Primer, probe and kit for detecting EHV-1 and EHV-4 viruses

Abstract

The invention discloses a primer, a probe and a kit for detecting EHV-1 and EHV-4 viruses, and relates to the technical field of virus detection.The primer and the probe comprise a cross primer, a pair of probe primers and a pair of outer primers, the nucleotide sequence of the cross primer is shown as SEQ ID NO: 1, the nucleotide sequence of the probe primers is shown as SEQ ID NO: 2-3, and the nucleotide sequence of the outer primers is shown as SEQ ID NO: 2-3. The nucleotide sequences of the outer primers are as shown in SEQ ID NO: 4-5. The EHV-1 / 4 CPA-strip detection technology established by the invention does not need complex instruments, is simple and convenient to operate, can carry out in-situ detection, and provides convenience for the detection of EHV-1 and EHV-4.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a primer, a probe and a kit for detecting EHV-1 and EHV-4 viruses. Background Art

[0002] Equine rhinopneuminitis (ER) is a general term for highly contagious diseases among equine animals caused by equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4), and is a Category III epidemic disease. The infection causes primary respiratory diseases, and in severe cases can also cause neurological symptoms such as miscarriage in pregnant horses, paralysis, death of newborn foals, and fatal equine herpesvirus encephalitis (Equineherpesvirus myeloencephalopathy, EHM). The infected horses excrete toxins through nasal secretions, feces, aborted fetuses, etc., causing the pathogen to spread widely in the horse herd. The disease not only threatens the health of horses, but also brings serious harm to the industrial chain such as horse breeding, horse racing, and affiliated entertainment horse industries.

[0003] At present, there is no effective treatment for the disease, nor is there a vaccine with an ideal immune effect. Early screening is the key to preventing equine rhinopneumonitis. Screening methods include virus isolation, immunofluorescence, PCR, etc., but they have the disadvantages of complex operation, long time consumption, dependence on instruments, strict requirements on experimental conditions, and easy contamination. Therefore, establishing a rapid and accurate clinical detection method is of great significance for clinical screening, monitoring and epidemiological investigation.

[0004] Cross-primer isothermal amplification (CPA) technology can achieve in vitro nucleic acid amplification at a constant temperature. It is inexpensive, easy and fast to operate, and has high sensitivity and specificity. It has been used in the detection of pathogens in various fields and is suitable for areas where EHV-1 and EHV-4 are prevalent but resources are limited. Summary of the invention

[0005] The existing detection methods for EHV-1 and EHV-4 cannot meet the needs of early screening and early prevention of diseases, and it is even more difficult to achieve early screening of mixed infection cases. Based on this, the present invention aims to provide a primer, probe and kit for detecting EHV-1 and EHV-4 viruses, so as to achieve the goal of rapid and accurate detection of EHV-1 and EHV-4.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The invention provides a CPA-EHV-1 / 4 primer and a probe for cross primer amplification detection of EHV-1 and EHV-4 viruses. The primer and the probe include a cross primer, a pair of probe primers, and a pair of outer primers. The nucleotide sequence of the cross primer is shown in SEQ ID NO: 1, the nucleotide sequence of the probe primer is shown in SEQ ID NO: 2-3, and the nucleotide sequence of the outer primer is shown in SEQ ID NO: 4-5.

[0007] The CPA-EHV-1 / 4 primers for cross-primer amplification detection of EHV-1 and EHV-4, the EHV-1 / 4 primers selected as shown in SEQ ID NO: 1~5 are used as primers for final screening.

[0008] The present invention also provides a kit for cross-primer amplification detection of EHV-1 and EHV-4 viruses, the kit comprising the primers and probes according to claim 1, the nucleotide sequences of the primers and probes are shown in SEQ ID NOs: 1 to 5.

[0009] Furthermore, the kit also includes a basic buffer solution, dNTPs, Mg 2+ , Betaine, nuclease-free water, Bst DNA polymerase.

[0010] The kit comprises the following components: CPA-EHV-1 / 4: 10x Bst Reaction Buffer: 2.5 μL, dNTPs (10mM): 2.5 μL, Mg 2+ (100mM): 1.5 μL, Betaine (0.5 M): 2.5 μL, Nuclease-free water: 7.5 μL, Bst DNA polymerase: 1.0 μL; cross primer 1s-2a (10 μM): 2.5 μL, upstream outer primer 4a (10 μM): 0.5 μL, downstream outer primer 5s (10 μM): 0.5 μL, probe primer (FITC) 3s (10 μM): 1.5 μL, probe primer (Biotin) 1s (10 μM): 1.5 μL, template: 1.0 μL.

[0011] A method for detecting EHV-1 and EHV-4 based on CPA technology, comprising the following steps: mixing the above primer combination with a sample to be tested, preparing a CPA reaction system, and performing an amplification reaction at 63°C for 40 min; taking 5.0 μL of the amplified product, adding it to 45 μL of ddH2O for dilution, and then dripping it onto the end of the sample pad of a lateral flow chromatography test strip, reacting for 5 min, and then reading the result.

[0012] The lateral flow chromatography test strip has anti-FITC mouse monoclonal antibody on the binding pad, goat anti-mouse antibody on the quality control line, streptavidin on the Biotin detection line, and FITC mouse monoclonal antibody is used to mark colloidal gold.

[0013] The judgment of CPA-EHV-1 / 4 results is as follows: (1) When bands appear on both the T test line and the quality control line, it indicates that the test sample contains equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), or a mixed infection of equine herpesvirus type 1 and type 4; (2) When a band appears on the control line but no band appears on the test line, it indicates that the result is negative, i.e., the sample does not contain equine herpesvirus type 1, equine herpesvirus type 4, or mixed infection of equine herpesvirus type 1 and type 4, or the content of equine herpesvirus is lower than the detection limit of the CPA-EHV-1 / 4 method; (3) If no bands appear on the test line or the quality control line, the test result is invalid.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The primers, probes, kits, and test strips provided by the present invention for cross-primer amplification detection of EHV-1 and EHV-4 have high stability, specificity, and sensitivity. The sensitivity of the CPA-EHV-1 / 4 test strip reaches 1.44×10 3 Copies μL -1 and 1.02×10 3 Copies μL -1 times; (2) The primers, probes, kits and test strips for cross-primer amplification detection of EHV-1 and EHV-4 provided by the present invention have the advantages of being rapid, specific, sensitive, visual and simple. They are less dependent on instruments and are suitable for real-time visual detection of equine herpesvirus type 1 and equine herpesvirus type 4 infection at the grassroots or on-site, and can be used as a good method for carrying out detection in farms, grassroots laboratories and other places.

[0015] In summary, the EHV-1 / 4 CPA-strip detection technology established in the present invention does not require complex instruments, is easy to operate, and can be used for on-site detection, which provides convenience for the detection of EHV-1 and EHV-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is the plasmid electrophoresis result diagram and CPA result diagram in Example 3 of the present invention; Figure A is the EHV-1 / 4 ICP4 plasmid electrophoresis result diagram; Figure B is the EHV-1 / 4 ICP4 CPA result diagram; in Figure A and Figure B: lane M is DNA Marker DL 2000, lane N is a negative control, lane 1 is EHV-1 ICP4, and lane 2 is EHV-4 ICP4 respectively; Figure 2 It is a diagram of the optimization result of the cross primer 1s-2a in Example 3 of the present invention; Figure A is a diagram of the optimization result of the cross primer of the EHV-1 ICP4 plasmid, and Figure B is a diagram of the optimization result of the cross primer of the EHV-4 ICP4 plasmid; in Figures A and B: lane M is DNAMarker DL 2000, lane N is a negative control, and lanes 1 to 6 are added with 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 μL of cross primer 1s-2a, respectively; Figure 3 It is a probe primer 1s / 3s optimization result diagram in Example 3 of the present invention; Figure A is an EHV-1 ICP4 plasmid probe primer optimization result diagram, and Figure B is an EHV-4 ICP4 plasmid probe primer optimization result diagram; in Figure A and Figure B: lane M is DNAMarker DL 2000, lane N is a negative control, and lanes 1 to 6 are added with 0.5, 0.75, 1.0, 1.25, 1.5 and 1.75 μL of probe primer 1s / 3s, respectively; Figure 4 It is a diagram of the optimization result of the outer primer 4a / 5s in Example 3 of the present invention; Figure A is a diagram of the optimization result of the outer primer of the EHV-1 ICP4 plasmid, and Figure B is a diagram of the optimization result of the outer primer of the EHV-4 ICP4 plasmid; in Figures A and B: lane M is DNA MarkerDL DL 2000, lane N is a negative control, and lanes 1 to 6 are added with 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 μL of outer primer 4a / 5s, respectively; Figure 5 It is a diagram of the optimization result of Betaine in Example 3 of the present invention; Figure A is a diagram of the optimization result of EHV-1 ICP4 plasmid Betaine, and Figure B is a diagram of the optimization result of EHV-4 ICP4 plasmid Betaine; in Figures A and B: lane M is DNA Marker DLDL 2000, lane N is a negative control, and lanes 1 to 6 are added with 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μL Betaine, respectively; Figure 6 is Mg in Example 3 of the present invention 2+ Optimization results diagram; Figure A is EHV-1 ICP4 plasmid Mg 2+ Optimization results, Figure B is EHV-4 ICP4 plasmid Mg2+ Optimization results: In Figure A and Figure B, lane M is DNA Marker DL 2000, lane N is negative control, and lanes 1 to 6 are added with 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 μL Mg 2+ ; Figure 7 It is a graph of the dNTPs optimization results in Example 3 of the present invention; Figure A is a graph of the EHV-1 ICP4 plasmid dNTPs optimization results, and Figure B is a graph of the EHV-4 ICP4 plasmid dNTPs optimization results; in Figures A and B: lane M is DNA Marker DL 2000, lane N is a negative control, and lanes 1 to 6 are 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 μL dNTPs, respectively; Figure 8 It is a graph of the reaction temperature optimization result in Example 3 of the present invention; Figure A is a graph of the reaction temperature optimization result of the EHV-1 ICP4 plasmid, and Figure B is a graph of the reaction temperature optimization result of the EHV-4 ICP4 plasmid; in Figures A and B: lane M is DNA MarkerDL 2000, lane N is a negative control, and lanes 1 to 5 are 59, 61, 63, 65 and 67° C., respectively; Fig. 9 It is a graph of the reaction time optimization result in Example 3 of the present invention; Figure A is a graph of the EHV-1 ICP4 plasmid reaction time optimization result, and Figure B is a graph of the EHV-4 ICP4 plasmid reaction time optimization result; in Figures A and B: lane M is DNA MarkerDL 2000, lane N is a negative control, and lanes 1 to 5 are 20, 40, 60, 80 and 100 min, respectively; Fig.10 It is the specific test result of CPA-EHV-1 / 4 in Example 4 of the present invention; Figure A is a diagram of the comparison result of EHV-1 / 4 ICP4 sequences, and Figure B is a specific analysis of EHV-1 / 4; in Figure B, M represents DNA Marker 2000, N represents a blank control with ddH2O as a template, and 1 to 11 represent EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, EHV-6, EHV-8, EHV-9, PRV, BHV-1, and CpHV-1 ICP4 recombinant plasmids; Fig.11 The results of the CPA-EHV-1 / 4 sensitivity test in Example 5 of the present invention; Figure A is the EHV-1 / 4 PCR sensitivity analysis, Figure B is the EHV-1 / 4 CPA sensitivity analysis, and Figure C is the EHV-1 / 4 Real Time PCR sensitivity analysis; in Figures A and B: 1 to 11 represent 10 10 , 10 9 , 108 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , M represents DNA Marker 2000, N represents negative control; in Figure C, from left to right represent 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , NTC is the negative control; Fig.12 is the feasibility verification result of the CPA-EHV-1 / 4 test strip in Example 6 of the present invention, 1 and 2 are EHV-1 and EHV-4, respectively, and N is a negative control; Fig.13 The results of the order of adding potassium carbonate and antibody in Example 6 of the present invention are verified. 1 is a blank control group; 2 is first adding FITC antibody and mixing, and then adding potassium carbonate solution; 3 is first adding potassium carbonate solution, and then adding FITC antibody; Fig.14 It is a graph showing the pH optimization result of colloidal gold labeled antibody in Example 6 of the present invention; Figure A is a graph showing the pH optimization result of colloidal gold, and Figure B is a graph showing the pH optimization test strip result; in Figures A and B: the volumes of potassium carbonate added are 4, 6, 8, 10, and 12 μL, respectively; Fig.15 It is a graph showing the optimization result of the FITC antibody labeling amount in Example 6 of the present invention; Figure A is a graph showing the optimization result of the colloidal gold FITC antibody labeling amount, and Figure B is a graph showing the optimization result of the FITC antibody labeling amount test strip; in Figures A and B: the volumes of the added FITC antibody are 1, 2, 3, 4, and 5 μL, respectively; Fig.16 This is a diagram showing the optimization results of the amount of amplification product added in Example 6 of the present invention; the volumes of the amplification product added are 1, 5, 10, 15, and 20 μL, respectively; Fig.17 It is the CPA-EHV-1 / 4 sensitivity result diagram in Example 7 of the present invention; Figure A is the sensitivity result diagram of the EHV-1 test strip, and Figure B is the sensitivity result diagram of the EHV-4 test strip; in Figures A and B: N is a negative control, 1 to 11 are 10 10 , 109 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 Plasmid concentration; Fig.18 It is the CPA-EHV-1 / 4 specific result diagram in Example 8 of the present invention; N is a negative control, and numbers 1 to 11 are EHV-1, EHV-2, EHV-3, EHV-4, EHV-5, EHV-6, EHV-8, EHV-9, PRV, BHV-1, and CpHV-1 ICP4 recombinant plasmids; Fig.19 This is a graph of the reproducibility of CPA-EHV-1 / 4 in Example 9 of the present invention. Three different concentrations of equine herpes virus type 1 and type 4 ICP4 recombinant plasmids (10 8 , 10 7 , 10 6 copies / μL) to verify the intra-group and inter-group repeatability. DETAILED DESCRIPTION

[0017] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0018] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0019] Example 1 Construction of standard plasmids of EHV-1 ICP4 and EHV-4 ICP4 According to the EHV-1 ICP4 and EHV-4 ICP4 gene sequences registered in NCBI, the sequences were aligned using DNA MAN software, and highly homologous conserved regions were selected. The selected conserved fragments were commissioned to Shanghai Qingke Company for synthesis and construction into the pcDNA3.1 vector, and the EHV-1 ICP4 and EHV-4 ICP4 plasmids were extracted according to the instructions of the Tiangen plasmid extraction kit, and the concentration was determined and the copy number of the plasmid was calculated as the CPA positive standard plasmid.

[0020] The specific method is: a. Centrifuge the synthesized ampicillin-resistant plasmid powder to the bottom of the tube and add an appropriate amount of ddH2O to dissolve and mix. Thaw the DH5α competent medium on ice, add the plasmid to the competent medium at a ratio of 10:1, flick to mix, and place on ice for 30 min.

[0021] b. Heat shock at 42℃ for 90 seconds and place on ice for 3 min~5 min.

[0022] c. Add 900 μL LB liquid culture medium and culture at 37°C in a shaker at 180 rpm for 60 min.

[0023] d. Take 100 μL of bacterial solution and spread it evenly on LB solid medium with ampicillin resistance, place it in a 37℃ incubator and culture it for 12 h~16 h, and select single colonies of appropriate size to pick bacteria.

[0024] e. Inoculate the picked single colony into 5 mL LB liquid culture medium containing 0.5 μL Amp (100 μM) antibiotic resistance, shake the culture at 37°C and 180 rpm for 12 h~16 h, extract the standard positive plasmid according to the instructions of Tiangen Plasmid DNA Mini Reagent Extraction Kit, determine the concentration of the recombinant plasmid with a UV spectrophotometer, and calculate the copy number according to the following formula.

[0025]

[0026] Example 2 Primer pair and probe design for CPA experiment of EHV-1 / 4 The ORF64 and ICPF4 gene sequences of equine herpesvirus type 1 and type 4 were downloaded from NCBI as targets for primer design. DNAMAN software was used for sequence alignment to find the conserved sequences. SnapGene was used to design CPA primers and probes according to the CPA primer design principles. The primer and probe sequences are shown in Table 1.

[0027] The main operation steps are as follows: CPA basic reaction system is operated according to the instructions and references ( Bst DNA Polymerase, Large Fragment, Shanghai Bio-Tech). According to the instructions and references, the basic reaction system was constructed 25 μL; 10x Bst Reaction Buffer 2.5 μL, dNTPs (10mM) 2.5 μL, Mg 2+ (100 mM) 1.5 μL, Betaine (0.5M) 2.5 μL, Nuclease-free water 9.0 μL, BstDNA polymerase 1.0 μL; cross primer 1s-2a (10 μM) 2.0 μL, upstream outer primer 4a (10 μM) 0.5 μL, downstream outer primer 5s (10 μM) 0.5 μL, probe primer (FITC) 3s (10 μM) 1.0 μL, probe primer (Biotin) 1s (10 μM) 1.0 μL, DNA template: 1.0 μL. Amplification conditions were 63°C for 60 min. The amplification product was verified by 1.5% agarose gel electrophoresis.

[0028] Table 1 EHV-1 / 4 primer sequences

[0029] Example 3 Establishment of CPA-EHV-1 / 4 reaction system and optimization of conditions (1) Establishment of CPA-EHV-1 / 4 reaction system Reference cross primer isothermal amplification literature and Bst DNA polymerase instructions, set up CPA-EHV-1 / 4 25 μL basic reaction system as shown in Table 2, nuclease-free water as negative control, use EHV-1, EHV-4 recombinant plasmids with ICP4 gene as positive templates. In the EHV-1 / 4 CPA basic reaction system, select the initial reaction temperature of 63°C, and amplify isothermally in a constant temperature metal bath for 60 min. At the same time, use 1.5% concentration agarose electrophoresis to detect and verify the amplification results of the reaction product to identify whether the reaction has occurred. Figure 1 If the result is established, the basic reaction system is used for subsequent optimization of other components. On this basis, the cross primers, outer primers, and probe primers are optimized in turn. The working concentrations of other components remain unchanged, and a negative control is set up for each group. The reaction temperature (59℃~67℃), reaction time (20 min~100 min) and the components in the system (Betaine, Mg 2+ , dNTPs and Bst The optimized conditions were selected by 1.5% agarose gel electrophoresis to verify the optimization results of each group.

[0030] Table 2 CPA-EHV-1 / 4 basic reaction system

[0031] (2) Optimization of cross primer 1s-2a The experiment first determined the basic reaction system, and then optimized the cross primers. The concentrations of each component in the basic reaction system were all working concentrations, and the working concentrations of the primers used were all 10 μmol·L -1The volumes of cross primers 1s-2a added to the system were 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 μmol·L -1 If the system was less than 25 μL, nuclease-free water was used to make up the difference. The reaction was carried out at 63°C for 60 min and at 80°C for 20 min. After the reaction was completed, 1.5% agarose gel electrophoresis was used to verify the reaction. The results showed that the cross primer 1s-2a was 1.0 μmol·L -1 The optimal cross primer concentration is Figure 2 ).

[0032] (3) Optimization of probe primer 1s / 3s After the optimal concentration of the cross primers was determined, the volumes of probe primers 1s and 3s added to the system were selected to be 0.5, 0.75, 1.0, 1.25, 1.5, and 1.75 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 μmol·L, respectively. -1 If the system was less than 25 μL, nuclease-free water was used to make up the volume. The reaction was carried out at a temperature of 63°C for 60 min and at an inactivation temperature of 80°C for 20 min. After the reaction was completed, 1.5% agarose gel electrophoresis was performed to verify that the optimal reaction concentration of the probe primer 1s / 3s was 0.6 μmol·L -1 ( Figure 3 ).

[0033] (4) Optimization of outer primers 4a / 5s After the optimal concentration of the probe primers was determined, the volumes of the external primers 4a and 5s added to the system were selected to be 0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 μmol·L, respectively. -1 If the system was less than 25 μL, nuclease-free water was used to make up the remaining amount. The reaction was carried out at 63°C for 60 min and at 80°C for 20 min. After the reaction was completed, 1.5% agarose gel electrophoresis was used to verify the reaction. The results showed that the optimal reaction concentration of the outer primers 4a / 5s was 0.2 μmol·L -1 ( Figure 4 ).

[0034] (5) Betaine Optimization In the experiment, the cross primer, probe primer and outer primer were added to the optimal working concentration volume determined in the previous experiment, and the volume of each component added in the other basic reaction system remained unchanged (Table 2). The concentration of Betaine selected in the system was 5 mol·L -1 The volumes of the reagents were 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mol·L -1 If the system is less than 25 μL, use nuclease-free water to make up, react at 63°C for 60 min, inactivate at 80°C for 20 min, and use 1.5% agarose gel electrophoresis to verify after the reaction is completed. The results show that the optimal reaction concentration of Betaine is 0.5 mol·L -1 ( Figure 5 ).

[0035] (6) Mg 2+ optimization After determining the optimal concentrations of cross primers, probe primers, outer primers and Betaine, the other components in the system remain unchanged and Mg is added. 2+ The volumes of the reagents were 0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mmol·L -1 When the system was less than 25 μL, nuclease-free water was used to make up. The reaction temperature was 63°C for 60 min and the inactivation temperature was 80°C for 20 min. After the reaction was completed, 1.5% agarose gel electrophoresis was used to verify that Mg 2+ The optimal reaction concentration was 6.0 mmol·L -1 ( Figure 6 ).

[0036] (7) dNTPs optimization Determine the cross primer, probe primer, outer primer, Betaine and Mg 2+ After the optimal concentration, the other components in the system remained unchanged, and the volumes of dNTPs added to the system were selected to be 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 μL, respectively, and their corresponding working concentrations in the basic reaction system were 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mmol·L, respectively. -1When the system was less than 25 μL, nuclease-free water was used to make up the difference. The reaction temperature was 63°C for 60 min and the inactivation temperature was 80°C for 20 min. After the reaction was completed, 1.5% agarose gel electrophoresis was used to verify that the optimal concentration of dNTPs was 1.0 mmol·L -1 ( Figure 7 ).

[0037] (8) Reaction temperature optimization The reaction temperatures were selected as 59, 61, 63, 65 and 67 °C, respectively. According to the optimized CPA-EHV-1 / 4 reaction system shown in Table 3, the reaction temperature was 59-67 °C for 60 min, and the inactivation temperature was 80 °C for 20 min. After the reaction amplification was completed, 1.5% agarose gel electrophoresis was used for verification. The results showed that the optimal reaction temperature was 63 °C ( Figure 8 ).

[0038] (9) Optimization of response time The reaction time of the selected test was 20, 40, 60, 80, and 100 min, respectively. According to the optimized CPA-EHV-1 / 4 reaction system shown in Table 3, the reaction temperature was 63 ° C for 20-100 min, and the inactivation temperature was 80 ° C for 20 min. After the reaction amplification was completed, 1.5% agarose gel electrophoresis was used for verification. The results showed that the optimal reaction time was 40 min ( Fig. 9 ).

[0039] Table 3 CPA-EHV-1 / 4 optimal reaction system

[0040] Example 4 Specificity analysis of CPA-EHV-1 / 4 Sequence alignment results showed that the EHV-1 / 4 ICP4 target fragment gene was significantly different from other herpes virus ICP4 genes and the homology of the conserved region was poor, see Fig.10A. The results showed that the EHV-1 / 4 ICP4 target fragment gene was significantly different from other herpesvirus ICP4 genes and the conserved region homology was poor. The ICP4 genes of equine herpesvirus type 1, equine herpesvirus type 2, equine herpesvirus type 3, equine herpesvirus type 4, equine herpesvirus type 5, equine herpesvirus type 6, equine herpesvirus type 8, equine herpesvirus type 9, pseudorabies virus PRV, bovine herpesvirus type 1 BHV-1 and goat herpesvirus CpHV-1 were all constructed into the pcDNA3.1 vector and synthesized by the company. The obtained recombinant plasmid was used as a template for specific detection. At the same time, the recombinant plasmid carrying the EHV-1 / 4 ICP4 gene was used as a positive template, and a negative control without adding a template was set. The EHV-1 / 4 CPA optimal reaction system was used for amplification at 63°C and 40 min. The products were verified by agarose gel electrophoresis at a concentration of 1.5%. The results showed that only EHV-1 ICP4 and EHV-4 The ICP4 recombinant plasmid showed a typical ladder-shaped amplification band, while the amplification products of the ICP4 recombinant plasmids of EHV-2, EHV-3, EHV-5, EHV-6, EHV-8, EHV-9, PRV, CpHV-1, and BHV-1 and the negative control showed no obvious amplification bands after gel electrophoresis, indicating that the established EHV-1 / 4 CPA detection technology has good specificity ( Fig.10 B).

[0041] Example 5 Sensitivity Analysis of CPA-EHV-1 / 4 The concentrations of EHV-1 ICP4 and EHV-4 ICP4 recombinant plasmids were measured using an ultraviolet spectrophotometer, and the copy numbers were calculated according to the copy number formula. The results were 1.44×10 10 Copies μL -1 and 1.02×10 10 Copies μL -1 The recombinant plasmid was diluted with nuclease-free water for 10 10 -10 0 copies / μL gradient dilution, select 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0Plasmid concentration was determined by setting nuclease-free water as negative control, and CPA-EHV-1 / 4 reaction was performed. It was compared with PCR and Real Time PCR (the latter two were amplified with external primers). The EHV-1 / 4 CPA optimal reaction system was used for amplification at 63°C for 40 min. The amplified products were verified by 1.5% agarose gel electrophoresis. The results showed that the established EHV-1 / 4 CPA minimum detection limits were 1.44×10 3 Copies μL -1 and 1.02×10 3 Copies μL -1 ( Fig.11 B). The results of conventional PCR amplification showed that the sensitivity was only 10 times lower than that of EHV-1 / 4 CPA, that is, the minimum detection limit of conventional PCR was 1.44×10 4 Copies μL -1 and 1.02×10 4 Copies μL -1 ( Fig.11 A). Real-time PCR results showed that the sensitivity was basically consistent with that of EHV-1 / 4 CPA, and the minimum detection limit was 1.44×10 3 Copies μL -1 and 1.02×10 3 Copies μL -1 ( Fig.11 C).

[0042] Example 6 Preparation of CPA-EHV-1 / 4 Test Strip (1) Feasibility verification of CPA-EHV-1 / 4 test strip Take 5 μL of CPA-EHV-1 / 4 amplification product and dilute it with 45 μL of 10 mM PB. Add the diluted solution to the sample pad of the lateral flow chromatography test strip and wait for 5-10 minutes to observe the test results. The results show that the line of the test strip is the same as Figure 1 The electrophoresis results confirm each other's feasibility. The premise of the test strip showing the line is that the CPA system amplifies the product band ( Fig.12 ).

[0043] (2) Verification of the order of adding potassium carbonate and adding antibodies Take 1 mL of colloidal gold solution and add it to 1.5 mL centrifuge tubes, a total of three tubes. Set No. 1 as the control group; add 0.5 μL of 10 mg / mL FITC antibody to No. 2 and mix well, then add 10 μL of 0.1 M potassium carbonate solution to adjust the pH, and incubate at 4℃ for 30 min; first add 10 μL of 0.1 M potassium carbonate solution to No. 3, then add 0.5 μL of 10 mg / mL FITC antibody, and incubate at 4℃ for 30 min. The results showed that adding FITC antibody to No. 2 first and then adjusting the pH with potassium carbonate would reduce the stability of the colloidal gold solution, and a large amount of colloidal gold particles would aggregate and precipitate, and the color of the solution would change from wine red to lavender ( Fig.13 ).

[0044] (3) Optimization of pH value of colloidal gold labeled antibodies The experiment selected 0.1 M potassium carbonate solution as the pH regulator, took 1 mL of colloidal gold solution in a 1.5 mL centrifuge tube, and selected the volume of potassium carbonate added as 4, 6, 8, 10, and 12 μL, respectively. The optimal addition amount was selected based on the color change of the colloidal gold solution and the line display of the test strip. The results showed that when the added volume of potassium carbonate was 8 μL, the line display effect of the test strip was the best, and the binding efficiency of colloidal gold particles and FITC antibodies was good under this pH condition ( Fig.14 ).

[0045] (4) Optimization of FITC antibody labeling amount Take 1 mL of colloidal gold solution in a 1.5 mL centrifuge tube, add 8 μL of potassium carbonate to adjust the pH and mix well. The volume of adding 10 mg / mL FITC antibody is selected as 1, 2, 3, 4, and 5 μL in sequence. The optimal addition amount is selected based on the color change of the colloidal gold solution and the line display of the test strip. The results show that the colloidal gold particles have the best binding efficiency and high stability when the FITC antibody is added at 2 μL ( Fig.15 ).

[0046] (5) Optimization of the amount of amplification product added 10 mM phosphate buffer (PB) was selected as the loading buffer, and the amount of amplification product added was selected to be 1, 5, 10, 15, and 20 μL, and the loading buffer was supplemented to 50 μL. The optimal amount of amplification product was selected by observing the appearance of the test line (T line) of the test strip. The results showed that as the amount of amplification product increased, the color of the test line (T line) gradually became lighter. Therefore, the experiment finally selected the addition of 5 μL of amplification product as the amplification condition ( Fig.16 ).

[0047] Example 7 CPA-EHV-1 / 4 Test Strip Sensitivity Analysis Combined with the optimal conditions optimized in Example 6, the sensitivity test was performed using the EHV-1 / 4 CPA optimal reaction system at 63°C and 40 min combined with a test strip. The results showed that the minimum detection limit of EHV-1 / 4 CPA-strip for EHV-1 ICP4 recombinant plasmid was 1.44×10 3 Copies μL -1 , EHV-4 ICP4 recombinant plasmid was 1.02×10 3 Copies μL -1 .and Fig.10 The sensitivity of the test strips is consistent with that of CPA, 10 times higher than that of PCR, and basically consistent with the results of Real Time PCR ( Fig.17 ).

[0048] Example 8 CPA-EHV-1 / 4 Test Strip Specificity Analysis The recombinant plasmids of equine herpesvirus type 1, equine herpesvirus type 2, equine herpesvirus type 3, equine herpesvirus type 4, equine herpesvirus type 5, equine herpesvirus type 6, equine herpesvirus type 8, equine herpesvirus type 9, pseudorabies virus PRV, bovine herpesvirus type 1 BHV-1 and caprine herpesvirus CpHV-1 ICP4 were used as templates for CPA-EHV-1 / 4 reaction, and the amplification results were analyzed using lateral flow chromatography test strips to verify their specificity. The results showed that the EHV-1 / 4 CPA-strip and EHV-1 CPA-stirp had good specificity ( Fig.18 ).

[0049] Example 9 Repeatability Analysis of CPA-EHV-1 / 4 Test Strip Three different concentrations of equine herpesvirus type 1 and type 4 ICP4 recombinant plasmids (10 8 , 10 7 , 10 6 copies / μL) to verify the intra-group and inter-group repeatability. The results showed that there was no significant change in the detection line of the same concentration ( Fig.19 ).

[0050] Example 10 CPA-EHV-1 / 4 kit assembly The CPA-EHV-1 / 4 kit was assembled with the best reaction system in Example 3: cross primer 1s-2a (10 μM): 2.5 μL, upstream outer primer 4a (10 μM): 0.5 μL, downstream outer primer 5s (10 μM): 0.5 μL, probe primer (FITC) 3s (10 μM): 1.5 μL, probe primer (Biotin) 1s (10 μM): 1.5 μL; 10x BstReaction Buffer: 2.5 μL, dNTPs (10 mM): 2.5 μL, Mg 2+ (100 mM): 1.5 μL, Betaine (0.5 M): 2.5 μL, Nuclease-free water: 7.5 μL, Bst DNA polymerase: 1.0 μL; Template: 1.0 μL; Test strip prepared in Example 6.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer and a probe for detecting EHV-1 and EHV-4 viruses, characterized in that: The primers and probes include a cross primer, a pair of probe primers, and a pair of outer primers. The nucleotide sequence of the cross primer is shown in SEQ ID NO: 1, the nucleotide sequence of the probe primer is shown in SEQ ID NO: 2-3, and the nucleotide sequence of the outer primer is shown in SEQ ID NO: 4-5.

2. A kit for detecting EHV-1 and EHV-4 viruses, characterized in that: The kit comprises the primers and probes according to claim 1, and the nucleotide sequences of the primers and probes are shown in SEQ ID NOs: 1 to 5.

3. A kit for detecting EHV-1 and EHV-4 viruses according to claim 2, characterized in that: The kit also includes a basic buffer solution, dNTPs, Mg 2+ , Betaine, nuclease-free water, Bst DNA polymerase.

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