Primer group, kit and method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus

Abstract

The invention discloses a primer group, a kit and a method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus. Belongs to the technical field of molecular biological detection. According to the method, simultaneous detection of the spike protein, the envelope protein and the transmembrane protein of the bovine coronavirus is realized, and compared with the prior art, the sensitivity, the specificity and the repeatability of detection are greatly improved, so that the method has important significance in preventing and controlling prevalence of the bovine coronavirus.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection technology, and more specifically to a primer set, a kit and a method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus. Background Art

[0002] Bovine coronavirus (BCoV) is a virus that can cause a variety of diseases, mainly affecting cattle. It can cause diarrhea (CD) in newborn calves, which is an important disease caused by the virus. In addition, BCoV can also cause respiratory tract infection (RCV) in cattle and winter dysentery (WD) in adult cattle. Since its first report in the United States in 1973, BCoV has been shown to be widely distributed worldwide, affecting cattle herds in many countries and regions. BCoV belongs to the genus Coronavirus in the family Coronaviridae. It is an enveloped single-stranded positive-sense RNA virus with a genome size of 31Kb and a particle diameter of 120-160nm. It encodes five structural proteins: spike protein (S), envelope protein (E), transmembrane protein (M), hemagglutinin esterase protein (HE) and nucleocapsid protein (N). S protein is an important protein for host cell attachment, virus entry and pathogenesis, and is involved in the adsorption and membrane fusion process of the virus and host cells. E protein contains a transmembrane region and is the smallest structural protein of BCoV. It is mainly involved in the composition of the coronavirus particle envelope. Studies have shown that when the virus infects cells, a large amount of E protein is produced. This protein is involved in the assembly and budding of virus particles. The lack of E protein will significantly reduce the titer and maturation of the virus; E protein is also involved in the regulation of biological processes such as cell inflammation, apoptosis, and autophagy by the virus. M protein is the most abundant transmembrane transport protein on the surface of the virus envelope. By comparing the M gene sequences of different BCoV strains published by NCBI, it was found that its homology is more than 95%. It is often used as a target gene for establishing nucleic acid diagnostic technology or serological diagnosis. M protein can be divided into three parts: the short glycosylated N-terminal region exposed outside the virus envelope, the large C-terminal region in the virus envelope, and the middle α-helical region. Studies have shown that M protein plays an important role in stimulating the host body to produce immune responses, stimulates the body to produce interferon, is responsible for the transportation of nutrients, and also plays a role in the assembly and budding of virus particles.

[0003] In summary, the S, M, and E gene segments of bovine coronavirus (BCoV) contain important information encoding viral surface proteins and membrane proteins, which are crucial for the construction of bovine coronavirus virus-like particle vaccines. Based on this, providing a method for simultaneously detecting bovine coronavirus S, M, and E proteins is crucial for the construction of bovine coronavirus particle-like vaccines. However, there are few studies on the simultaneous detection of the above three proteins in the prior art.

[0004] Therefore, how to provide a primer set, kit and method for simultaneously detecting bovine coronavirus spike protein, envelope protein and transmembrane protein with high detection sensitivity and strong specificity is a technical problem that needs to be urgently solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a primer set, a kit and a method for simultaneously detecting the spike protein, envelope protein and transmembrane protein of bovine coronavirus, which can realize the simultaneous detection of the bovine coronavirus spike protein (S), envelope protein (E) and transmembrane protein (M) genes, and has high detection sensitivity, strong specificity and good repeatability, and is suitable for popularization and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A primer set for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, the primer set and nucleotide sequence are as follows:

[0008] SF: 5'-CCTTCCATCTCCACTGACAC-3', SEQ ID NO.1;

[0009] SR: 5'-GTACCCTTCAGAGCCATGTT-3', SEQ ID NO.2;

[0010] EF: 5'-GGTTGAACACGTAGATGGAA-3', SEQ ID NO.3;

[0011] ER: 5'-ATCATCGTGTGTGGTGGCTTT-3', SEQ ID NO.4;

[0012] MF: 5'-ACACTGGACCGCTGACGAA-3', SEQ ID NO.5;

[0013] MR: 5'-ACGAACATGGAGCGGGAGGT-3', SEQ ID NO. 6.

[0014] Another object of the present invention is to provide: a kit for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, comprising the above-mentioned primer set.

[0015] Another object of the present invention is to provide: the use of the above-mentioned primer set or the above-mentioned kit in the detection of bovine coronavirus spike protein, envelope protein and transmembrane protein.

[0016] Another object of the present invention is to provide: a qPCR method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, using the above primer set or kit for detection, comprising the following steps: extracting DNA of the sample to be tested as a template, using the above primer set or kit for detection, if a 166 bp band appears in the amplified product, it means that the bovine coronavirus spike protein is positive; if a 150 bp band appears, it means that the bovine coronavirus envelope protein is positive; if a 180 bp band appears, it means that the bovine coronavirus transmembrane protein is positive;

[0017] The methods are not intended for disease detection and / or treatment.

[0018] Preferably, the qPCR detection system is as follows:

[0019] 2×ChamQ Universal SYBR qPCR MasterMix 10μL;

[0020] SF 0.5 μL;

[0021] SR 0.5 μL;

[0022] EF 0.5 μL;

[0023] ER 0.5 μL;

[0024] MF 0.5 μL;

[0025] MR 0.5 μL;

[0026] DNA template 2 μL;

[0027] RNase-free water 5μL;

[0028] The concentrations of the primers SF, SR, EF, ER, MF and MR are all 500 nmol / L.

[0029] Preferably, the qPCR detection procedure is as follows: pre-denaturation at 95°C for 30s; denaturation at 95°C for 20s, annealing and extension at 54.5°C for 30s, and 40 cycles.

[0030] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0031] Compared with the prior art, the present invention can simultaneously detect the bovine coronavirus spike protein (S), envelope protein (E) and transmembrane protein (M) genes, and has high detection sensitivity, strong specificity and good repeatability, is suitable for popularization and application, and is of great significance for preventing and controlling the spread of bovine coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 : The results of ordinary PCR amplification of S gene; M: marker; 1: negative control; 2-4: S gene amplification.

[0034] Figure 2 : The result of common PCR amplification of E gene; M: marker; 1: negative control; 2-4: E gene amplification.

[0035] Figure 3 : The result of ordinary PCR amplification of M gene; wherein, M: marker; 1: negative control; 2-4: M gene amplification.

[0036] Figure 4 : S gene primer concentration optimization results, the bold curve is the optimal concentration; 1-6: 10μmol / L, 5μmol / L, 2.5μmol / L, 1μmol / L, 500nmol / L, 100nμmol / L; 7: negative control.

[0037] Figure 5 : E gene primer concentration optimization results, the bold curve is the optimal concentration; 1-6: 10μmol / L, 5μmol / L, 2.5μmol / L, 1μmol / L, 500nmol / L, 100nμmol / L; 7: negative control.

[0038] Figure 6 : M gene primer concentration optimization results, the bold curve is the optimal concentration; 1-6: 10μmol / L, 5μmol / L, 2.5μmol / L, 1μmol / L, 500nmol / L, 100nμmol / L; 7: negative control.

[0039] Figure 7 is: S fluorescence quantitative PCR amplification curve; 1-7: 1×10 7 copies / μL~1×10 1 copies / μL; 8: negative control.

[0040] Figure 8 Figure 3: Standard curve of S fluorescence quantitative PCR.

[0041] Fig. 9 E is the fluorescence quantitative PCR amplification curve; 1-7: 1×10 7 copies / μL~1×10 1 copies / μL; 8: negative control.

[0042] Fig.10 Figure : E. Fluorescence quantitative PCR standard curve.

[0043] Fig.11 M fluorescence quantitative PCR amplification curve; 1-7: 1×10 7 copies / μL~1×10 1 copies / μL; 8: negative control.

[0044] Fig.12 M is the standard curve of fluorescence quantitative PCR.

[0045] Fig.13 =S fluorescence quantitative PCR sensitivity test results; 1-7: 1×10 7 ~1×10 1 copies / μL; 8: negative control.

[0046] Fig.14 E: Results of fluorescence quantitative PCR sensitivity experiment; 1-7: 1×10 7 ~1×10 1 copies / μL; 8: negative control.

[0047] Fig.15 M fluorescence quantitative PCR sensitivity test results; 1-7: 1×10 7 ~1×10 1 copies / μL; 8: negative control.

[0048] Fig.16 : S fluorescence quantitative PCR specific experimental results; 1: S gene; 2: negative control, BRV, BVDV, IBRV, BRSV, Mh, Pm A, Pm B, Escherichia coli, and Salmonella.

[0049] Fig.17 : E fluorescence quantitative PCR specific experimental results; 1: E gene; 2: negative control, BRV, BVDV, IBRV, BRSV, Mh, Pm A, Pm B, Escherichia coli, and Salmonella.

[0050] Fig.18: M fluorescence quantitative PCR specific experimental results; 1: M gene; 2: negative control, BRV, BVDV, IBRV, BRSV, Mh, Pm A, Pm B, Escherichia coli, and Salmonella.

[0051] Fig.19 is: S fluorescence quantitative PCR repeatability experiment results; 1-4: 1×10 6 ~1×10 3 copies / μL; 5: negative control.

[0052] Fig. 20 E: Results of the repeatability experiment of fluorescence quantitative PCR; 1-4: 1×10 6 ~1×10 3 copies / μL; 5: negative control.

[0053] Fig.21 M fluorescence quantitative PCR repeatability test results; 1-4: 1×10 6 ~1×10 3 copies / μL; 5: negative control.

[0054] Fig. 22 These are: the optimization results of triple fluorescence quantitative PCR primer concentrations for BCoV S, E and M genes; 1-6: 10μmol / L, 5μmol / L, 2.5μmol / L, 1μmol / L, 500nmol / L, 100nμmol / L; 7: negative control.

[0055] Fig.23 is: triple fluorescence quantitative PCR amplification curve; 1-9: 1×10 9 copies / μL~1×10 1 copies / μL; 10: negative control.

[0056] Fig.24 Figure 3: Standard curve of triple fluorescence quantitative PCR.

[0057] Fig.25 is: triple fluorescence quantitative PCR sensitivity curve; 1-8: 1×10 8 copies / μL~1×10 1 copies / μL; 8: negative control.

[0058] Fig.26 : Specific results of triple fluorescence quantitative PCR; 1: S gene; 2: M gene; 3: E gene; 4: negative control, BRV, BVDV, IBRV, BRSV, Mh, Pm A, Pm B, Escherichia coli, and Salmonella.

[0059] Fig. 27 The results of the triple fluorescence quantitative PCR repeatability test; 1-3: 1×10 6 ~10 4 copies / μL; 4: negative control. DETAILED DESCRIPTION

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1

[0062] Primer design and synthesis

[0063] According to the S, E, and M gene sequences of the bovine coronavirus isolate Mebus (GenBank accession number: U00735), LUN isolate (GenBank accession number: AF391542.1), NM45 isolate (GenBank accession number: MZ711356.1), DB2 isolate (GenBank accession number: DQ811784.2), and E-AH187 isolate (GenBank accession number: EF424619.1) published on GenBank, multiple sequence alignment was performed using the MegAlign program in Lasergener software, and specific primers were designed and synthesized by selecting the conserved sequences on the S, E, and M gene fragments (see Table 1). The upstream and downstream primers were named SF and SR, EF and ER, MF and MR, respectively, and the designed primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0064] Table 1 Primer sequences of spike protein (S), envelope protein (E) and transmembrane protein (M) of bovine coronavirus

[0065]

[0066] Example 2

[0067] Acquisition of virus-positive plasmid

[0068] (1) Viral nucleic acid extraction: BCoV total RNA was extracted using the RNAsimple total RNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific experimental steps were performed according to the instructions;

[0069] (2) Reverse transcription of BCoV total RNA was performed using a reverse transcription kit to prepare cDNA. The reaction system and procedure are shown in Table 2:

[0070] Table 2 Reverse transcription reaction system and reaction procedure

[0071]

[0072] (3) The cDNA obtained in step 2 was used as a template and conventional PCR amplification was performed using the primers designed in Example 1. The amplified products were subjected to agarose gel electrophoresis. After the electrophoresis was completed, the results were observed. The electrophoresis bands were consistent with the size of the target gene fragment (e.g. Figure 1-Figure 3 ), using gel recovery to obtain recovered bovine coronavirus S gene, E gene and M gene recovery products;

[0073] The common PCR amplification reaction system (25 μL) is: Premix TaqTM 12.5 μL, primers (10 μmol / L) 1.0 μL each, template cDNA 2.0 μL, ddH 2 2. 4% dHO 8.5 μL.

[0074] The common PCR amplification reaction parameters are: 94°C for 3 min; 94°C for 20 s, 56°C for 20 s, 72°C for 20 s, 30 cycles; 72°C for 7 min.

[0075] (4) The S, E and M genes of the bovine coronavirus were recovered and respectively mixed with pMD TM The ligation product was obtained by incubating at 16°C for 30 min.

[0076] The ligation system (10 μL) was as follows: pMD19-T Vector 1 μL, gel recovery product 4.0 μL, Solution I 5.0 μL.

[0077] The ligation product was transformed into Takara DH5α competent cells stored at -80°C, positive clones were obtained by colony PCR, and plasmids were extracted after shaking culture in LB liquid culture medium for 12-16 hours. The plasmids were sequenced and compared to obtain positive recombinant plasmids, which were stored at -20°C for future use.

[0078] (5) Calculation and dilution of recombinant plasmid DNA copy number

[0079] After extracting the plasmid, 1 μL was taken from each sample. The concentrations of the S, E, and M standard positive plasmids were measured to be 153.1 ng / μL, 140.3 ng / μL, and 123.4 ng / μL, respectively. The calculation formula was: plasmid copy number (copies / μL) = 6.02 × (recombinant plasmid concentration ng / μL × 10 -9 )×10 23 / (660×number of bases of recombinant plasmid) to calculate the plasmid copy number. The copy numbers of S, E and M plasmids were 8.41×10 11 copies / μL、8.53×10 11 copies / μL and 6.25×10 11 When preparing the positive standard, use enzyme-free water for 10-fold dilution.

[0080] Example 3

[0081] Detection of S, E and M genes of bovine coronavirus by single-plex fluorescence quantitative PCR

[0082] (1) Optimization of reaction parameters

[0083] In order to explore the effects of different reaction conditions on single-plex fluorescence quantitative PCR amplification of S gene, E gene and M gene, it is necessary to explore and optimize the PCR conditions to obtain the best reaction conditions and provide an appropriate optimization range for the optimization of each condition of triple fluorescence quantitative PCR.

[0084] Positive plasmid DNA was used as a template, and the final volume of 0.5 μL of upstream and downstream primers was respectively drawn, and the loading concentrations were 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1 μmol / L, 500 nmol / L, and 100 nmol / L. The concentration with the highest fluorescence value and the smallest Ct value was selected as the optimal concentration. If the two conditions cannot be met at the same time, the Ct value is given priority.

[0085] The results are as follows Figure 4-Figure 6 As shown, when the S primer concentration is 500nmol / L, the Ct value is the smallest, and the amplification curve is good; when the E primer concentration is 1μmol / L, the Ct value is the smallest, and the amplification curve is good; when the M primer concentration is 500nmol / L, the Ct value is the smallest, and the amplification curve is good; therefore, in this experiment, the optimal primer volume for S and M is 0.5μL (500nmol / L), and the optimal primer volume for E is 0.5μL (1μmol / L).

[0086] (2) Establishment of standard curves for single-plex fluorescence quantitative PCR of S, E and M

[0087] Template selection 1×10 8 copies / μL~1×10 1The positive plasmid was prepared at 8 concentrations (100 copies / μL), and each template was repeated 3 times to set up a negative control. The primers were reacted according to the optimization results, and the correlation coefficient and amplification efficiency were obtained according to the software analysis, and the standard curve equation of the single quantitative PCR of the bovine coronavirus S gene, E gene and M gene was drawn. The results are shown in Figure 7-Figure 12 shown.

[0088] Result analysis: The standard curve regression equation of S is y=-3.3405x+28.782, and the correlation coefficient is R 2 The standard curve regression equation of E is y = -3.1845x + 27.693, and the correlation coefficient R 2 The standard curve regression equation of M is y = -3.4083x + 28.625, and the correlation coefficient R 2 Reach 0.9902;

[0089] (3) S, E and M single-plex fluorescence quantitative PCR sensitivity test

[0090] The template is 1×10 8 copies / μL~1×10 1 8 standard positive plasmids were prepared with 3 replicates / μL, and negative controls were set up. At the same time, the detection was carried out according to the conventional PCR and the optimized fluorescence quantitative PCR method to obtain the minimum detection concentration of the quantitative PCR detection method.

[0091] The results are as follows Figure 13-Figure 15 As shown, the minimum copy number detected by fluorescence quantitative PCR for S, E, and M was 1×10 1 copies / μL.

[0092] (4) Single-plex fluorescence quantitative PCR specificity test of S, E and M genes

[0093] The reaction system and procedure remained unchanged. The nucleic acids of bovine rotavirus (BRV), bovine viral diarrhea / mucosal virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), Mannheimia haemolytica (Mh), Pasteurella multocida type A (PmA), Pasteurella multocida type B (Pm B), Escherichia coli, and Salmonella extracted by DNA / RNA extraction kit (all commercially available pathogens) were used as templates. 7 The S, E, and M standard positive plasmids were used as positive controls, and negative controls were set up for specificity verification. The results were as follows Figure 16-Figure 18 Only S, E and M genes showed amplification curves, indicating that S, E and M single-plex fluorescence quantitative PCR had good specificity

[0094] (5) Repeatability test of single-plex fluorescence quantitative PCR of S, E and M

[0095] Template 1×10 6 copies / μL~1×10 3 copies / μL positive plasmid, repeated three times or more, set up negative control, analyzed the experimental data, and verified the stability of the fluorescence quantitative PCR method. The results are as follows Figure 19-21 As shown, it shows that the S, E and M single-plex fluorescence quantitative PCR have good repeatability.

[0096] Example 4

[0097] Establishment of a triple fluorescence quantitative PCR method for bovine coronavirus S, E and M genes

[0098] (1) Optimization of triple fluorescence quantitative PCR reaction system for bovine coronavirus S, E and M genes

[0099] Amplification was performed according to the optimized reaction conditions in the single-plex fluorescence quantitative PCR method (reaction system: 20 μL).

[0100] Primers with concentrations of 10 μmol / L, 5 μmol / L, 2.5 μmol / L, 1 μmol / L, 500 nmol / L, and 100 nmol / L were mixed in a ratio of 1:1:1 for RT-PCR reaction, and the optimal primer concentration was selected according to the Ct value.

[0101] The results are as follows Fig. 22 As shown in the figure, when the concentration is 500 nmol / L and the primers are in a ratio of 1:1:1, the amplification curve is good. Therefore, the optimal loading amount of primers S, E and M is determined to be 0.5 μL (500 nmol / L).

[0102] (2) Establishment of triple fluorescence quantitative PCR standard curve for S, E and M genes

[0103] The primer sequences are shown in Table 1 ;

[0104] The reaction system is 20 μL, as follows:

[0105] 2×ChamQ Universal SYBR qPCR MasterMix 10μL

[0106] SF 0.5 μL (500 nmol / L);

[0107] SR 0.5 μL (500 nmol / L);

[0108] EF 0.5 μL (500 nmol / L);

[0109] ER 0.5 μL (500 nmol / L);

[0110] MF 0.5 μL (500 nmol / L);

[0111] MR 0.5 μL (500 nmol / L);

[0112] DNA template 2 μL;

[0113] Add RNase-free water to 20 μL.

[0114] The reaction procedure is as follows:

[0115] Pre-denaturation at 95°C for 20 s; denaturation at 95°C for 20 s, annealing and extension at 54.5°C for 30 s, 40 cycles.

[0116] Template selection 1×10 9 copies / μL~1×10 1 copies / μL positive plasmid, each template was repeated 3 times, and a negative control was set up. The results were as follows Fig.23 and Fig.24 shown.

[0117] The standard curve regression equation is y = -2.9842x + 32.633, and the correlation coefficient is R 2 Reaching 0.9757.

[0118] (3) Sensitivity test

[0119] The amplification system and procedure were the same as those for triple fluorescence quantitative PCR standard curve establishment, and the template was 1×10 8 copies / μL~1×10 1 copies / μL standard positive plasmid. The results are shown in the attached Fig.25 The minimum detection concentration of the bovine coronavirus S gene, E gene and M gene triple fluorescence quantitative PCR is 1×10 1 copies / μL.

[0120] (4) Specificity test

[0121] The amplification system and procedure were the same as those for the triple fluorescence quantitative PCR standard curve establishment, and the template was the same as that for the single fluorescence quantitative PCR specificity test. The results were as follows Fig.26 Only the positive plasmids of S gene, E gene and M gene showed amplification curves.

[0122] (5) Repeatability test

[0123] The amplification system and procedure were the same as those for triple fluorescence quantitative PCR standard curve establishment, 1×10 6 copies / μL-1×104 copies / μL positive plasmid as template, the results are as follows Fig. 27 As shown, the Ct values ​​of the same template of S gene, E gene and M gene are basically the same, and the amplification curves are also roughly the same; the coefficient of variation of the repeated in the group is less than 2%, indicating that the test has high repeatability and good stability.

[0124] Example 5

[0125] Primer combination application

[0126] The nucleic acid samples extracted from 30 fecal samples were tested using the primer combination in Table 1 and the triple fluorescent quantitative PCR detection method for bovine coronavirus S gene, E gene and M gene established in Example 3. The results showed that 10 S genes, 8 E genes and 8 M genes were detected in 20 fecal samples, and 6 were triple positive.

[0127] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0128] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer set for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, characterized in that: The primer set and nucleotide sequence are as follows: SF: 5'-CCTTCCATCTCCACTGACAC-3', SEQ ID NO.1; SR: 5'-GTACCCTTCAGAGCCATGTT-3', SEQ ID NO.2; EF: 5'-GGTTGAACACGTAGATGGAA-3', SEQ ID NO.3; ER: 5'-ATCATCGTGTGTGGTGGCTTT-3', SEQ ID NO.4; MF: 5'-ACACTGGACCGCTGACGAA-3', SEQ ID NO.5; MR: 5'-ACGAACATGGAGCGGGAGGT-3', SEQ ID NO.

6.

2. A kit for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, characterized in that: Comprising the primer set of claim 1.

3. Use of the primer set described in claim 1 or the kit described in claim 2 in the detection of bovine coronavirus spike protein, envelope protein and transmembrane protein.

4. A qPCR method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus, characterized in that: Detection using the primer set of claim 1 or the kit of claim 2 comprises the following steps: extracting DNA of the sample to be tested as a template, and detecting with the primer set of claim 1 or the kit of claim 2, if a 166 bp band appears in the amplified product, it means that the bovine coronavirus spike protein test is positive; if a 150 bp band appears, it means that the bovine coronavirus envelope protein test is positive; if a 180 bp band appears, it means that the bovine coronavirus transmembrane protein test is positive; The methods are not intended for disease detection and / or treatment.

5. The qPCR method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus according to claim 4, characterized in that: The detection system of the qPCR amplification is as follows: 2×ChamQ Universal SYBR qPCR MasterMix 10μL; SF 0.5 μL; SR 0.5 μL; EF 0.5 μL; ER 0.5 μL; MF 0.5 μL; MR 0.5 μL; DNA template 2 μL; RNase-free water 5μL; The concentrations of the primers SF, SR, EF, ER, MF and MR are all 500 nmol / L.

6. The qPCR method for simultaneously detecting spike protein, envelope protein and transmembrane protein of bovine coronavirus according to claim 4, characterized in that: The detection procedure of the qPCR amplification is as follows: pre-denaturation at 95°C for 30s; denaturation at 95°C for 20s, annealing and extension at 54.5°C for 30s, and 40 cycles.

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