A multiple real-time fluorescent quantitative kit for detecting bovine diarrhea associated virus

Abstract

The application discloses a multi-link real-time fluorescent quantitative kit for detecting bovine diarrhea associated viruses. The application provides a primer composition for detecting bovine diarrhea associated viruses and a probe composition for detecting bovine diarrhea associated viruses, and further develops a multi-link real-time fluorescent quantitative PCR kit capable of simultaneously detecting six bovine diarrhea associated viruses. The kit provided by the application can be used to simultaneously detect six viruses of BToV, BEV, BNoV, BCoV, BRV and BVDV by using two reaction systems of the same reaction procedure, and has the advantages of simple operation, short time, high specificity, high sensitivity, good repeatability and the like, and can rapidly and accurately realize quantitative detection. The kit has important significance and wide application prospect in the monitoring of calf diarrhea epidemic diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection, and more specifically, to a multi-unit real-time fluorescence quantitative reagent kit for detecting bovine diarrhea-associated viruses. Background Technology

[0002] Calf diarrhea is a significant disease affecting the global cattle industry. Pathogens causing calf diarrhea include parasites, bacteria, and viruses.

[0003] Torovirus (ToV) is a single-stranded positive-sense RNA virus belonging to the genus Torovirus, subfamily Torovirinae, family Tobaniviridae, order Nidovirales. As a pathogen affecting the digestive tract, it has been detected in multiple species, including cattle, pigs, horses, goats, and humans. Bovine torovirus (BToV) was first discovered in 1982 during an outbreak of diarrhea in the United States. Studies have shown widespread infection in calves with diarrhea, particularly those under three weeks of age. Currently, BToV has been detected in 17 countries worldwide, and its harmful effects are receiving increasing attention.

[0004] Bovine enterovirus (BEV) is a non-enveloped, single-stranded, positive-sense RNA virus belonging to the Enterovirus genus. Initially, the virus was thought to be non-pathogenic, but current research shows that while most infected cattle exhibit subclinical symptoms, several strains have been isolated that cause severe clinical symptoms, including diarrhea, bloody stools, respiratory symptoms, reduced milk production, and reproductive disorders, resulting in significant economic losses.

[0005] Bovine norovirus (BNoV) belongs to the genus Norovirus in the family Caliciviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus. Norovirus is an important pathogen causing diarrhea, leading to severe diarrhea in multiple species, including pigs, cattle, sheep, dogs, cats, rodents, and humans. Bovine norovirus was first reported in the UK in 1976, and has since been reported in many other countries worldwide, including the US, Germany, Italy, South Korea, and China. Regression studies have confirmed that bovine norovirus infection causes severe diarrheal symptoms and significant intestinal lesions. The virus primarily causes diarrhea in calves, and BNoV can be detected in the feces of adult cows, young cattle, and asymptomatic calves.

[0006] Bovine coronavirus (BCoV) belongs to the order Nidoviraes, family Coronaviridae, and genus β-coronavirus. It is a single-stranded positive-sense RNA virus. It can cause diarrhea in newborn calves, winter dysentery in adult cattle, and respiratory diseases. Bovine coronavirus was first discovered and isolated in 1973, and has since been detected globally.

[0007] Bovine rotavirus (BRV) is a member of the genus Rotavirus in the family Reoviridae. Rotaviruses comprise seven distinct groups (AG). Diarrhea in calves is primarily caused by group A rotavirus, with calves under 7 days old being most susceptible. It mainly affects calves aged 15–45 days, causing symptoms such as diarrhea, dehydration, and lethargy. This results in high infection and mortality rates.

[0008] Bovine viral diarrhea virus (BVDV) is a single-stranded, positive-sense, enveloped RNA virus belonging to the genus *Phosphovirus* in the family Flaviviridae. It is a significant infectious disease affecting the cattle industry, causing immune disorders and hemorrhagic infections in cattle, leading to severe symptoms such as erosion and necrosis of the digestive tract mucosa, gastroenteritis, and diarrhea. In pregnant cows, BVDV infection can cause reproductive disorders, including decreased milk production, abortion, slow fetal growth, and fetal malformations. Cattle of all ages can be infected with BVDV, but calves are most susceptible. BVDV was first discovered and isolated in the United States in 1946 and has since been reported in many countries worldwide.

[0009] Currently, there are no commercially available vaccines in China for BToV, BEV, BNoV, BCoV, and BRV, making disease control quite challenging. Control primarily relies on biosecurity measures, including monitoring cattle herds, culling infected cattle, and purging the herd. While a commercial vaccine exists for BVDV, most cattle farms still opt for culling infected cattle and purging the herd. Therefore, a rapid and sensitive method for detecting these six pathogens is needed for herd monitoring. However, there are currently no reports of kits or methods using multi-unit real-time quantitative PCR to simultaneously detect BToV, BEV, BNoV, BCoV, BRV, and BVDV. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multi-linked real-time quantitative PCR primer, probe and kit for detecting six bovine viruses.

[0011] The first objective of this invention is to provide a primer composition for detecting bovine diarrhea-associated viruses.

[0012] A second objective of this invention is to provide a probe composition for detecting bovine diarrhea-associated viruses.

[0013] The third objective of this invention is to provide a multi-linked real-time quantitative PCR detection system for detecting bovine diarrhea-associated viruses.

[0014] The fourth objective of this invention is to provide a multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated viruses.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0016] A primer composition for detecting bovine diarrhea-associated virus, the primer composition comprising primer set 1 and primer set 2,

[0017] Primer set 1 contains primers with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8;

[0018] Primer set 2 contains primers with nucleotide sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17;

[0019] The bovine diarrhea-associated viruses are one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

[0020] A probe composition for detecting bovine diarrhea-associated viruses, the probe composition comprising probe group 1 and probe group 2, wherein the nucleotide sequences of probe group 1 are shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9, and the nucleotide sequences of probe group 2 are shown in SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18, and the bovine diarrhea-associated virus is one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus and bovine viral diarrhea virus.

[0021] Preferably, each probe 3' end in probe group 1 is provided with an MGB, and the fluorescent groups labeled on the 5' ends of each probe in probe group 1 are different.

[0022] More preferably, the fluorescent groups labeled at the 5' ends of each probe in the probe group 1 are CY5, VIC and FAM, respectively.

[0023] Preferably, each probe in probe group 1 has an MGB at its 3' end and a different fluorescent group at its 5' end.

[0024] More preferably, the fluorescent groups labeled at the 5' ends of each probe in the probe group 2 are CY5, VIC and FAM, respectively.

[0025] The application of the primer composition and / or probe composition in the preparation of a kit for detecting bovine diarrhea-associated viruses should also be within the scope of protection of this invention. The bovine diarrhea-associated viruses are one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

[0026] A multi-linked real-time quantitative PCR detection system for detecting bovine diarrhea-related viruses, comprising reaction system 1 and reaction system 2, wherein reaction system 1 contains primer set 1 and reaction system 2 contains primer set 2.

[0027] Preferably, the reaction system 1 further includes the probe group 1.

[0028] More preferably, a single reaction in the reaction system 1 comprises 1.2–4.8 μL of the primer set 1, 0.3–1.2 μL of the probe set 1, 2 μL of the reaction template to be tested, 10 μL of 2×qPCR Probe Master Mix, and 2–6.5 μL of deionized water.

[0029] Most preferably, a single reaction in reaction system 1 comprises 2.8 μL of primer set 1, 1 μL of probe set 1, 2 μL of reaction template to be tested, 10 μL of 2×qPCR Probe Master Mix and 4.2 μL of deionized water.

[0030] More preferably, in the reaction system 1, a single reaction comprises

[0031] BToV-F with the nucleotide sequence of SEQ NO.1: 0.4 μL (10 μM);

[0032] BToV-R with nucleotide sequence SEQ NO.2: 0.4 μL (10 μM);

[0033] BToV-MGB with nucleotide sequence SEQ NO.3: 0.4 μL (10 μM);

[0034] BEV-F with nucleotide sequence SEQ NO.4: 0.5 μL (10 μM);

[0035] BEV-F with nucleotide sequence SEQ NO.5: 0.5 μL (10 μM);

[0036] BEV-MGB with nucleotide sequence SEQ NO.6: 0.3 μL (10 μM);

[0037] BNoV-F with nucleotide sequence SEQ NO.7: 0.5 μL (10 μM);

[0038] BNoV-R with nucleotide sequence SEQ NO.8: 0.5 μL (10 μM);

[0039] BNoV-MGB with nucleotide sequence SEQ NO.9: 0.3 μL (10 μM);

[0040] 2 μL of the test reaction template, 10 μL of 2×qPCR Probe Master Mix, and 4.2 μL of deionized water.

[0041] Preferably, the reaction system 2 further includes the probe group 2.

[0042] More preferably, a single reaction in the reaction system 2 comprises 1.2–4.8 μL of the primer set 2, 0.3–1.2 μL of the probe set 2, 2 μL of the reaction template to be tested, 10 μL of 2×qPCR Probe Master Mix, and 2–6.5 μL of deionized water.

[0043] Most preferably, a single reaction in reaction system 2 comprises 2.6 μL of primer set 2, 0.8 μL of probe set 2, 2 μL of reaction template to be tested, 10 μL of 2×qPCR Probe Master Mix and 4.6 μL of deionized water.

[0044] More preferably, in the reaction system 2, a single reaction comprises

[0045] BCoV-F with the nucleotide sequence SEQ NO.10: 0.3 μL (10 μM);

[0046] BCoV-R with nucleotide sequence SEQ NO.11: 0.3 μL (10 μM);

[0047] BCoV-MGB with nucleotide sequence SEQ NO.12: 0.3 μL (10 μM);

[0048] BRV-F with nucleotide sequence SEQ NO.13: 0.4 μL (10 μM);

[0049] BRV-F with nucleotide sequence SEQ NO.14: 0.4 μL (10 μM);

[0050] BRV-MGB with nucleotide sequence SEQ NO.15: 0.3 μL (10 μM);

[0051] BVDV-F with nucleotide sequence SEQ NO.16: 0.6 μL (10 μM);

[0052] BVDV-R with nucleotide sequence SEQ NO.17: 0.6 μL (10 μM);

[0053] BVDV-MGB with the nucleotide sequence SEQ NO.18: 0.2 μL (10 μM)

[0054] The test reaction template consisted of 2 μL, 2×qPCR Probe Master Mix 10 μL, and deionized water 4.6 μL.

[0055] The application of the aforementioned multi-linked real-time quantitative PCR detection system in the preparation of a kit for detecting bovine diarrhea-related viruses should also be within the scope of protection of this invention. The bovine diarrhea-related viruses are one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

[0056] A multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated viruses, wherein the bovine diarrhea-associated viruses are one or more selected from bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

[0057] The multi-unit real-time quantitative PCR kit includes:

[0058] Component A: Primer set 1 in the primer composition;

[0059] Component B: Primer set 2 in the primer composition.

[0060] Preferably, component A further contains probe group 1 from the probe composition.

[0061] More preferably, the amount of component A used in a single reaction is as follows:

[0062] BToV-F with the nucleotide sequence SEQ NO.1: 0.4 μL (10 μM);

[0063] BToV-R with nucleotide sequence SEQ NO.2: 0.4 μL (10 μM);

[0064] BToV-MGB with nucleotide sequence SEQ NO.3: 0.4 μL (10 μM);

[0065] BEV-F with nucleotide sequence SEQ NO.4: 0.5 μL (10 μM);

[0066] BEV-F with nucleotide sequence SEQ NO.5: 0.5 μL (10 μM);

[0067] BEV-MGB with nucleotide sequence SEQ NO.6: 0.3 μL (10 μM);

[0068] BNoV-F with nucleotide sequence SEQ NO.7: 0.5 μL (10 μM);

[0069] BNoV-R with nucleotide sequence SEQ NO.8: 0.5 μL (10 μM);

[0070] BNoV-MGB with nucleotide sequence SEQ NO.9: 0.3 μL (10 μM), total 3.8 μL.

[0071] Preferably, component B further contains probe group 2 from the probe composition.

[0072] More preferably, the amount of component B used in a single reaction is as follows:

[0073] BCoV-F with the nucleotide sequence SEQ NO.10: 0.3 μL (10 μM);

[0074] BCoV-R with nucleotide sequence SEQ NO.11: 0.3 μL (10 μM);

[0075] BCoV-MGB with nucleotide sequence SEQ NO.12: 0.3 μL (10 μM);

[0076] BRV-F with nucleotide sequence SEQ NO.13: 0.4 μL (10 μM);

[0077] BRV-F with nucleotide sequence SEQ NO.14: 0.4 μL (10 μM);

[0078] BRV-MGB with nucleotide sequence SEQ NO.15: 0.3 μL (10 μM);

[0079] BVDV-F with nucleotide sequence SEQ NO.16: 0.6 μL (10 μM);

[0080] BVDV-R with nucleotide sequence SEQ NO.17: 0.6 μL (10 μM);

[0081] BVDV-MGB with nucleotide sequence SEQ NO.18: 0.2 μL (10 μM), total 3.4 μL.

[0082] Preferably, the multi-unit real-time quantitative PCR kit further contains

[0083] Component C: Real-time PCR reagent;

[0084] Component D: Positive control 1: Three recombinant positive plasmid DNAs linked with bovine circovirus M gene, bovine enterovirus 5'UTR gene and bovine norovirus Rdrp gene;

[0085] Component E: Positive control 2: Three recombinant positive plasmid DNAs linked with the bovine coronavirus N gene, bovine rotavirus NSP5 gene, and bovine viral diarrhea virus 5'UTR gene.

[0086] More preferably, component C is 2×qPCR Probe Master Mix.

[0087] More preferably, the multi-unit real-time quantitative PCR kit also contains component F: deionized water.

[0088] The method of using the multi-unit real-time quantitative PCR kit for purposes other than disease diagnosis and / or treatment includes the following steps:

[0089] S1. Extract RNA from fecal or anal swab samples of diarrheal calves, and then reverse transcribe it into cDNA;

[0090] S2. Using the cDNA prepared in step S1 as the template for the test reaction, prepare the reaction system using the aforementioned multi-link real-time quantitative PCR kit;

[0091] S3. Real-time quantitative PCR amplification was performed using the reaction system prepared in step S3. The amplification program was as follows: 95℃ for 5 min; 95℃ for 10 s; 57~62℃ for 30 s, 45 cycles, with fluorescence signal acquisition at 60℃.

[0092] S4. Determine the results based on the number of amplification reaction cycles in step S3: Reactions with fewer than 35 reaction cycles and a good amplification curve are judged as positive for virus infection; reactions with 35 to 40 reaction cycles and an amplification curve are judged as suspicious and need to be tested again; reactions with no reaction cycles and no amplification curve are judged as negative for virus infection.

[0093] Preferably, the reaction system in step S2 is divided into reaction system 1, reaction system 2, positive control reaction system 1, and positive control reaction system 2;

[0094] Reaction system 1 contains 3.8 μL of component A, 2 μL of the reaction template to be tested, 10 μL of component C, and 4.2 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction.

[0095] Reaction system 2 contains 3.4 μL of component B, 2 μL of the reaction template to be tested, 10 μL of component C, and 4.6 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction.

[0096] The positive control reaction system 1 contains 3.8 μL of component A, 2 μL of component D, 10 μL of component C and 4.2 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction;

[0097] The positive control reaction system 2 contained 3.4 μL of component B, 2 μL of component E, 10 μL of component C, and 4.6 μL of deionized water, totaling 20 μL, which was the amount used for a single reaction.

[0098] Compared with the prior art, the present invention has the following beneficial effects:

[0099] This invention designs six pairs of specific primers and six specific probes based on conserved regions of the BNoV Rdrp gene, BEV 5'UTR gene, BToV M gene, BCoV N gene, BRV NSP5 gene, and BVDV 5'UTR gene. The specific probes are TaqMan minor groove binder (MGB) probes. TaqMan MGB probes are dual-labeled probes: a fluorescent reporter group is labeled at the 5' end, and a non-fluorescent quencher group (NFQ) is labeled at the 3' end. The 3' end also contains a minor groove binder (MGB). The MGB increases the probe's annealing temperature (Tm), making the probe binding to the template more stable. Furthermore, the TaqMan MGB probe is significantly shorter than conventional probes, thus improving the flexibility of sequence identification and making it applicable to a wider range of detection templates. Because the TaqMan MGB probe quenches the signal generated by the fluorescent reporter group at the other end through the NFQ group, the background signal is lower than that of other conventional probes, improving detection sensitivity and accuracy.

[0100] Furthermore, the inventors designed primers by selecting conserved regions of the BToV, BEV, BNoV, BCoV, BRV, and BVDV sequences, and selected MGB probes to further improve their specificity, which helps to ensure the accuracy and reliability of the detection results. The six pairs of specific primers and six specific probes claimed in this invention all have high sensitivity, and the detection limits against BToV, BEV, BNoV, BCoV, BRV, and BVDV positive recombinant plasmids are 1.91 copies / μL, 96.0 copies / μL, 12.8 copies / μL, 16.4 copies / μL, 18.2 copies / μL, and 65.3 copies / μL, respectively.

[0101] Using a kit containing the six pairs of specific primers and six specific probes claimed in this invention for calf disease monitoring allows for the detection of six pathogens using two reaction systems under the same reaction procedure. This is simple to operate and effectively avoids contamination caused by multiple sample additions. Moreover, the entire reaction procedure takes no more than 1 hour, which is short. There is no need for gel electrophoresis; the results can be determined simply by observing the Ct value and amplification curve, making result interpretation easy.

[0102] Therefore, it can be seen that the primers and probes for which protection is sought in this invention have high specificity and good sensitivity; the kit for which protection is sought in this invention is easy to operate, takes short time, and is easy to judge results. It can simultaneously and rapidly detect BToV, BEV, BNoV, BCoV, BRV and BVDV, and has broad application prospects in the rapid clinical detection of calf diarrhea. Attached Figure Description

[0103] Figure 1 The results are PCR electrophoresis detection results for BToV, BEV, BNoV, BCoV, BRV, and BVDV standard plasmids. Lane M is the DL2000 Marker; lane 1 is the BToV standard plasmid; lane 2 is the BEV standard plasmid; lane 3 is the BNoV standard plasmid; lane 4 is the BCoV standard plasmid; lane 5 is the BRV standard plasmid; lane 6 is the BVDV standard plasmid; and lane 7 is the negative control.

[0104] Figure 2 Figure 1 shows the alignment results of BToV, BEV, BNoV, BCoV, BRV, and BVDV plasmids. Figure 2 shows the alignment results of the BToV standard plasmid; Figure 3 shows the alignment results of the BEV standard plasmid; Figure 4 shows the alignment results of the BNoV standard plasmid; Figure 5 shows the alignment results of the BCoV standard plasmid; Figure 6 shows the alignment results of the BRV standard plasmid; and Figure 7 shows the alignment results of the BVDV standard plasmid.

[0105] Figure 3The figures show the standard curves for multiplex real-time quantitative PCR of BToV, BEV, BNoV, BCoV, BRV, and BVDV. Figure A shows the standard curve for BNoV; Figure B shows the standard curve for BEV; Figure C shows the standard curve for BToV; Figure D shows the standard curve for BRV; Figure E shows the standard curve for BCoV; and Figure F shows the standard curve for BVDV.

[0106] Figure 4 Figure 1 shows the amplification curves of multiple real-time quantitative PCR for BToV, BEV, BNoV, BCoV, BRV, and BVDV. Figure A shows the amplification curve of BNoV; Figure B shows the amplification curve of BEV; Figure C shows the amplification curve of BToV; Figure D shows the amplification curve of BRV; Figure E shows the amplification curve of BCoV; and Figure F shows the amplification curve of BVDV.

[0107] Figure 5 Figure 1 shows the sensitivity amplification curves of multiplex real-time quantitative PCR for BToV, BEV, BNoV, BCoV, BRV, and BVDV. Figure A shows the sensitivity amplification curve for BNoV; Figure B shows the sensitivity amplification curve for BEV; Figure C shows the sensitivity amplification curve for BToV; Figure D shows the sensitivity amplification curve for BRV; Figure E shows the sensitivity amplification curve for BCoV; and Figure F shows the sensitivity amplification curve for BVDV.

[0108] Figure 6 Figure 1 shows the specific amplification curves of multi-unit real-time quantitative PCR for BToV, BEV, BNoV, BCoV, BRV, and BVDV. Figure A shows the amplification curve of reaction system 1 containing BToV, BEV, and BNoV, while Figure B shows the amplification curve of reaction system 2 containing BCoV, BRV, and BVDV.

[0109] Figure 7 The figures show the sensitivity results of PCR amplification for BToV, BEV, BNoV, BCoV, BRV, and BVDV. Figure A shows the sensitivity amplification results for BNoV; Figure B shows the sensitivity amplification results for BEV; Figure C shows the sensitivity amplification results for BToV; Figure D shows the sensitivity amplification results for BRV; Figure E shows the sensitivity amplification results for BCoV; and Figure F shows the sensitivity amplification results for BVDV. Detailed Implementation

[0110] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0111] Example 1: Determination of primers and probes for BToV, BEV, BNoV, BCoV, BRV, and BVDV

[0112] I. Experimental Methods

[0113] Based on the full sequences of bovine circovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) published in Genebank, the most conserved genes were identified as BNoV Rdrp gene (gene number: MN480761.1), BEV 5'UTR gene (gene number: MK639928.1), BToV M gene (gene number: MN882587.1), BCoV N gene (gene number: ON142320.1), BRV NSP5 gene (gene number: JN831208.1), and BVDV 5'UTR gene (gene number: MN417910.1). Specific primers and MGB probes were designed using the most conserved fragments of these genes.

[0114] II. Experimental Results

[0115] The obtained primer and probe sequences for the six viruses are shown in Table 1.

[0116] Table 1 Primers and probes for BToV, BEV, BNoV, BCoV, BRV, and BVDV

[0117]

[0118]

[0119] Example 2: The Influence of Multiplex Quantitative PCR Reaction System and Procedure on Amplification Results

[0120] I. Preparation of BToV, BEV, BNoV, BCoV, BRV and BVDV positive recombinant plasmids

[0121] 1. Experimental Methods

[0122] The amplified products of the BNoV Rdrp gene, BEV 5'UTR gene, BToV M gene, BCoV N gene, BRV NSP5 gene, and BVDV 5'UTR gene were ligated into the pMD18-T vector and transformed into DH-5α competent cells. Positive single colonies were picked, cultured, and sequenced. The correctly sequenced recombinant positive plasmids were named pMD18-T-BToV, pMD18-T-BEV, pMD18-T-BNoV, pMD18-T-BCoV, pMD18-T-BRV, and pMD18-T-BVDV, respectively. The correctly sequenced recombinant positive plasmids were extracted and stored at -20℃ for subsequent experiments.

[0123] 2. Experimental Results

[0124] Electrophoresis results of positive recombinant plasmids are shown in Figure 1 Sequencing results are shown in Table 2 and... Figure 2 The results indicate that the constructed plasmids are BToV, BEV, BNoV, BCoV, BRV, and BVDV, respectively. Positive recombinant plasmids pMD18-T-BToV, pMD18-T-BEV, pMD18-T-BNoV, pMD18-T-BCoV, pMD18-T-BRV, and pMD18-T-BVDV were extracted and their concentrations were determined. The copy numbers of the standard plasmids were calculated using the formula. The copy numbers of the standard plasmids were 1.28 × 10⁻⁶. 11 Copies / μL, 9.60×10 10 Copies / μL, 1.91×10 11 Copies / μL, 1.82×10 10 Copies / μL, 1.64×10 10 Copies / μL, 6.53×10 10 Copies / μL.

[0125] Table 2 Sequencing results after culturing positive single colonies

[0126]

[0127] II. The Influence of Primer and Probe Concentration on Amplification Results

[0128] 1. Experimental Methods

[0129] Seven different concentration gradients were set for different primers: 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, and 0.4 μM. Seven different concentration gradients were set for different probes: 0.05 μM, 0.075 μM, 0.1 μM, 0.125 μM, 0.15 μM, 0.175 μM, and 0.2 μM.

[0130] Using the controlled variable method, the effect of different primer concentrations on amplification results was investigated at a probe concentration of 0.1 μM. After determining the optimal primer ratio of outer and inner primers, the effect of different probe concentrations on amplification results was investigated using the controlled variable method.

[0131] 2. Experimental Results

[0132] Based on the results in Tables 3 and 4, the concentration with the lowest Ct value was selected as the optimal primer and probe concentration, thus determining the optimal primer ratio.

[0133] The optimal primer and probe concentrations for BToV are 0.2 μM and 0.2 μM, respectively, meaning the optimal primer and probe concentration ratio for BToV is 1:1.

[0134] The optimal primer and probe concentrations for BEV are 0.25 μM and 0.15 μM, respectively, which means the optimal primer and probe concentration ratio for BEV is 5:3.

[0135] The optimal primer and probe concentrations for BNoV are 0.25 μM and 0.15 μM, respectively, which means the optimal primer and probe concentration ratio for BNoV is 5:3.

[0136] The optimal primer and probe concentrations for BCoV are 0.15 μM and 0.15 μM, respectively, meaning the optimal primer and probe concentration ratio for BCoV is 1:1.

[0137] The optimal primer and probe concentrations for BRV are 0.2 μM and 0.15 μM, respectively, which means the optimal primer and probe concentration ratio for BRV is 4:3.

[0138] The optimal primer and probe concentrations for BVDV are 0.3 μM and 0.1 μM, respectively, which means the optimal primer and probe concentration ratio for BVDV is 3:1.

[0139] Table 3. Effect of primer concentration on amplification efficiency

[0140]

[0141] Table 4. Effect of probe concentration on amplification efficiency

[0142]

[0143]

[0144] III. The Influence of Multiple Reaction System Combinations on Amplification Results

[0145] 1. Experimental Methods

[0146] BEV, BToV, and BNoV were processed in the same system, while BCoV, BRV, and BVDV were processed in another system. Both systems were amplified using the same reaction procedure. The reaction volume was 20 μL, which included 10 μL of qPCR Probe Master mix, three pairs of primers, three probes, template, and sterile double-distilled water.

[0147] Based on the influence of primer and probe concentrations on amplification results, the multi-linked real-time quantitative PCR reaction systems for BEV, BToV, and BNoV are shown in Table 5; the multi-linked real-time quantitative PCR reaction systems for BCoV, BRV, and BVDV are shown in Table 6.

[0148] Table 5. Optimal reaction systems for BEV, BToV, and BNoV real-time quantitative PCR.

[0149]

[0150] Table 6. Optimal Multi-Link Real-Time Quantitative PCR Reaction Systems for BCoV, BRV, and BVDV

[0151]

[0152]

[0153] 2. Experimental Results

[0154] Based on the emerging pathogens and the traditional common pathogens, six bovine diarrhea-related viruses were divided into two systems for amplification. The amplification efficiency was calculated according to the standard curve expression. The amplification efficiency of the six pathogens in both reaction systems was between 90% and 110%, and both systems met the minimum amplification efficiency requirements.

[0155] The optimal multiplex real-time quantitative PCR reaction systems for BEV, BToV, and BNoV are detailed in Table 5; the optimal multiplex real-time quantitative PCR reaction systems for BCoV, BRV, and BVDV are detailed in Table 6.

[0156] IV. Effect of Annealing Temperature on Amplification Efficiency

[0157] 1. Experimental Methods

[0158] use Amplification and analysis were performed using a 480Ⅱ real-time quantitative PCR instrument. The reaction program was: denaturation (95℃) for 5 min; denaturation (95℃) for 10 s; annealing for 30 s; for a total of 45 cycles. Fluorescence signals were collected at 60℃. Six annealing temperatures were set: 57℃, 58℃, 59℃, 60℃, 61℃, and 62℃.

[0159] 2. Experimental Results

[0160] Based on the results in Table 7, the annealing temperature with the smallest Ct value is selected as the optimal annealing temperature, which is 60℃.

[0161] The optimal reaction program is: 95℃ for 5 min; 95℃ for 10 s; 60℃ for 30 s; for a total of 45 cycles; fluorescence signal is collected at 60℃.

[0162] Table 7 Effect of annealing temperature on amplification efficiency

[0163]

[0164]

[0165] V. Determination of Multiplex Quantitative PCR Reaction System and Procedure

[0166] The reaction system is divided into reaction system 1 and reaction system 2. Reaction system 1 contains...

[0167] BToV-F with the nucleotide sequence SEQ NO.1: 0.4 μL (10 μM);

[0168] BToV-R with nucleotide sequence SEQ NO.2: 0.4 μL (10 μM);

[0169] BToV-MGB with nucleotide sequence SEQ NO.3: 0.4 μL (10 μM);

[0170] BEV-F with nucleotide sequence SEQ NO.4: 0.5 μL (10 μM);

[0171] BEV-F with nucleotide sequence SEQ NO.5: 0.5 μL (10 μM);

[0172] BEV-MGB with nucleotide sequence SEQ NO.6: 0.3 μL (10 μM);

[0173] BNoV-F with nucleotide sequence SEQ NO.7: 0.5 μL (10 μM);

[0174] BNoV-R with nucleotide sequence SEQ NO.8: 0.5 μL (10 μM);

[0175] BNoV-MGB with nucleotide sequence SEQ NO.9: 0.3 μL (10 μM), 10 μL of 2×Probe Master mix, 2 μL of template DNA to be tested, and 4.3 μL of ddH2O;

[0176] Reaction system 2 contains

[0177] BCoV-F with the nucleotide sequence SEQ NO.10: 0.3 μL (10 μM);

[0178] BCoV-R with nucleotide sequence SEQ NO.11: 0.3 μL (10 μM);

[0179] BCoV-MGB with nucleotide sequence SEQ NO.12: 0.3 μL (10 μM);

[0180] BRV-F with nucleotide sequence SEQ NO.13: 0.4 μL (10 μM);

[0181] BRV-F with nucleotide sequence SEQ NO.14: 0.4 μL (10 μM);

[0182] BRV-MGB with nucleotide sequence SEQ NO.15: 0.3 μL (10 μM);

[0183] BVDV-F with nucleotide sequence SEQ NO.16: 0.6 μL (10 μM);

[0184] BVDV-R with nucleotide sequence SEQ NO.17: 0.6 μL (10 μM);

[0185] BVDV-MGB with nucleotide sequence SEQ NO.18: 0.2 μL (10 μM), 10 μL of 2×Probe Master mix, 2 μL of template DNA to be tested, and 4.6 μL of ddH2O.

[0186] The reaction program was 95℃ for 5 min; 95℃ for 10 s; 60℃ for 30 s, for 45 cycles, with fluorescence signal acquisition performed at 60℃.

[0187] Example 3: Establishment of a standard curve for multi-unit real-time quantitative PCR

[0188] I. Experimental Methods

[0189] The DNA from the six recombinant positive plasmids obtained in Example 2 was extracted and serially diluted 10-fold, resulting in a plasmid DNA concentration of 10. 8 ~10 0 Copies / μL. Following the reaction system and procedure determined in Example 2, serially diluted plasmid DNA was added sequentially for amplification. A standard curve for the multiplex real-time quantitative PCR method was established by plotting the logarithm of the number of recombinant positive plasmid DNA starting templates on the X-axis and the Ct of the number of cycles in the multiplex real-time quantitative PCR on the Y-axis.

[0190] II. Experimental Results

[0191] The standard curve for multi-unit real-time quantitative PCR is shown below. Figure 3 The multi-unit real-time quantitative PCR amplification curves are shown in the figure. Figure 4 Select 10 with a good linear relationship. 8 ~10 2 A standard curve was established based on the amplification results of standard plasmid DNA diluted in copies / μL. A linear relationship expression was established based on the linear relationship between the initial template number logarithm (X-axis) and the Ct value (Y-axis). Expressions for BToV, BEV, BNoV, BCoV, BRV, and BVDV were also established, along with their correlation coefficients R. 2The amplification efficiency E is shown in Table 4. As can be seen from Table 4, the primer reaction of the multiplex real-time quantitative PCR is sensitive and reliable, and can be used to accurately quantify the amount of virus carried in bovine diarrhea samples.

[0192] Table 8. Expressions and correlation coefficients R for BToV, BEV, BNoV, BCoV, BRV, and BVDV. 2 and amplification efficiency E

[0193] Bovine virus name expression <![CDATA[Expression correlation coefficient R 2 > Amplification efficiency E BNoV Y = -3.555X + 38.54 0.999 91.11％ BEV Y = -3.264X + 42.79 0.991 102.48％ BToV Y = -3.307X + 42.01 0.987 100.63％ BRV Y = -3.422X + 42.64 0.996 95.99％ BCoV Y = -3.335X + 42.44 0.998 99.46％ BVDV Y = -3.120X + 42.81 0.993 109.18％

[0194] Example 4: A multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated viruses

[0195] I. Composition

[0196] A multi-unit real-time quantitative PCR kit for simultaneous detection of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus, comprising the following components:

[0197] Component A: 2×qPCR Probe Master Mix;

[0198] Component B: Deionized water;

[0199] Component C: The composition of the amount used in a single reaction is as follows:

[0200] BToV-F with the nucleotide sequence SEQ NO.1: 0.4 μL (10 μM);

[0201] BToV-R with nucleotide sequence SEQ NO.2: 0.4 μL (10 μM);

[0202] BToV-MGB with nucleotide sequence SEQ NO.3: 0.4 μL (10 μM);

[0203] BEV-F with nucleotide sequence SEQ NO.4: 0.5 μL (10 μM);

[0204] BEV-F with nucleotide sequence SEQ NO.5: 0.5 μL (10 μM);

[0205] BEV-MGB with nucleotide sequence SEQ NO.6: 0.3 μL (10 μM);

[0206] BNoV-F with nucleotide sequence SEQ NO.7: 0.5 μL (10 μM);

[0207] BNoV-R with nucleotide sequence SEQ NO.8: 0.5 μL (10 μM);

[0208] BNoV-MGB with nucleotide sequence SEQ NO.9: 0.3 μL (10 μM), total 3.8 μL;

[0209] Component D: The composition of component D in a single reaction is as follows:

[0210] BCoV-F with the nucleotide sequence SEQ NO.10: 0.3 μL (10 μM);

[0211] BCoV-R with nucleotide sequence SEQ NO.11: 0.3 μL (10 μM);

[0212] BCoV-MGB with nucleotide sequence SEQ NO.12: 0.3 μL (10 μM);

[0213] BRV-F with nucleotide sequence SEQ NO.13: 0.4 μL (10 μM);

[0214] BRV-F with nucleotide sequence SEQ NO.14: 0.4 μL (10 μM);

[0215] BRV-MGB with nucleotide sequence SEQ NO.15: 0.3 μL (10 μM);

[0216] BVDV-F with nucleotide sequence SEQ NO.16: 0.6 μL (10 μM);

[0217] BVDV-R with nucleotide sequence SEQ NO.17: 0.6 μL (10 μM);

[0218] BVDV-MGB with nucleotide sequence SEQ NO.18: 0.2 μL (10 μM), total 3.4 μL;

[0219] Component E: Positive control tube 1 containing positive recombinant plasmid DNA from bovine circovirus, bovine enterovirus, and bovine norovirus;

[0220] Component F: Positive control tube 2 containing positive recombinant plasmid DNA of bovine coronavirus, bovine rotavirus and bovine viral diarrhea virus.

[0221] II. Experimental Methods

[0222] (1) Extract RNA from fecal or anal swab samples of diarrheal calves and then reverse transcribe it into cDNA;

[0223] (2) Using the cDNA prepared in step (1) as the template for the test reaction, prepare the reaction system. The reaction system is divided into reaction system 1, reaction system 2, positive control reaction system 1 and positive control reaction system 2.

[0224] Reaction system 1 contains 3.8 μL of component C, 2 μL of the reaction template to be tested, 10 μL of component A, and 4.2 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction.

[0225] Reaction system 2 contains 3.4 μL of component D, 2 μL of the reaction template to be tested, 10 μL of component A, and 4.6 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction.

[0226] The positive control reaction system 1 contains 3.8 μL of component C, 2 μL of component E, 10 μL of component A and 4.2 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction.

[0227] The positive control reaction system 2 contains 3.4 μL of component D, 2 μL of component F, 10 μL of component A and 4.6 μL of deionized water, totaling 20 μL, which is the amount used for a single reaction;

[0228] (3) Use the reaction system prepared in step (2) to perform real-time fluorescence quantitative PCR amplification. The amplification program is: 95℃ for 5 min; 95℃ for 10 s; 60℃ for 30 s, 45 cycles, and fluorescence signal acquisition is performed at 60℃.

[0229] (4) The results are determined according to the number of cycles of the multi-link real-time fluorescence quantitative PCR amplification reaction in step (3): the reaction with a reaction cycle number of less than 35 and a good amplification curve is judged as positive for virus infection; the reaction with a reaction cycle number of 35 to 40 and an amplification curve is judged as suspicious and needs to be tested again; the reaction with no reaction cycle number and no amplification curve is judged as negative for virus infection.

[0230] Example 5: Sensitivity of a multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated viruses

[0231] I. Experimental Methods

[0232] The DNA from the six recombinant positive plasmids obtained in Example 2 was extracted and serially diluted 10-fold, resulting in a plasmid DNA concentration of 10. 5 ~10 0 Copies / μL. The serially diluted plasmid DNA was amplified sequentially using the kit from Example 3, with double-distilled water as a negative control, for sensitivity testing. The minimum detectable copy number was determined using Ct < 40.

[0233] II. Experimental Results

[0234] Sensitive amplification curves are shown below. Figure 5 , with 10 5 ~10 0Six recombinant positive plasmid DNAs (copies / μL) were added as templates to the corresponding reaction systems for sensitivity testing. The limits of detection (LOD) were 1.91 copies / μL for BNoV, 96.0 copies / μL for BEV, 12.8 copies / μL for BToV, 16.4 copies / μL for BRV, 18.2 copies / μL for BCoV, and 65.3 copies / μL for BVDV. No amplification was observed in the negative controls of all primer sets, indicating that this multi-combination real-time quantitative PCR kit for detecting bovine diarrhea-related viruses has high sensitivity.

[0235] Example 6: Repeatability and stability of a multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated virus.

[0236] I. Experimental Methods

[0237] DNA from the six recombinant positive plasmids obtained in Example 2 was extracted and serially diluted 10-fold. 7 Copies / μL, 10 6 Copies / μL, 10 5 Using Copies / μL plasmid DNA as a template, three intra-batch and inter-batch experiments were performed using the kit in Example 3. The mean Ct value, standard deviation, and coefficient of variation were calculated to analyze the repeatability and stability of the detection method.

[0238] II. Experimental Results

[0239] Three intra-batch and three inter-batch assays were performed using different concentrations and plasmids as templates. The results are shown in Table 5. Table 5 shows that the coefficients of variation (CV) for the intra-batch and inter-batch assays of BToV were 0.55%–1.37%; for BEV, 0.44%–2.38%; for BNoV, 0.25%–1.91%; for BCoV, 0.11%–2.27%; for BRV, 0.01%–0.32%; and for BVDV, 0.02%–0.20%. All CV values ​​were less than 3%, indicating that the kit for detecting bovine diarrhea-related viruses using real-time quantitative PCR has good reproducibility and stability.

[0240] CV = (SD / MN) × 100%

[0241] Table 9 Results of repeatability tests for multi-unit real-time quantitative PCR

[0242]

[0243]

[0244] Example 7: Specificity of a multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-associated viruses.

[0245] I. Experimental Methods

[0246] Using cDNA or DNA of BEV, BToV, BNoV, BCoV, BRV, and BVDV as positive controls and sterile double-distilled water as a negative control, cDNA or DNA of bovine ephemeral fever virus (BEFV), bovine nodular dermatitis virus (LSDV), and bovine respiratory syncytial virus (BRSV) was extracted for specificity evaluation of the multi-linked real-time quantitative PCR method. The kit described in Example 3 was used to sequentially detect the amplification of cDNA or DNA of different bovine viruses for specificity testing.

[0247] II. Experimental Results

[0248] Specific test results are shown in Figure 6 In the same reaction procedure, different cDNA and DNA were added to the two systems for amplification, such as... Figure 6 As shown, only BToV, BEV, and BNoV showed fluorescence signals in reaction system 1, and the amplification curves were good. Only BCoV, BRV, and BVDV showed fluorescence signals in reaction system 2. The wells containing interfering DNA and the negative control showed no fluorescence signal or amplification curve. These results indicate that the kit for detecting bovine diarrhea-related viruses using real-time quantitative PCR has good specificity, and no non-specific amplification was observed.

[0249] Example 8: Sensitivity Comparison of a Multi-Unit Real-Time Quantitative PCR Kit for Detecting Bovine Diarrhea-Associated Virus and PCR Method

[0250] I. Experimental Methods

[0251] The BToV, BEV, BNoV, BCoV, BRV, and BVDV positive recombinant plasmid DNAs prepared in Example 2 were extracted and serially diluted 10-fold to obtain a plasmid DNA concentration of 10. 8 ~10 1Copies / μL. The serially diluted plasmid DNA was amplified using primer sequences from SEQ NO.1, SEQ NO.2; SEQ NO.4, SEQ NO.5; SEQ NO.7, SEQ NO.8; SEQ NO.10, SEQ NO.11; SEQ NO.13, SEQ NO.14; SEQ NO.16, and SEQ NO.17, respectively. Double-distilled water was used as a negative control. The amplification system is shown in Table 10. The sensitivity was compared with that of the multi-unit real-time quantitative PCR kit described in Example 4.

[0252] Table 10 PCR amplification system

[0253]

[0254] II. Experimental Results

[0255] The results of PCR sensitivity tests for BToV, BEV, BNoV, BCoV, BRV, and BVDV positive recombinant plasmid DNA are shown in the figure. Figure 7 The detection limits of PCR for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 × 10⁻⁶. 4 Copies / μL, 9.6×10 4 Copies / μL, 1.28×10 4 Copies / μL, 1.64×10 5 Copies / μL, 1.82×10 4 Copies / μL and 6.53×10 5 Copies / μL.

[0256] A comparison of the sensitivity results of the multi-unit real-time quantitative PCR kit for detecting bovine diarrhea-related viruses obtained in Example 5 shows that the sensitivity of the multi-unit real-time quantitative PCR kit claimed in this invention is 1,000 to 10,000 times higher than that of PCR.

[0257] Example 9: Clinical Sample Detection Results

[0258] I. Experimental Methods

[0259] A total of 295 samples were collected from Guangdong Province, China. The samples included feces and anal swabs from diarrheal calves, used as clinical samples. RNA was extracted from the samples using the RNAfast200 Total RNA Rapid Extraction Kit, and then reverse-engineered into cDNA using the HiScript III All-in-one RT SuperMix for subsequent experiments. The reverse-engineered cDNA was sequentially detected using the kit described in Example 3. Each cDNA sample was divided into two portions: one portion was detected using the multi-unit real-time quantitative PCR kit described in Example 4; the other portion was detected using the detection method established in the reference, with each sample tested in triplicate. The results obtained using the multi-unit real-time quantitative PCR kit described in Example 4 were analyzed and compared with the results of the published detection method to determine the positive and negative concordance rates.

[0260] The primer sequences and sources disclosed in the references are shown in Table 13.

[0261] II. Experimental Results

[0262] The suitability of a multi-linked real-time quantitative PCR kit for detecting bovine diarrhea-related viruses was evaluated by testing 295 calf diarrhea samples. The results of multi-linked real-time quantitative PCR versus PCR are shown in Table 6. From the 295 diarrhea samples, 1 sample was positive for BToV, 18 for BEV, 2 for BNoV, 4 for BCoV, 32 for BRV, and 6 for BVDV. The positive rates were 0.34% for BToV, 6.44% for BEV, 0.68% for BNoV, 1.36% for BCoV, 10.85% for BRV, and 2.03% for BVDV. BRV and BEV showed the highest positive rates among the tested samples. This is the first time BToV and BNoV have been detected in Guangdong Province, indicating that bovine norovirus and bovine circovirus are prevalent in Guangdong.

[0263] As shown in Table 12, compared with the PCR detection results, the kit described in Example 3 showed a higher positive detection rate. The negative controls for each batch of tests were valid. Sequencing of the detected positive samples revealed positive results for viral infection, consistent with the detection results of the kit described in Example 3. Therefore, the kit described in Example 3 demonstrates higher sensitivity and more accurate detection results in practical applications.

[0264] Table 11 Results of multi-sample real-time quantitative PCR.

[0265]

[0266] Table 12 Comparison results of multi-link real-time quantitative PCR and PCR

[0267]

[0268] Table 13 lists the sequences and sources of the primers used.

[0269]

[0270] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a composition in the preparation of a kit for detecting bovine diarrhea-associated viruses, characterized in that, The composition comprises primer set 1, probe set 1, primer set 2, and probe set 2, wherein primer set 1 contains primers with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8; The nucleotide sequences of probe group 1 are shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9; Primer set 2 contains primers with nucleotide sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; The nucleotide sequences of probe group 2 are shown in SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18; The bovine diarrhea-associated viruses are one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

2. The application of a multi-unit real-time quantitative PCR detection system in the preparation of a kit for detecting bovine diarrhea-related viruses, characterized in that, The multi-linked real-time quantitative PCR detection system includes reaction system 1 and reaction system 2. Reaction system 1 includes primer set 1 and probe set 1, and reaction system 2 includes primer set 2 and probe set 2. Primer set 1 contains primers with nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8; The nucleotide sequences of probe group 1 are shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9; Primer set 2 contains primers with nucleotide sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 17; The nucleotide sequences of probe group 2 are shown in SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18; The bovine diarrhea-associated viruses are one or more of bovine circovirus, bovine enterovirus, bovine norovirus, bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus.

3. The application according to claim 2, characterized in that, The reaction system 1 and reaction system 2 also contain real-time PCR reagents; The reaction system 1 also includes positive control 1: three recombinant positive plasmid DNAs linked with bovine circovirus M gene, bovine enterovirus 5'UTR gene and bovine norovirus Rdrp gene; The reaction system 2 also includes positive control 2: three recombinant positive plasmid DNAs linked with the bovine coronavirus N gene, the bovine rotavirus NSP5 gene, and the bovine viral diarrhea virus 5'UTR gene.

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