Method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescence bicolor ELISA

Abstract

The invention relates to the technical field of medical biology, in particular to a method for identifying and diagnosing PEDV (Porcine Epidemic Diarrhea Virus) and PoRV-A. Colloidal gold is used as a carrier, and a monoclonal antibody (mAb) for resisting PEDV and PoRV-A and an initiator are respectively modified on colloidal gold (Au) to prepare a probe (Au-I-mAb). An ELISA hole is coated with a monoclonal antibody (mAb), when a target virus exists, a probe is added to form a double-antibody sandwich compound (mAb-antibody-mAb-Au-I), a hairpin is added, and an initiation chain on the compound recognizes the matched hairpin and induces the hairpin to cross to generate HCR. Due to the fact that the hairpins are marked by different fluorophores, when H1 is in a closed hairpin structure, fluorescence on H1 is quenched by a quenching group, after H1 is subjected to ring opening through HCR, the fluorophores are away from the quenching group, and fluorescence is recovered. Fluorescence excitation is carried out by using corresponding emission wavelengths, and two target pathogens are finally detected at the same time by detecting output signals of different fluorophores.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical biotechnology, and in particular to a method for differentially diagnosing PEDV and PoRV-A based on HCR fluorescent dual-color ELISA. Background Art

[0002] Virus detection refers to the process of identifying and confirming the presence and type of a virus through specific methods and technical means. Porcine epidemic diarrhea is a highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV); porcine group A rotavirus (PoRV-A) is the main serotype of PoRV that causes severe gastroenteritis in piglets. The clinical manifestations of these two viral infections are very similar, and these two diseases often cause mixed infections, making it difficult to make differential diagnosis based on clinical symptoms. At present, the common detection methods for diagnosing these two viruses, PEDV and PoRV-A, are immunological detection technology and molecular biological detection technology. Immunological detection technology is not sensitive enough and has certain difficulties in achieving multiple detection. Molecular biological detection technology is cumbersome to operate and requires extraction of viral nucleic acid. At present, multiplex PCR or multiplex real-time fluorescence quantitative PCR can be used to simultaneously differentiate and diagnose these two pathogens (PEDV and PoRV-A), but the detection process requires enzyme participation, has high requirements for reaction conditions, and is often accompanied by essential steps such as pre-enrichment and nucleic acid extraction. The operation is cumbersome and requires professional operating technicians and well-equipped laboratory conditions. Summary of the invention

[0003] In view of the above-mentioned defects and problems, the present invention provides a method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA. By combining the advantages of immunological detection technology and molecular biological detection technology, a new detection method with simple operation, strong specificity and the ability to simultaneously detect PEDV and PoRV-A is constructed.

[0004] The solution adopted by the present invention to solve the technical problem is: a method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA, comprising the following steps: S1. Using freeze-thaw method, thiol-modified oligonucleotide DNA AI and BI are fixed to the surface of gold particles to prepare colloidal gold-nucleic acid complexes Au-AI and Au-BI for use; S2, colloidal gold-nucleic acid complexes Au-AI and Au-BI prepared by freeze-thaw method, and their preparation effects were tested by HCR experiment; S3, preparing colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method for use; S4. Prepare colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method, and test the preparation effect by performing HCR experiment; S5. Construction of HCR fluorescent dual-color ELISA differential diagnosis method for PEDV and PoRV-A; S6. Conduct specific analysis and clinical analysis; S7. Process and verify the experimental data.

[0005] Furthermore, the specific steps of preparing the colloidal gold-nucleic acid complex in S1 are as follows: S11, take 1mL of 20nm colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2mol / L K2CO3; S12, activate 8 μL AI and BI with 4 μL 2 mol / L TCEP, mix well and react at room temperature for 30 min; S13, adding the activated AI and BI dropwise into 200 μL of colloidal gold solution; S14, placing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, taking it out to melt, and centrifuging it in a centrifuge at 13,000 r / min for 5 minutes; S15, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S16. After discarding the supernatant, dissolve it with 400 μL 0.01 mol / L PBS and prepare it as colloidal gold-nucleic acid complexes Au-AI and Au-BI.

[0006] Furthermore, in S2, the detection steps are as follows: S21, take a black ELISA plate, add 50 μL Au-AI, 1 μL each of A-H1 and A-H2 to the first well, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S22, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the second well, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S23, add 1 μL of A-H1 and A-H2 to well 3, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S24, react in a 37°C constant temperature incubator for 1 hour. After the reaction is completed, the results are detected using a multifunctional microplate reader and the result data are collected.

[0007] Furthermore, the specific operation steps for preparing the colloidal gold-nucleic acid-antibody complex in S3 are as follows: S31, take 1mL of 20nm colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2mol / L K2CO3; S32, activate 8 μL AI and BI with 4 μL 2mmol / L TCEP, mix well and react at room temperature for 30 min; S33, adding the activated AI and BI dropwise to 200 μL of colloidal gold solution, freezing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, and then taking it out to melt; S34, add 4 μL of anti-PEDV S mAb to the melted colloidal gold-nucleic acid mixture Au-AI, add 4 μL of anti-PoRV-A VP6 mAb to the melted colloidal gold-nucleic acid mixture Au-BI, and incubate at room temperature with shaking for 4 h; S35. After the incubation, 22uL of 10% BSA solution was fully mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30min to block the excess sites; S36. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS. The product is then centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS for use as colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2.

[0008] Furthermore, in S4, the detection steps are as follows: S41, take a black ELISA plate, add 50 μL Au-AI-mAb1, 1 μL A-H1 and A-H2 to the first well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S42, add 50 μL Au-AI-mAb1, 50 μL Au-BI-mAb2, 1 μL each of A-H1 and A-H2, B-H1 and B-H2 to the second well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S43, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the third well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S44, add 1 μL of A-H1 and A-H2 to well 4, and use 5 nM Mg 2+ The volume was filled to 150 μL with PBS buffer and B-H1 and B-H2 were treated in the same manner; S45, react in a 37°C constant temperature incubator for 1 hour. After the reaction is completed, the results are detected using a multifunctional microplate reader.

[0009] Furthermore, in S5, the construction method is as follows: S51, anti-PEDV S monoclonal antibody (mAb1) and anti-PoRV-A VP6 monoclonal antibody (mAb2) were mixed and coated in a black ELISA plate, incubated at 4°C overnight, washed three times with PBST and patted dry; S52, incubate with rapid blocking solution I at room temperature for 30 min, wash the plate three times with PBST and pat dry; S53, add 50uL of PBS, PEDV, PoRV-A virus solution or a mixture of PEDV and PoRV-A virus to the wells respectively, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S54, add 50uL Au-AI-mAb1 and 50uL Au-BI-mAb2 to the wells, mix, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S55, add 1 μL each of A-H1, A-H2, B-H1, B-H2, and 5 nM Mg 2+ The volume was filled to 100 μL with PBS buffer and reacted in a 37°C constant temperature incubator for 1 h. The results were detected using a multifunctional microplate reader and the result data were collected.

[0010] Furthermore, in S6, the method of specific analysis is: using two target viruses PEDV and PoRV-A and five non-target viruses TGEV, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine parvovirus, and classical swine fever virus as the objects to be tested, using 0.01 mol / L PBS as the negative control, and conducting the experiment according to the method.

[0011] Furthermore, in said S6, the method of clinical analysis is: taking 3 samples of clinical fluid that is positive for PEDV and PoRV-A by PCR, centrifuging and taking the supernatant for actual sample testing; using 0.01 mol / L PBS as a negative control, conducting experiments according to the method, and evaluating the applicability of the method in actual clinical practice.

[0012] Furthermore, in S24, S45, and S55, the fluorescence parameters are set as follows: A: the excitation wavelength is 494 nm, and the emission wavelength is 522 nm; B: the excitation wavelength is 540 nm, and the emission wavelength is 570 nm.

[0013] Beneficial effects of the invention: The invention combines the advantages of immunological detection technology and molecular biological detection technology to establish a fluorescent two-color ELISA method with simple operation, strong specificity, and rapid identification and detection of PEDV and PoRV-A at the same time. It can not only detect the two viruses at the same time, but also does not require a nucleic acid extraction step, and has the advantages of constant temperature, enzyme-free, strong specificity, and good tolerance to sample matrices and enzyme inhibitors. Compared with immunological detection technology and molecular biological detection technology, it has the advantages of not requiring nucleic acid extraction, simple operation, and being able to detect multiple viruses at one time. This method is of great significance for the rapid diagnosis of porcine viral diarrhea. The establishment of this method can also provide new ideas for the simultaneous detection of other viral diseases. With the development of science and technology and the reduction of material and instrument costs, this method will become a way to quickly detect multiple pathogens in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a preparation effect diagram of colloidal gold-nucleic acid complex, where A is the preparation effect of Au-AI, with an excitation wavelength of 494 nm and an emission wavelength of 522 nm; B is the preparation effect of Au-BI, with an excitation wavelength of 540 nm and an emission wavelength of 570 nm; Figure 2 The figure is a preparation effect diagram of colloidal gold-nucleic acid-antibody complex, wherein A is the preparation effect of Au-AI-mAb1, with an excitation wavelength of 494 nm and an emission wavelength of 522 nm; B is the preparation effect of Au-BI-mAb2, with an excitation wavelength of 540 nm and an emission wavelength of 570 nm; Figure 3 This is a graph showing the results of detecting PEDV and PoRV-A based on HCR fluorescent dual-color ELISA; Figure 4 is the specificity evaluation diagram; Figure 5 These are clinical evaluation diagrams, where A is the PEDV clinical detection and analysis, with an excitation wavelength of 494 nm and an emission wavelength of 522 nm; B is the preparation effect of PoRV-A clinical detection and analysis, with an excitation wavelength of 540 nm and an emission wavelength of 570 nm. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0016] See also Figure 1-5The present invention provides a technical solution for a method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA. Colloidal gold is used as a carrier, and monoclonal antibodies (mAb) and initiators against PEDV and PoRV-A are modified on colloidal gold (Au) to prepare probes (Au-I-mAb). The monoclonal antibody (mAb) is coated on the ELISA well. When the target virus is present, a double antibody sandwich complex (mAb-pathogen-mAb-Au-I) is formed after the probe is added. When a hairpin is added, the initiator chain on the complex will recognize the matching hairpin and induce the hairpin hybridization to occur HCR. Since each hairpin is labeled with a different fluorescent group, when H1 is in a closed hairpin structure, the fluorescence on it is quenched by the quenching group. After HCR occurs to open the ring of H1, the fluorescent group and the quenching group are separated and the fluorescence is restored. Fluorescence excitation is performed using the corresponding emission wavelength, and the output signals of different fluorescent groups are detected to ultimately achieve simultaneous detection of the two target pathogens.

[0017] Example 1: This example provides a method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA The main reagents and materials for the experiment include: Gold nanoparticles (20 nm) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; anti-PEDV S monoclonal antibody and anti-PoRV-A VP6 monoclonal antibody were purchased from Tracing Source and Biological Company; A set of oligonucleotide hybridization sequences (including AI, A-H1, A-H2) and B set of oligonucleotide hybridization sequences (including BI, B-H1, B-H2) were prepared by Shanghai Sangon Biotechnology Co., Ltd., China, and the purification method was HPLC. The sequences are shown in Table 2; Rapid blocking solution I, 20×ELISA coating buffer, 10×PBST WB rinse solution, and black ELISA plate were purchased from Shanghai Sangon Biotechnology Co., Ltd., China; PEDV, PoRV-A virus solution and sample solution were stored in this experiment. All reagents and solvents were of analytical grade, and all solutions and reagents used in the experiment were prepared with ultrapure water.

[0018]

[0019] The main instruments used in the experiment are shown in Table 2:

[0020] The detection method and steps are as follows: Step 1: Prepare colloidal gold-nucleic acid complexes: Use freeze-thaw method to fix thiol-modified oligonucleotides AI and BI (DNA) to the surface of gold particles to prepare colloidal gold-nucleic acid complexes Au-AI and Au-BI for use. The specific steps are as follows: S11, take 1mL of colloidal gold (20nm) into a glass bottle and adjust its pH to 7.5 using 0.2mol / L K2CO3; S12, activate 8 μL AI and BI with 4 μL 2 mol / L TCEP, mix well and react at room temperature for 30 min; S13, add the activated AI and BI dropwise into 200 μL colloidal gold solution (pH=7.5); S14, placing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, taking it out to melt, and centrifuging it in a centrifuge at 13,000 r / min for 5 minutes; S15, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S16. After discarding the supernatant, dissolve it with 400 μL 0.01 mol / L PBS and prepare it as colloidal gold-nucleic acid complexes Au-AI and Au-BI.

[0021] Step 2: Detect the preparation effect of colloidal gold-nucleic acid complex: The colloidal gold-nucleic acid complexes Au-AI and Au-BI prepared by freeze-thaw method are tested by HCR experiment. The specific operation steps are as follows: S21. Take a black ELISA plate, add 50 μL Au-AI, 1 μL each of A-H1 and A-H2 to the first well, and wash with PBS buffer (5 nM Mg 2+ ) were filled to 100 μL and group B was treated in the same way; S22, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the second well, and wash with PBS buffer (5 nM Mg 2+ ) were filled to 100 μL and group B was treated in the same way; S23, add 1 μL of A-H1 and A-H2 to well 3 and wash with PBS buffer (5 nM Mg 2+ ) were filled to 100 μL and group B was treated in the same way; The reaction was carried out in a constant temperature incubator at S24 and 37°C for 1 hour. After the reaction was completed, the results were detected using a multifunctional microplate reader. The fluorescence parameters were set as follows: A: excitation wavelength was 494 nm, emission wavelength was 522 nm; B: excitation wavelength was 540 nm, emission wavelength was 570 nm.

[0022] Step 3, preparation of colloidal gold-nucleic acid-antibody complex: prepare colloidal gold-nucleic acid-antibody complex Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method for standby use. The specific operation steps are as follows: S31, take 1mL (20nm) colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2mol / L K2CO3; S32, activate 8 μL AI and BI with 4 μL 2mmol / L TCEP, mix well and react at room temperature for 30 min; S33, add the activated AI and BI dropwise into 200 μL colloidal gold solution (pH = 7.5), freeze the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, then take it out and melt it; S34, add 4 μL of anti-PEDV S mAb to the melted colloidal gold-nucleic acid mixture Au-AI, add 4 μL of anti-PoRV-A VP6 mAb to the melted colloidal gold-nucleic acid mixture Au-BI, and incubate at room temperature with shaking for 4 h; S35. After the incubation, 22uL of 10% BSA solution was fully mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30min to block the excess sites; S36. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS. The product is centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS to prepare the colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2. Au-AI-mAb1 is colloidal gold particles connecting AI and anti-PEDV S mAb, and Au-BI-mAb2 is colloidal gold particles connecting BI and anti-PoRV-A VP6 mAb.

[0023] Step 4: Detect the preparation effect of colloidal gold-nucleic acid-antibody complex: prepare colloidal gold-nucleic acid-antibody complex Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method, and detect the preparation effect by performing HCR experiment. The specific operation steps are as follows: S41. Take a black ELISA plate, add 50 μL Au-AI-mAb1, 1 μL A-H1 and 1 μL A-H2 to the first well, and wash with PBS buffer (containing 5 nM Mg 2+ ) were filled to 150 μL and treated in the same way as group B; S42, add 50 μL Au-AI-mAb1, 50 μL Au-BI-mAb2, 1 μL each of A-H1 and A-H2, B-H1 and B-H2 to the second well, and wash with PBS buffer (containing 5 nM Mg 2+) were filled to 150 μL and treated in the same way as group B; S43, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the third well, and wash with PBS buffer (containing 5 nM Mg 2+ ) were filled to 150 μL and treated in the same way as group B; S44, add 1 μL of A-H1 and A-H2 to well 4 and wash with PBS buffer (containing 5 nM Mg 2+ ) Fill to 150 μL and treat B-H1 and B-H2 in the same way; S45, react in a 37℃ constant temperature incubator for 1h. After the reaction is completed, the results are detected using a multifunctional microplate reader. The fluorescence parameters are set as follows: A: excitation wavelength is 494nm, emission wavelength is 522nm; B: excitation wavelength is 540nm, emission wavelength is 570nm.

[0024] Step 5: Constructing the HCR fluorescent dual-color ELISA differential diagnosis method for PEDV and PoRV-A: S51. Mix anti-PEDV S monoclonal antibody (mAb1) and anti-PoRV-A VP6 monoclonal antibody (mAb2) (30ug / mL, 50 uL each) and coat them in a black ELISA plate. Incubate at 4°C overnight. Wash the plate three times with PBST and pat dry. S52, incubate with fast blocking solution I (200 uL / well) at room temperature for 30 min, wash the plate three times with PBST and pat dry; S53, add 50uL of PBS, PEDV, PoRV-A virus solution or a mixture of PEDV and PoRV-A virus to the wells respectively, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S54, add 50uL Au-AI-mAb1 and 50uL Au-BI-mAb2 to the wells, mix, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S55, add 1 μL of A-H1, A-H2, B-H1, B-H2, and wash with PBS buffer (containing 5 nM Mg 2+ ) was made up to 100 μL, and the reaction was carried out in a 37°C constant temperature incubator for 1 h. The results were detected by a multifunctional microplate reader. The fluorescence parameters were set as follows: A: excitation wavelength was 494 nm, emission wavelength was 522 nm; B: excitation wavelength was 540 nm, emission wavelength was 570 nm, and the result data were collected.

[0025] Step 6. Specificity analysis: In order to evaluate the specificity of this method, two target viruses (PEDV and PoRV-A) and five non-target viruses TGEV, porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), and classical swine fever virus (CSFV) were used as analytes, and 0.01 mol / L PBS was used as a negative control. The experiment was carried out according to the above method.

[0026] Step 7, clinical analysis: Take 3 samples of the clinical fluid that tested positive for PEDV and PoRV-A by PCR, centrifuge and take the supernatant for actual sample testing. Use 0.01 mol / L PBS as a negative control, perform the experiment according to the above method, and evaluate the applicability of this method in actual clinical practice. Result judgment: The average value of negative plus 3 times the standard deviation is positive.

[0027] Step 8. Data processing: The experiment was repeated three times, and the results are the mean ± standard deviation (SD) of the three replicates. SPSS software (version 18.0) was used for one-way analysis of variance and Bonferroni post hoc test. P < 0.05 was considered statistically significant.

[0028] Based on the combination of HCR and immunological reaction, this study constructed a HCR-based fluorescent two-color ELISA method for detecting PEDV and PoRV-A with PEDV and PoRV-A as targets.

[0029] The present invention successfully prepared colloidal gold-nucleic acid-antibody complex (Au-AI-mAb) by freeze-thaw method and electrostatic adsorption method, the antibody can bind to the corresponding antigen, and the DNA oligonucleotide can trigger the hairpin to perform HCR and produce strong fluorescence. The colloidal gold-nucleic acid-antibody complex prepared in this study has good quality and can be applied to ELISA method; The present invention successfully constructs a method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent two-color ELISA. Relying on the different output fluorescence signals, the method achieves simultaneous and specific detection of PEDV and PoRV-A. The method has good applicability in clinical sample detection and can be extended to the clinical detection of other viruses.

[0030] Example 2: This example provides the detection effect of the detection method provided in Example 1 1. Preparation effect of colloidal gold-nucleic acid complex: The colloidal gold-nucleic acid complex Au-AI and Au-BI prepared by freeze-thaw method were tested by HCR experiment. Figure 1As shown, the negative control has very low fluorescence intensity because it has no initiator chain, A hairpin (A-H1 and A-H2), and B hairpin (B-H1 and B-H2); the positive controls AI and BI can trigger HCR of the corresponding hairpins and produce strong fluorescence. The colloidal gold-nucleic acid complexes (Au-AI and Au-BI) in the experimental group can also trigger HCR of the corresponding hairpins and produce strong fluorescence, but their fluorescence values ​​are weaker than those of the positive control. The results show that the colloidal gold-nucleic acid complexes (Au-AI and Au-BI) were successfully prepared by the freeze-thaw method, and the original biological properties were maintained.

[0031] 2. Preparation effect of colloidal gold-nucleic acid-antibody complex: Colloidal gold-nucleic acid-antibody complex Au-AI-mAb1 and Au-BI-mAb2 were prepared by freeze-thaw method and electrostatic adsorption method, and the preparation effect was tested by HCR experiment. Figure 2 As shown in the figure, the negative control has very low fluorescence intensity produced by hairpin A (A-H1 and A-H2) and hairpin B (B-H1 and B-H2) due to the absence of initiator chain; the positive controls AI and BI can trigger HCR of the corresponding hairpins and produce strong fluorescence. The colloidal gold-nucleic acid-antibody complexes in the experimental group (Au-AI-mAb1, Au-BI-mAb2) can also trigger HCR of the corresponding hairpins and produce strong fluorescence, but their fluorescence values ​​are weaker than those of the positive control. When the two colloidal gold-nucleic acid-antibody complexes are mixed, Au-AI-mAb1 and Au-BI-mAb2 can still trigger HCR of the corresponding hairpins and produce strong fluorescence, but their fluorescence values ​​are slightly weakened compared with the single (unmixed) ones. The results show that the colloidal gold-nucleic acid-antibody complexes (Au-AI-mAb1, Au-BI-mAb2) were successfully prepared by freeze-thaw method and electrostatic adsorption method, and the original biological properties were maintained.

[0032] 3. Detection results of PEDV and PoRV-A based on HCR fluorescence dual-color ELISA: In order to explore whether this method can achieve simultaneous detection of PEDV and PoRV-A, PEDV, PoRV-A, and PEDV + PoRV-A were added to the ELISA wells as the target objects to be tested, and the final fluorescence detection was performed with an excitation wavelength of 494 nm, an emission wavelength of 522 nm and an excitation wavelength of 540 nm, and an emission wavelength of 570 nm. Figure 3As shown, when the target to be tested contains only PEDV, the fluorescence signal can be detected only at the emission wavelength of 494nm; when the target to be tested contains only PoRV-A, the fluorescence signal can be detected only at the emission wavelength of 540nm, indicating that the detection method is specific; when the target to be tested contains PEDV and PoRV-A, the fluorescence signal can be detected at the emission wavelengths of 494nm and 540nm, and it is different from the negative control. The results show that the detection method can achieve the simultaneous detection of PEDV and PoRV-A by specifically outputting the corresponding fluorescence signals.

[0033] 4. Specificity analysis: PEDV and PoRV-A were used as target viruses and five other non-target viruses for specificity analysis. Figure 4 As shown, when only two target viruses are present, obvious fluorescent signals are generated, while other non-target viruses have no obvious signals and cannot be distinguished from the negative control sample, indicating good specificity.

[0034] 5. Clinical analysis: Take 3 samples of the clinical fluid that is positive for PEDV and PoRV-A by PCR, centrifuge and take the supernatant for actual sample testing. Figure 5 As shown in the figure, compared with the results of PCR nucleic acid detection, the accuracy of this method in detecting PEDV was 100% (3 / 3) and the accuracy of detecting PoRV-A was 100% (3 / 3). The results showed that this method has good applicability in clinical samples.

[0035] In summary, the present invention successfully prepared two sets of colloidal gold-nucleic acid-antibody complexes, combined the double antibody sandwich ELISA with HCR, took PEDV and PoRV-A as targets, and constructed a method for detecting PEDV and PoRV-A by fluorescent dual-color ELISA based on HCR. Relying on the different output fluorescence signals, this method achieves simultaneous and specific detection of PEDV and PoRV-A. This method does not require a nucleic acid extraction step and has the advantages of constant temperature, enzyme-free, and strong specificity. In addition, this method has good applicability in clinical sample detection and can be extended to the clinical detection of other viruses.

[0036] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA, characterized in that: The following steps are involved: S1. Using freeze-thaw method, thiol-modified oligonucleotide DNA AI and BI are fixed to the surface of gold particles to prepare colloidal gold-nucleic acid complexes Au-AI and Au-BI for use; S2, colloidal gold-nucleic acid complexes Au-AI and Au-BI prepared by freeze-thaw method, and their preparation effects were tested by HCR experiment; S3, preparing colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method for use; S4. Prepare colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2 by freeze-thaw method and electrostatic adsorption method, and test the preparation effect by performing HCR experiment; S5. Construction of HCR fluorescent dual-color ELISA differential diagnosis method for PEDV and PoRV-A; S6. Conduct specific analysis and clinical analysis; S7. Process and verify the experimental data.

2. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: The specific steps for preparing the colloidal gold-nucleic acid complex in S1 are as follows: S11, take 1mL of 20nm colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2mol / L K2CO3; S12, activate 8 μL AI and BI with 4 μL 2 mol / L TCEP, mix well and react at room temperature for 30 min; S13, adding the activated AI and BI dropwise into 200 μL of colloidal gold solution; S14, placing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, taking it out to melt, and centrifuging it in a centrifuge at 13,000 rpm for 5 minutes; S15, discard the supernatant and re-dissolve with 200 μL 0.01 mol / L PBS, and then centrifuge at 13000 r / min for 5 min; S16. After discarding the supernatant, dissolve it with 400 μL 0.01 mol / L PBS and prepare it as colloidal gold-nucleic acid complexes Au-AI and Au-BI.

3. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: In S2, the detection steps are as follows: S21, take a black ELISA plate, add 50 μL Au-AI, 1 μL each of A-H1 and A-H2 to the first well, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S22, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the second well, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S23, add 1 μL of A-H1 and A-H2 to well 3, and use 5 nM Mg 2+ The volume of the cells was filled to 100 μL with PBS buffer and group B was treated in the same way; S24, react in a 37°C constant temperature incubator for 1 hour. After the reaction is completed, the results are detected using a multifunctional microplate reader and the result data are collected.

4. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: The specific steps for preparing the colloidal gold-nucleic acid-antibody complex in S3 are as follows: S31, take 1mL of 20nm colloidal gold into a glass bottle, and adjust its pH to 7.5 using 0.2mol / L K2CO3; S32, activate 8 μL AI and BI with 4 μL 2mmol / L TCEP, mix well and react at room temperature for 30 min; S33, adding the activated AI and BI dropwise to 200 μL of colloidal gold solution, freezing the colloidal gold-nucleic acid mixture in a -20°C refrigerator for 1 hour, and then taking it out to melt; S34, add 4 μL of anti-PEDV S mAb to the melted colloidal gold-nucleic acid mixture Au-AI, add 4 μL of anti-PoRV-AVP6 mAb to the melted colloidal gold-nucleic acid mixture Au-BI, and incubate at room temperature with shaking for 4 h; S35. After the incubation, 22uL of 10% BSA solution was fully mixed with the colloidal gold-nucleic acid-antibody mixture and mixed at room temperature for 30min to block the excess sites; S36. After the sealing and mixing is completed, the reaction product is placed in a centrifuge and centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS. The product is then centrifuged at 13000 r / min for 5 min. The supernatant is discarded and the product is re-dissolved with 200 μL 0.01 mol / L PBS for use as colloidal gold-nucleic acid-antibody complexes Au-AI-mAb1 and Au-BI-mAb2.

5. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: In S4, the detection steps are as follows: S41, take a black ELISA plate, add 50 μL Au-AI-mAb1, 1 μL A-H1 and A-H2 to the first well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S42, add 50 μL Au-AI-mAb1, 50 μL Au-BI-mAb2, 1 μL each of A-H1 and A-H2, B-H1 and B-H2 to the second well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S43, add 1 μL AI, 1 μL A-H1 and 1 μL A-H2 to the third well, and use 5 nM Mg 2+ The volume of group B was filled to 150 μL with PBS buffer and treated in the same manner; S44, add 1 μL of A-H1 and A-H2 to well 4, and use 5 nM Mg 2+ The volume was filled to 150 μL with PBS buffer and B-H1 and B-H2 were treated in the same manner; S45, react in a 37°C constant temperature incubator for 1 hour. After the reaction is completed, the results are detected using a multifunctional microplate reader.

6. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: In the S5, the construction method is as follows: S51, anti-PEDV S monoclonal antibody (mAb1) and anti-PoRV-A VP6 monoclonal antibody (mAb2) were mixed and coated in a black ELISA plate, incubated at 4°C overnight, washed three times with PBST and patted dry; S52, incubate with rapid blocking solution I at room temperature for 30 min, wash the plate three times with PBST and pat dry; S53, add 50uL PBS, PEDV, PoRV-A virus solution or a mixture of PEDV and PoRV-A virus to the wells respectively, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S54, add 50uL Au-AI-mAb1 and 50uL Au-BI-mAb2 to the wells, mix, incubate at room temperature for 1h, wash the plate 5 times with PBST and pat dry; S55, add 1 μL each of A-H1, A-H2, B-H1, B-H2, and 5 nM Mg 2+ The volume was filled to 100 μL with PBS buffer and reacted in a 37°C constant temperature incubator for 1 h. The results were detected using a multifunctional microplate reader and the result data were collected.

7. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: In S6, the method of specific analysis is: using two target viruses PEDV and PoRV-A and five non-target viruses TGEV, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine parvovirus, and classical swine fever virus as the test objects, using 0.01 mol / L PBS as the negative control, and conducting the experiment according to the method.

8. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 1, characterized in that: In S6, the method of clinical analysis is: take 3 samples of clinical fluid that is positive for PEDV and PoRV-A by PCR, centrifuge and take the supernatant for actual sample testing; use 0.01 mol / L PBS as a negative control, conduct experiments according to the method, and evaluate the applicability of this method in actual clinical practice.

9. The method for differential diagnosis of PEDV and PoRV-A based on HCR fluorescent dual-color ELISA according to claim 5, characterized in that: In S24, S45, and S55, the fluorescence parameters are set as follows: A: the excitation wavelength is 494 nm, and the emission wavelength is 522 nm; B: the excitation wavelength is 540 nm, and the emission wavelength is 570 nm.

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