and disadvantages due to different detection equipment requirements and detection accuracy. Because the ELISA method has good convenience, it is one of the most widely used detection methods in batch detection.

[0011] Magnetic nanoparticles (MNPs), also known as magnetic microspheres, have broad application prospects in medicine, pharmaceuticals, separation and purification, and wastewater treatment due to their biocompatibility, high specific surface area, and solution suspension.

[0012] In this application, Fe3O4 magnetic microspheres were used as a medium, and after coupling it with the N protein of PEDV, a magnetic nanoparticle MNPs-N probe was prepared (MNPs were used as a solid support, which could promote the rapid binding of antigens and antibodies through the action of a magnetic field). Further combined with the preparation of the McAb-HRP probe, a dual-probe competitive enzyme-linked immunosorbent (ELISA) method for PEDV was established. Rapid detection of PEDV can be achieved by optimizing relevant detection parameters and experimental conditions. Relevant experimental results show that the detection method provided in this application can obtain accurate results within 50 minutes, and has excellent inter-batch and intra-batch reproducibility. This effect has very important technical significance for the immediate detection and judgment of PED and further prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The electron microscopy (TEM) and particle size test results of MNPs and MNPs-N; in the figure: (a) is the TEM image of MNPs; (b) is the TEM image of MNPs-N; (c) is the particle size test results of MNPs and MNPs-N; Figure 2 The relevant test results of the prepared monoclonal antibody McAb; wherein: (a) is the test result of the specific IgG titer of BALB / c mice by indirect ELISA method during the preparation process; (b) is the Western blot test result of the reaction between the prepared McAb and the N protein monoclonal antibody; (c) is the IFA identification result of the prepared McAb; (d) is the subtype identification result of the prepared McAb; (e) is the SDS-Page electrophoresis test result of the purified McAb; Figure 3 The test results of the prepared McAb-HRP probe are shown in Figure 2; (a) is the UV-visible scanning spectrum result; (b) is the ELISA test result; Figure 4 The optimization results of some reaction conditions in the constructed dual-probe competitive ELISA method; wherein: (a) is the optimization result of the dilution of the sample to be tested; (b) is the optimization result of the reaction time; (c) is the optimization result of the color development time; Figure 5 To determine the critical value; Figure 6 Specific test results. DETAILED DESCRIPTION

[0014] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0015] Biomaterials: Positive standard sera for porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and pseudorabies virus (PRV) were purchased from China Veterinary Culture Collection (CVCC, Beijing, China); Porcine reproductive and respiratory syndrome virus (PRRSV) positive serum samples, classical swine fever virus (PEDV) negative serum samples and related clinical serum samples were all collected from relevant sample pigs in the pig farms of the applicant and the inventor's cooperative enterprise; because the applicant and the inventor belong to professional scientific research and teaching institutions, they have relevant sample collection and preservation conditions and facilities; PEDV (HNAY strain) is a common and frequently used strain in current porcine epidemic diarrhea (PED) research and can be obtained through public channels. The applicant and inventor belong to a professional scientific research and teaching institution and have preserved the strain for a long time.

[0016] The expression and purification of the N protein of PEDV is as follows (the specific operations are commonly used in existing molecular biology research and will not be repeated here): RNA of PEDV (HNAY strain) was extracted and reverse transcribed to prepare cDNA, and primers were designed and PCR amplified using the cDNA as a template to obtain the N gene of PEDV; Subsequently, pET28a was used as a vector to recombinantly construct an N-pET28a recombinant plasmid containing the N gene cloned above, and further bacterial liquid PCR and sequencing were performed to ensure that the recombinant plasmid was constructed correctly; Finally, the correctly identified recombinant plasmid N-pET28a was inoculated into LB medium for culture. When OD600 was about 1.0, IPTG was added to a final concentration of 1 mmol, and the expression was induced at 37°C and 220r for 5 h. The supernatant was collected and further purified using a nickel column to obtain purified N protein.

[0017] Based on the N protein of PEDV, the inventors further prepared monoclonal antibodies (McAb). The specific preparation and screening process of McAb against N protein is as follows: After 30 μg (concentration 1 μg / μl) of purified N protein and immune adjuvant were mixed evenly at a volume ratio of 1:1, three BALB / c female mice (6 weeks old) were immunized intramuscularly; After the injection, the antibody production in mice was detected by indirect enzyme-linked immunosorbent assay, and mice with higher antibody levels were euthanized, and their spleens were removed, ground, and fused with SP2 / 0 cells; The hybridoma cells were tested by indirect ELISA, and the positive cells were subcloned three times by limiting dilution. The monoclonal cells that stably secreted N protein-specific antibodies were amplified and cultured, and ascites were prepared. Finally, the ascites was purified using a protein G affinity chromatography column to obtain purified McAb.

[0018] Experimental reagents and products: 1640 culture medium and DMEM culture medium were purchased from Shanghai Yuanpei; Immune adjuvant (QuickAntibody-Mouse5W), purchased from Biodragon; Morpholineethanesulfonic acid (MES), a product of Merck Millipor (Billerica, Massachusetts, USA); N-hydroxythiosuccinimide sodium salt (NHC) and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); horseradish peroxidase (HRP) was a product of Sigma-Aldrich (St. Louis, MO, USA); 96-well magnetic labeling plate was purchased from Nanjing Dongna Biotechnology Co., Ltd. (Nanjing, China); Magnetic nanoparticles (MNPs) (the magnetic nanoparticles in the product are uniformly dispersed in the protective solution with a concentration of 10 mg / ml) were purchased from Nanjing Dongna Biotechnology Co., Ltd. (Nanjing, China).

[0019] Example 1 The main technical idea of ​​the present application is to establish a competitive enzyme-linked immunosorbent (ELISA) method based on dual probes. Therefore, the construction of relevant probes is the basis of the technical solution of the present application. This embodiment briefly introduces the preparation of relevant probes as follows.

[0020] (I) Preparation of magnetic nanoparticle probes MNPs-N The specific preparation process of magnetic nanoparticle probe MNPs-N is as follows: Take 0.2 ml of MNPs (10 mg / ml) solution, obtain MNPs nanoparticle material after magnetic separation, wash twice with MES (0.015 M, pH = 5.5) solution, and resuspend with 0.015 ml MES; After resuspension, add 0.2 mg of EDC and 0.2 mg of NHC (EDC and NHC are both 10 mg / ml solutions, take the corresponding volume), and shake the reaction at room temperature (about 25°C) for 30 minutes; After the reaction, the mixture was separated by magnetic separation, the supernatant was discarded, the MNPs were washed once with MES solution, and resuspended with 0.015 ml MES. After resuspension, 40 μg of purified N protein of PEDV was added, and incubated at room temperature (about 15-25°C) with shaking for 4 h; After the incubation, the MNPs were separated by magnetic force and washed with PBST buffer (pH = 7.4) (the coupling efficiency of MNPs and N protein was detected at the same time); Then, 1% BSA (solvent is PBST) was added and incubated at room temperature with shaking for about 1 h to block the unbound sites on the MNPs; After blocking the unbound sites on the MNPs, the coupled MNPs-N were washed with PBST buffer and resuspended with 0.2 mL of PBST buffer, sampled for detection and stored at 4°C for future use.

[0021] When testing the samples, transmission electron microscopy and laser particle size analyzer were used to characterize the MNPs-N, and direct enzyme-linked immunosorbent assay was used to test the stability of the prepared MNPs-N probe.

[0022] The main idea of ​​preparing the MNPs-N probe is: carboxyl-modified MNPs first react with EDC and NHC solutions to form an activated ester solution, which then reacts with the amino groups on the N protein to form an MNPs-N complex as a probe. The prepared MNPs-N was examined by TEM. The results are shown in Figure 1 As shown. The analysis shows that: The average particle size of the original MNPs is about 500 nm, which is similar to that of MNPs ( Figure 1 (a)) compared with MNPs-N( Figure 1 (b)) There are tiny protrusions on the surface, and the average particle size is about 800nm, which indicates that the N protein has successfully attached to the MNPs. Further particle size test results ( Figure 1 (c)) shows that the particle size of MNPs-N is significantly larger than that of MNPs, which further indicates that MNPs formed a stable complex with N protein and the MNPs-N probe was successfully prepared.

[0023] (II) Preparation of McAb-HRP probe Before preparing the probe, the prepared McAb was tested for its reactivity to the N protein by Western blotting (WB) and indirect immunofluorescence assay (IFA), and the subtype of the monoclonal antibody was determined by a mouse monoclonal antibody subtype kit.

[0024] The specific McAb-HRP probe preparation process is as follows.

[0025] Take 5 mg of HRP and dissolve it in 1 mL of deionized water, add 200 μL of 0.1 M NaIO4 solution, stir at room temperature in the dark for about 20 min, put it into a dialysis bag (dialysis molecular weight is 8000-14000D), and dialyze it in 1 mM sodium acetate buffer (pH=4.4) at 4°C for 12 h; After dialysis, add 200 μL of 0.2 M (pH = 9.6) CB solution to the dialysate, then quickly add 1 M NaOH (to enolize HRP), adjust the pH to about 9.5, and then quickly add 10 mg of purified antibody McAb (concentration of 4 mg / ml, PBS as solvent), and treat at 4°C with gentle stirring for about 12 hours; After the stirring treatment was completed, 235 μL of freshly prepared 4 mg / mL NaBH4 solution was added to the above mixture (47 μL of NaBH4 was added for every 1 mg of HRP), mixed well, and allowed to stand at 4°C for 2 h; After standing, add an equal volume of saturated ammonium sulfate solution, stand at 4°C for 30 min, centrifuge at 4°C and 12000 r / min for 30 min, discard the supernatant, resuspend the precipitate with PBS, and dialyze in PBS at 4°C for about 12 h; After the dialysis, the dialysate was centrifuged at 12000 r / min for 10 min, the supernatant was sampled (the supernatant was the McAb-HRP probe) for detection, and the rest was stored at -80°C for future use.

[0026] For the prepared samples, the absorption peak of McAb-HRP probe was detected by UV spectrophotometer, and the McAb-HRP probe was characterized by direct ELISA method.

[0027] Before preparing the McAb-HRP probe, the results of the McAb test showed that the serum potency of the immunized mice could reach a dilution factor of 1:2048000 ( Figure 2 (a)), this result shows that the prepared McAb has a good antibody titer for N protein. Further Western blot ( Figure 2 (b)) and IFA( Figure 2 (c)) also verified that the prepared McAb had good reactivity with N protein and could be used for subsequent probe preparation and detection applications. Further subtype detection results of the prepared McAb showed (using a triple mouse monoclonal antibody subtype identification kit) that the subtype of the prepared McAb was IgG1 with Kappa chain ( Figure 3 (d)). The results of purification test of the prepared McAb are as follows Figure 3 As shown in (e), it can be seen that the prepared McAb has a high purity and can better meet the needs of subsequent probe preparation.

[0028] The prepared McAb-HRP probe was detected by UV spectrophotometer and direct enzyme-linked immunosorbent assay. Figure 3 As shown. The analysis shows that: The absorption peak curve of McAb-HRP combines the absorption peaks of McAb and HRP together, and this result indicates that McAb and HRP form a stable conjugate; The ELISA test results showed that the monoclonal antibody in the synthetic probe maintained a high affinity for the N protein, indicating that the synthesis method did not affect the affinity of the monoclonal antibody for the N protein, nor did it affect the catalytic ability of HRP.

[0029] Example 2 Based on the probe prepared in Example 1, the inventors constructed a further competitive ELISA detection method and further optimized and explored the relevant reaction conditions and reaction parameters. The specific process is briefly described as follows.

[0030] (I) Prepare the plate. Use the ELISA plate as the test container, add 200 μL of 5% BSA solution to each well, and incubate at 37°C for 1 hour. After incubation, pat dry and proceed directly to subsequent tests, or store at 4°C for later use. (II) Sampling and Testing Add 50 μL of MNPs-N probe, 50 μL of McAb-HRP probe, and 50 μL of PEDV-positive serum (or sample to be tested) to each well; PBS was used as blank control; After adding the sample, shake well at room temperature (about 15-28°C) and let it stand for 30 minutes; (III) Color development and determination After the reaction, the ELISA plate was magnetically separated for 10 seconds, the supernatant was discarded, and the precipitate was washed 4 times with PBST buffer; after washing, 100 μL of TMB colorimetric solution was added, and the colorimetric reaction was carried out at room temperature in the dark. After the reaction was completed, 50 μL of reaction stop solution was added (to stop the color development); Finally, the OD value was measured at 450 nm with PBS buffer as the control, and the inhibition percentage (PI) was calculated. PI = [(1 - OD450 nm positive value (P)) / negative value (N)] x 100%.

[0031] Based on the above reaction operation process, reaction parameters and reaction conditions optimization process, the specific optimization design parameters and results are briefly introduced as follows.

[0032] (1) Optimization of probe concentration Based on the principle of the “chessboard method”, the MNPs-N probe was diluted with PBS solution to five concentration gradients of 0.2 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL; The McAb-HRP probe prepared above was diluted with PBS solution at a ratio of 1:500, 1:1000, 1:2000, 1:4000, 1:6000, and 1:8000 respectively; Referring to the above operation, PEDV positive serum was used as the test sample (PEDV positive serum was undiluted, reacted for 30 minutes, and developed for 20 minutes), and PI was detected and calculated after the reaction was completed.

[0033] The PI results under different combinations are shown in Table 1.

[0034] Table 1. PI results of different MNPs-N probes and different McAb-HRP probe dilution combinations From the results in Table 1 above, it can be seen that when the concentration of MNPs-N probe is 1 μg / mL and the dilution ratio of McAb-HRP probe is 1:2000, the PI value is the highest. In other words, this concentration ratio combination is the best probe concentration ratio combination for detecting PEDV.

[0035] (2) Optimization of serum dilution to be tested Referring to the above “(1) Probe concentration optimization” process, the MNPs-N probe concentration was 1 μg / mL and the McAb-HRP probe dilution ratio was 1:2000 for detection. At the same time, the dilution ratio of PEDV-positive serum was adjusted (the dilution ratios with PBS were: 1:1, 1:2, 1:5, 1:10, 1:20, with undiluted serum as the control), and the PI values ​​of PEDV-positive serum at different dilution ratios were determined.

[0036] The results are as follows Figure 4 ( Figure 4 (a)). It can be seen that the PI of undiluted serum is the highest. That is, in actual detection applications, the sample to be tested can be directly tested without dilution.

[0037] (3) Optimization of response time Referring to the above process of "(2) Optimization of serum dilution to be tested", the MNPs-N probe concentration was 1 μg / mL and the McAb-HRP probe dilution ratio was 1:2000 for detection. PEDV-positive serum (undiluted) was used as the test sample, and the reaction time (incubation time) was adjusted to 5 min, 10 min, 15 min, 20 min, 25 min, 30 min and 35 min, respectively.

[0038] The results are as follows Figure 4 ( Figure 4 (b)). It can be seen that after the reaction time exceeds 25 minutes, the PI value is basically stable, but from the perspective of operational convenience and uniformity, 30 minutes can be the optimal reaction time.

[0039] (4) Optimization of color development time Referring to the above “(3) Reaction time optimization” process, the detection was performed with a MNPs-N probe concentration of 1 μg / mL and a McAb-HRP probe dilution ratio of 1:2000. PEDV-positive serum (undiluted) was used as the test sample. The reaction time (incubation time) was 30 min, and the color development time was adjusted to 5 min, 10 min, 15 min, 20 min, and 25 min, respectively.

[0040] The PI calculation results are as follows: Figure 4 ( Figure 4 (c)). It can be seen that after the color development time exceeds 15 minutes, the PI value is basically stable, but from the perspective of operational convenience and uniformity, 20 minutes can be the optimal color development time.

[0041] (5) Detection critical value Based on the above results of "(4) Optimization of color development time", the MNPs-N probe concentration was 1 μg / mL and the McAb-HRP probe dilution ratio was 1:2000 for detection, with a reaction time (incubation time) of 30 min and a color development time of 20 min as reaction parameters, and 30 PEDV-negative sera stored in the laboratory as test samples. The dual-probe competitive ELISA method constructed in this application was used for detection, and the PI value was calculated, and the average inhibition rate (X) and standard deviation (SD) of the samples were calculated. It is set that when the sample inhibition rate PI≤X+2SD, it is judged as negative, when PI≥X+3SD, it is judged as positive, and when the result is between the two, it is suspected.

[0042] The results are as follows Figure 5 The results show that the average (X) sample inhibition rate is 21.83% and the standard deviation (SD) is 7.34%. Therefore, results less than X+2SD=36.51% are considered negative, results greater than X+3SD=43.85% are considered positive, and results between 36.51% and 43.85% are considered suspicious.

[0043] (6) Repeatability and specificity evaluation Referring to the optimized reaction conditions in "(5) Detection critical value", for repeatability evaluation, 7 randomly selected laboratory-stored PEDV-positive sera were used as test samples for detection, and the inter-batch and intra-batch coefficients of variation (coefficient of variation = standard deviation SD / mean value Mean × 100%) were calculated to evaluate the reproducibility of the competitive ELISA constructed in this application.

[0044] With reference to the optimized reaction conditions in “(5) Detection critical value”, PCV-positive serum, PRRSV-positive serum, PRV-positive serum and ASFV-positive serum were used as test samples, and PEDV-positive serum was used as control to test the specificity of the method.

[0045] The results of the repeatability test are shown in Table 2. Figure 6 shown.

[0046] Table 2. Summary of results of repeatability test From Table 2 above and Figure 6 The results show that the ELISA detection method constructed in this application has good specificity for the detection of PEDV and can ensure the accuracy of the detection results. At the same time, the intra-batch and inter-batch coefficients of variation are both less than 10%, which indicates that the results of the detection method have good repeatability and high stability.

[0047] (7) Comparison of test results with commercial test products in the prior art A commercial kit for PEDV detection in the prior art (a porcine epidemic diarrhea virus ELISA (IgG) antibody detection kit produced by a biological company in Wuhan, which uses an indirect ELISA method to detect S protein antibodies of porcine epidemic diarrhea) was used as a control, and 101 randomly collected serum samples stored in the inventor's laboratory were used as test samples. With reference to the aforementioned optimal reaction conditions, the commercial kit and the ELISA detection method of the present application were used for detection. The specific test results are summarized in Table 3 below.

[0048] Table 3. Summary of clinical sample test results From the results in Table 3 above, it can be seen that the competitive ELISA constructed in the present application detected 79 positive sera and 22 negative sera, while the commercial kit detected 86 positive sera and 15 negative sera. The coincidence rate between the two is 93.07%. That is, the ELISA method constructed in the present application has a good accuracy rate and commercial prospects.

Claims

1. A porcine epidemic diarrhea virus detection method based on magnetic nanoparticles, characterized in that: This method belongs to a dual-probe competitive ELISA detection method, which specifically includes the following steps: (I) Spare board Use the ELISA plate as the detection container, add BSA solution to seal the plate for later use; (II) Sampling and Testing In the ELISA plate of step (i), each well is added with MNPs-N probe and McAb-HRP probe, and then with the sample to be tested; PBS sample solution was used as blank control; After adding the sample, shake well and let it stand; (III) Color development and determination After the reaction is finished, the ELISA plate is magnetically separated, the supernatant is discarded, and the precipitate is washed; Add TMB colorimetric solution, protect from light and develop color. After the reaction is completed, add reaction stop solution to stop the color development; Finally, PBS buffer was used as a control, and the OD value was measured at 450 nm to calculate the inhibition percentage PI. PI = [(1-OD450 nm positive value (P)) / negative value (N)] × 100%; The MNPs-N probe is prepared as follows: Take 2 mg of MNPs, wash them with MES solution, and then resuspend them in 0.015 ml of MES; After resuspension, add 0.2 mg of EDC and 0.2 mg of NHC and shake the reaction; After the reaction, the mixture was separated by magnetic separation, the supernatant was discarded, washed with MES solution and resuspended with 0.015 ml MES, and then the purified N protein of PEDV was added and incubated with shaking; After the incubation, the MNPs were separated magnetically and washed; Then, 1% BSA was added and incubated with shaking to block the unbound sites on MNPs. After the incubation, the coupled MNPs-N was washed and resuspended with PBST buffer to obtain a solution containing MNPs-N probes. The McAb-HRP probe is prepared as follows: First, a purified monoclonal antibody McAb against the N protein of PEDV was prepared; Subsequently, the McAb-HRP probe was prepared using the sodium periodate method.

2. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: In step (i), 200 μL of 5% BSA solution was added to each well of the ELISA plate, and the plate was incubated at 37° C. for 1 h. After incubation, the plate was patted dry to seal the plate.

3. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: In step (ii), 50 μL of MNPs-N probe and 50 μL of McAb-HRP probe were added to each well, and the amount of sample to be tested was 50 μL.

4. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: The preparation operation of the MNPs-N probe is as follows: 2 mg of MNPs were washed twice with MES solution and then resuspended with 0.015 ml of MES; the concentration of MES was 0.015 M and the pH was 5.5; After resuspension, add 0.2 mg of EDC and 0.2 mg of NHC, and shake at room temperature for 30 min; EDC and NHC are both 10 mg / ml solutions, and take the corresponding volume; After the reaction, the mixture was separated by magnetic separation, the supernatant was discarded, the MNPs were washed once with MES solution, and resuspended with 0.015 ml MES. After resuspension, 40 μg of purified N protein of PEDV was added, and incubated at room temperature with shaking for 4 h; After the incubation, the MNPs were separated by magnetic separation and washed with PBST buffer (pH = 7.4); Then, 1% BSA was added and incubated at room temperature with shaking for 1 h to block the unbound sites on MNPs; After blocking the unbound sites on the MNPs, the coupled MNPs-N were washed with PBST buffer and resuspended with 0.2 mL of PBST buffer to obtain a solution containing the MNPs-N probe.

5. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: The specific operation of the McAb-HRP probe is as follows: (I) Preparation of monoclonal antibodies against PEDV N protein The preparation method of the monoclonal antibody McAb against the N protein of PEDV is as follows: 30 μl of purified N protein with a concentration of 1 μg / μl was mixed evenly with the immune adjuvant at a volume ratio of 1:1, and then BALB / c female mice were immunized intramuscularly; After the injection, the antibody production in mice was detected by indirect enzyme-linked immunosorbent assay, and mice with higher antibody levels were euthanized, and their spleens were removed, ground, and fused with SP2 / 0 cells; The hybridoma cells are screened, the monoclonal cells that stably secrete N protein-specific antibodies are amplified and cultured, and ascites is prepared; finally, the ascites is purified using a protein G affinity chromatography column to obtain purified McAb; (II) Preparation of McAb-HRP probe Take 5 mg of HRP and dissolve it in 1 mL of deionized water. Add 200 μL of 0.1 M NaIO4 solution, stir at room temperature in the dark for 20 min, put it into a dialysis bag, and dialyze it in 1 mM sodium acetate buffer at 4 °C for 12 h. After dialysis, 200 μL of 0.2 M CB solution was added to the dialysate, and then 1 M NaOH was quickly added to adjust the pH to 9.5, followed by the rapid addition of 10 mg of purified antibody McAb, and the mixture was stirred at 4°C for 12 hours. After the stirring treatment was completed, 235 μL of freshly prepared 4 mg / mL NaBH4 solution was added to the above mixture, mixed evenly, and allowed to stand at 4°C for 2 h; After standing, add an equal volume of saturated ammonium sulfate solution, stand at 4°C for 30 min, centrifuge at 4°C and 12000 r / min for 30 min, discard the supernatant, resuspend the precipitate with PBS, and dialyze in PBS at 4°C for 12 h; After the dialysis, the dialysate was centrifuged at 12000 r / min for 10 min, and the supernatant was taken, which was the McAb-HRP probe solution.

6. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: In step (ii), when the concentration of MNPs-N probe is 1 μg / mL, the dilution ratio of McAb-HRP probe is 1:2000.

7. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: In step (ii), after adding the sample, shake well at room temperature and let it react for 30 minutes.

8. The porcine epidemic diarrhea virus detection method based on magnetic nanoparticles according to claim 1, characterized in that: In step (iii), the color development reaction is carried out for 20 minutes.

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