Primer probe combination for cattle mixed infection pathogen identification and application and kit thereof

Abstract

The invention relates to the technical field of virus detection, in particular to a primer probe combination for cattle mixed infection pathogen identification, application of the primer probe combination and a kit. When cattle pathogens are identified, the nucleotide sequence of an upstream primer of a used primer probe group is as shown in SEQ ID No.1, the nucleotide sequence of a downstream primer is as shown in SEQ ID No.2, and the nucleotide sequence of the probe is as shown in SEQ ID No.3. The bovine ephemeral fever virus, the bovine herpes virus type 4, the bovine rotavirus and the clostridium welchii can be detected and identified through four groups of primer probes. The primer probe combination provided by the invention can be used for identifying and diagnosing four common cattle pathogens through a tube reaction system by a fluorescent quantitative PCR method, can also be used for identifying a mixed infected sample, and has the advantages of accurate identification, high sensitivity, simplicity in operation, short time consumption and the like; and an effective technical means can be provided for cattle infectious disease pathogen diagnosis and disease prevention in China.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, in particular to a primer-probe combination for identifying mixed infection pathogens in cattle, an application thereof and a kit. Background Art

[0002] Cattle, as important economic animals, play a crucial role in global agriculture and animal husbandry. However, the occurrence and spread of infectious diseases in cattle cause serious economic losses to the livestock industry and pose a potential threat to public health and safety. Among the many infectious diseases in cattle, bovine ephemeral fever virus (BEFV), bovine herpesvirus 4 (BoHV4), bovine rotavirus (BRV), and Clostridium perfringens (CP) are four common pathogens. Infection with these pathogens can lead to different types of diseases, including respiratory diseases, gastrointestinal diseases, and systemic infections.

[0003] BEFV is an RNA virus that mainly causes bovine epidemic fever (also known as three-day fever). The disease is characterized by high fever, muscle pain, joint stiffness and loss of appetite, which can lead to a significant decline in cattle production performance. BoHV4 is a DNA virus that is associated with a variety of cattle diseases, including reproductive system diseases, mastitis and immunosuppression. Its infection is often latent and easily mixed with other pathogens, leading to complicated conditions. BRV is a double-stranded RNA virus belonging to the Reoviridae family. It is one of the main pathogens of calf diarrhea. Infection with this virus often leads to severe dehydration, electrolyte imbalance and even death, especially in young cattle with a high incidence and mortality rate. CP is a Gram-positive anaerobic bacterium that is widely present in the environment and in the intestines of cattle. Certain virulence factors (such as toxin A and toxin B) can cause acute enterotoxemia or necrotizing enteritis, which is common in intensive farms and causes large-scale deaths.

[0004] Co-infection with BRV and CP often leads to severe diarrhea and even death in calves, and the synergistic effect of BEFV and BoHV4 may aggravate respiratory symptoms. The risk of BEFV and BoHV4 transmission increases during the peak mosquito activity season in summer and autumn, while BRV and CP are more likely to break out under closed breeding conditions in winter. Because the above-mentioned mixed infection phenomenon is relatively common in cattle herds, whether it is vaccination, drug treatment or environmental management, the prevention and control measures for these four pathogens emphasize early detection, early isolation and early intervention. Therefore, it is particularly important to develop a technology that can detect the above pathogens simultaneously.

[0005] Fluorescence quantitative PCR (qPCR) has been widely used in pathogen detection due to its high sensitivity, high specificity, and rapidity. It can simultaneously detect up to four targets in a single reaction tube. Therefore, establishing a multiplex qPCR method for the simultaneous detection of these four important bovine pathogens would not only fill a gap in the detection of common bovine pathogens, shorten diagnostic cycles, and improve prevention and control efficiency, but also provide a new technical means for the precise prevention and control of bovine infectious diseases in my country and offer strong technical support for large-scale livestock farms. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a primer-probe combination for identifying mixed infection pathogens of cattle, its application and kit. The present invention designs a primer-probe group that can simultaneously identify four common pathogens of cattle, mixes the designed primer-probe groups to form a primer pool, and provides a multiplex real-time fluorescence quantitative PCR kit for identifying four common pathogens of cattle.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a primer-probe combination for identifying mixed infection pathogens in cattle, wherein the mixed infection pathogens in cattle include bovine typhus virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

[0009] When identifying the bovine fever virus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the probe is shown in SEQ ID No. 3;

[0010] When identifying the bovine herpesvirus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the probe is shown in SEQ ID No. 6;

[0011] When identifying the bovine rotavirus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 7, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 8, and the nucleotide sequence of the probe is shown in SEQ ID No. 9;

[0012] When identifying the Clostridium perfringens, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 10, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 11, and the nucleotide sequence of the probe is shown in SEQ ID No. 12.

[0013] Preferably, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0014] Preferably, the 5' end of the probe used to identify the bovine fever virus is modified with a ROX group and the 3' end is modified with MGB;

[0015] The 5' end of the probe used to identify the bovine herpes virus is modified with a Cy5 group and the 3' end is modified with BHQ2;

[0016] The 5' end of the probe used to identify the bovine rotavirus is modified with a FAM group and the 3' end is modified with an MGB group;

[0017] The 5' end of the probe used to identify Clostridium perfringens was modified with a VIC group, and the 3' end was modified with an MGB group.

[0018] Preferably, the detection target gene of the bovine fever virus is G;

[0019] The detection target gene of the bovine herpes virus is gB;

[0020] The detection target gene of the bovine rotavirus is VP6;

[0021] The detection target gene of Clostridium perfringens is cpa.

[0022] The present invention also provides the use of the primer-probe combination described in the above technical solution in preparing a reagent for identifying mixed pathogens of cattle infections.

[0023] Preferably, the cattle mixed infection pathogens include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

[0024] The present invention also provides a kit for identifying pathogens of mixed bovine infections, comprising a primer pool, wherein the primer pool includes primer probes for identifying bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens in the primer-probe combination described in the above technical solution.

[0025] Preferably, the concentrations of the upstream and downstream primers for identifying bovine fever virus in the primer pool are both 300 nM, and the concentration of the probe is 250 nM;

[0026] The concentrations of upstream and downstream primers for identifying bovine herpesvirus were both 100 nM, and the concentration of the probe was 250 nM;

[0027] The concentrations of upstream and downstream primers for identifying bovine rotavirus were both 300 nM, and the concentration of the probe was 250 nM;

[0028] The concentrations of the upstream and downstream primers for identifying Clostridium welchii were both 100 nM, and the concentration of the probe was 50 nM.

[0029] The present invention also provides the use of the kit described in the above technical solution in the preparation of a product for identifying mixed pathogenic infections in cattle.

[0030] Preferably, the cattle mixed infection pathogens include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

[0031] Beneficial effects of the present invention:

[0032] The present invention can detect and identify bovine effusion virus (BEFV), bovine herpesvirus type 4 (BoHV4), bovine rotavirus (BRV), and Clostridium perfringens (CP) using four sets of primer probes. Using the primer-probe combination provided by the present invention, a fluorescent quantitative PCR method can be used to differentially diagnose four common cattle pathogens using a single-tube reaction system. It can also identify mixed-infection samples. This method has the advantages of accurate identification, high sensitivity, simple operation, and short time consumption, and can provide an effective technical means for the diagnosis and prevention of bovine infectious disease pathogens in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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 are briefly introduced below.

[0034] Figure 1 Optimize the primer probe concentration for the primer pool;

[0035] Figure 2 Optimize the results for annealing temperature;

[0036] Figure 3 Standard curves drawn for the amplification of four target plasmids;

[0037] Figure 4 Amplification curves corresponding to the standard curves of the four target plasmids;

[0038] Figure 5 Figure 2 is the amplification curve for four target plasmids (8 replicates each) at a concentration of 5 copies / μl;

[0039] Figure 6 For 4 target plasmids (16 replicates each) in 10 5 Amplification curve at the concentration of copies / μl;

[0040] Figure 7 For 4 target plasmids (16 replicates each) in 10 2 Amplification curves at the concentration of copies / μl. DETAILED DESCRIPTION

[0041] The present invention provides a primer probe combination for identifying pathogens of mixed bovine infection, wherein the pathogens of mixed bovine infection include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens. When identifying the bovine typhoid virus, the nucleotide sequence of the upstream primer of the primer probe group used is shown as SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2, and the nucleotide sequence of the probe is shown as SEQ ID No. 3; when identifying the bovine herpes virus, the nucleotide sequence of the upstream primer of the primer probe group used is shown as SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown as SEQ ID No. 5, and the nucleotide sequence of the probe is shown as SEQ ID No. 6; when identifying the bovine rotavirus, the nucleotide sequence of the upstream primer of the primer probe group used is shown as SEQ ID No. 7, the nucleotide sequence of the downstream primer is shown as SEQ ID No. 8, and the nucleotide sequence of the probe is shown as SEQ ID No. 9; when identifying the Clostridium perfringens, the nucleotide sequence of the upstream primer of the primer probe group used is shown as SEQ ID No. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID No. The nucleotide sequence of the probe is shown as SEQ ID No.11, and the nucleotide sequence of the probe is shown as SEQ ID No.12.

[0042] The present invention preferably modifies the 5' end of the probe with a fluorescent group and the 3' end with a quencher group. In the present invention, the 5' end of the probe used to identify the bovine typhoid virus is preferably modified with a ROX group, and the 3' end is preferably modified with MGB; the 5' end of the probe used to identify the bovine herpes virus is preferably modified with a Cy5 group, and the 3' end is preferably modified with BHQ2; the 5' end of the probe used to identify the bovine rotavirus is preferably modified with a FAM group, and the 3' end is preferably modified with MGB; the 5' end of the probe used to identify Clostridium perfringens is preferably modified with a VIC group, and the 3' end is preferably modified with MGB.

[0043] In the present invention, the detection target gene of the bovine herpesvirus is preferably G; the detection target gene of the bovine herpesvirus is preferably gB; the detection target gene of the bovine rotavirus is preferably VP6; and the detection target gene of Clostridium perfringens is preferably cpa.

[0044] The present invention also provides the use of the primer-probe combination described in the above technical solution in preparing a reagent for identifying mixed bovine infection pathogens. In the present invention, the mixed bovine infection pathogens preferably include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

[0045] The present invention also provides a kit for identifying pathogens of mixed bovine infections, comprising a primer pool, wherein the primer pool includes primer probes for identifying bovine typhoid virus, bovine herpesvirus, bovine rotavirus, and Clostridium perfringens from the primer-probe combination described in the above technical solution. In the present invention, the concentrations of the upstream and downstream primers for identifying bovine typhoid virus in the primer pool are preferably both 300 nM, and the concentration of the probes is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying bovine herpesvirus are preferably both 100 nM, and the concentration of the probes is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying bovine rotavirus are preferably both 300 nM, and the concentration of the probes is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying Clostridium perfringens are preferably both 100 nM, and the concentration of the probes is preferably 50 nM.

[0046] The present invention also provides the use of the kit described in the above technical solution in the preparation of a product for identifying mixed bovine infection pathogens. In the present invention, the bovine pathogens preferably include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

[0047] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The following examples provide a primer probe set and a kit for identifying four common cattle pathogens, which can be used for one-step detection and identification of four common cattle pathogens: bovine effusion fever virus (BEFV), bovine herpesvirus type 4 (BoHV4), bovine rotavirus (BRV), and Clostridium perfringens (CP).

[0049] Example 1

[0050] 1. Design of specific primers and probes. Download the nucleic acid sequences of conserved genes or virulence genes from four pathogens prevalent in my country in recent years from Genbank. Perform multiple sequence alignment using sequence alignment software. Next, design specific primers and probes from regions that are conserved across strains of the pathogen but specific to other common bovine pathogens. Specifically, the primers and probes are shown in Table 1. The primers and probes were synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0051] Table 1 Primer and probe sequences for amplifying four common cattle pathogens

[0052]

[0053]

[0054] 2. Standard plasmid synthesis: Based on the four pairs of designed primer probes, the corresponding target fragments were synthesized and cloned into the pUC57 plasmid vector to obtain four standard plasmids. The standard plasmids were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0055] 3. Nucleic acid extraction and amplification kits. The nucleic acid extraction kit was purchased from Suzhou Youyi Landi Biotechnology Co., Ltd.: UE Body Fluid Viral DNA / RNA Mini-Prep Kit; the real-time fluorescence quantitative PCR kit was purchased from Yisheng Biotechnology Co., Ltd.: ⅢOne Step RT-qPCR Probe Kit. Specific extraction and amplification steps were performed according to the kit instructions.

[0056] 4. Verification of the effectiveness and specificity of primer probes: The four pairs of primer probes were mixed to form a primer pool. The mixing of the primer pool is shown in Table 2.

[0057] Table 2 qPCR primer pool for detecting four common cattle pathogens

[0058] Primer / probe name Final primer / probe concentration (μM) BEFV-F 2 BEFV-R 2 BEFV-P 1 BoHV4-F 2 BoHV4-R 2 BoHV4-P 1 BRV-F 2 BRV-R 2 BRV-P 1 CP-F 2 CP-R 2 CP-P 1

[0059] The four synthesized standard plasmids were diluted to 10 5 copies / μl as templates, and other bovine infectious pathogen nucleic acids stored in our laboratory were used as templates, and 8 pairs of primer probe sets were used. qPCR detection was performed using the III One Step RT-qPCR Probe Kit. The qPCR system is shown in Table 3.

[0060] Table 3 qPCR system

[0061]

[0062] RT-qPCR amplification was performed using a SLAN-96S fluorescence quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd. The RT-qPCR program was as follows: reverse transcription at 50°C for 15 minutes, pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 30 seconds, and extension at 60°C for 30 seconds, for 45 cycles. During the extension period, fluorescence signals were collected from four channels: FAM, VIC, ROX, and Cy5.

[0063] Results: When nucleic acid containing the target fragment was added, amplification curves were obtained with CT values between 19 and 22. No amplification was observed when nucleic acid containing no target fragment was added. The results are shown in Table 4, indicating that the primer pool had good effectiveness and specificity.

[0064] Table 4 Results of primer pool validity and specificity verification

[0065]

[0066]

[0067] Note: The value represents the CT value of the amplification curve -: RT-qPCR has no amplification curve

[0068] 5. Optimization of the optimal working concentration of primers and probes

[0069] (1) Optimization of BEFV-F / BEFV-R primer concentration

[0070] Primer mixtures containing BEFV-F / BEFV-R were prepared at working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. The concentrations of the other primer probes were fixed, and the plasmid containing the target fragment was used as a template to amplify the plasmid containing the target fragment. Three replicates were tested at each concentration. The qPCR reaction system is shown in Table 5.

[0071] Table 5 BEFV-F / BEFV-R primer concentration optimization

[0072]

[0073] The reaction conditions are the same as step 4.

[0074] (2) Optimization of primer concentrations for BoHV4-F / BoHV4-R, BRV-F / BRV-R, and CP-F / CP-R

[0075] The same method as the BEFV-F / BEFV-R primer concentration optimization method was used to prepare primer pairs with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. The plasmid containing the target fragment was amplified by fixing the concentrations of other primer probes.

[0076] (3) Optimization of BEFV-P, BoHV4-P, BRV-P, and CP-P probe concentrations

[0077] Similar to the primer concentration optimization method, probes with working concentrations of 50nM, 100nM, 150nM, 200nM, and 250nM were prepared respectively, and then the plasmid containing the target fragment was amplified according to the method of fixing the concentrations of other primers and probes.

[0078] (4) Primer probe concentration optimization results

[0079] After qPCR testing, each sample will obtain the amplification curve CT value and fluorescence signal intensity Rn. The three replicate values are averaged and the above primer and probe concentration optimization results are statistically summarized, as shown in Figure 2. Figure 1 As shown in the figure, the method for screening the optimal primer or probe concentration is to give priority to the primer or probe concentration with the smallest CT value. If the CT values are the same, the primer or probe concentration with the largest Rn value is selected. Finally, the optimal working concentrations of each primer and probe after optimization are:

[0080] BEFV-F:300nM, BEFV-R:300nM, BEFV-P:250nM;

[0081] BoHV4-F:100nM, BoHV4-R:100nM, BoHV4-P:250nM;

[0082] BRV-F: 300nM, BRV-R: 300nM, BRV-P: 250nM;

[0083] CP-F: 100nM, CP-R: 100nM, CP-P: 50nM.

[0084] 6. Annealing temperature optimization

[0085] Prepare 500 μl of each of two primer pools according to the optimal concentrations of primers and probes optimized in step 5, as shown in Table 6.

[0086] Table 6 Preparation of primer pool after concentration optimization

[0087]

[0088] The 10 5 The standard plasmids (100 copies / μl) were mixed and used as templates. qPCR was performed using a SLAN-96S instrument. Three replicates were tested for each condition. The optimal annealing temperature of the primer pool was screened. The reaction system was the same as in step 4. The reaction procedure is shown in Table 7.

[0089] Table 7 Annealing temperature optimization reaction program

[0090]

[0091] Results: The CT values of the three replicate wells after the experiment were averaged and the annealing temperature optimization results were statistically summarized, as follows: Figure 2 As shown in FIG, the annealing temperature with the lowest CT value and the highest Rn value was selected as the optimal annealing temperature. Finally, the optimal annealing temperature was selected to be 62°C.

[0092] According to the above optimization results of primer probe concentration and annealing temperature, the final reaction system and reaction procedure of the detection kit for four common cattle pathogens were determined. The primer pool was prepared as shown in step 6, the reaction system was consistent with step 4, and the reaction procedure was consistent with step 6. The annealing temperature was selected as 62°C, and the mixed standard plasmids (each plasmid concentration was about 10 5 copies / μl) was used as the positive control of this kit.

[0093] 7. Establishment of standard curve

[0094] The four standard plasmids with known copy numbers were diluted 10-fold in series and qPCR was performed using the kit prepared by the present invention and SLAN-96S instrument. Three replicates were detected for each concentration of plasmid. The standard curve was drawn using Graphpad Prism8 software with the CT value of the amplification curve as the ordinate and the logarithm of the template copy number as the abscissa. The standard curve is shown in Figure 1. Figure 3-Figure 4 As shown in the figure, the slopes of the standard curves for amplifying BEFV, BoHV4, BRV, and CP plasmids are -3.602, -3.351, -3.451, and -3.494, respectively. The amplification efficiencies are 89.51%, 98.80%, 94.88%, and 93.29%, respectively. 2 All of them were greater than 0.999, indicating that the primer pool had a high amplification efficiency.

[0095] 8. Sensitivity test

[0096] The standard plasmid was diluted to 10 copies / μl, 5 copies / μl, 1 copies / μl, and 0.5 copies / μl, and then mixed to obtain a mixed plasmid. The mixed plasmid was used as a template, and 8 replicates were detected at each concentration to determine the detection limit of the kit prepared by the present invention for each pathogen. The detection rate of the lowest detection repeat experiment reaching 100% was determined as the detection limit for the pathogen. The detection results of plasmids at various concentrations are shown in Table 8, which shows that the detection limit of the kit prepared by the present invention for all four pathogens can reach 5 copies / μl, proving the high sensitivity of the kit. The amplification curve when the plasmid concentration is 5 copies / μl is shown in Figure 8. Figure 5 shown.

[0097] Table 8 Sensitivity test results

[0098]

[0099] 9. Repeatability

[0100] like Figure 6-Figure 7 As shown, the standard plasmids were diluted to 10 5 copies / μl, 10 2 The plasmids were mixed and then used as templates to test 16 replicates at each concentration to evaluate the repeatability of the kit prepared by the present invention. After testing, the amplified CT value of each sample was obtained, and the average CT, standard deviation (SD), and coefficient of variation (CV) were calculated. The repeatability test results show (Table 9) that for 10 5 The amplified CT values ranged from 19 to 22, with a coefficient of variation of 0.13% to 2.26%. 2For a template with a volume of 100 copies / μl, the amplified CT value was between 30 and 33, and the coefficient of variation was between 0.87% and 2.18%, indicating that the kit prepared by the present invention has high repeatability.

[0101] Table 9 Repeatability test results

[0102]

[0103] 10. Clinical sample testing

[0104] To verify the detection effect of the kit prepared by the present invention on clinical samples, 51 swab samples collected from two cattle farms were tested: RNA was first extracted according to the method described in step 2, and then qPCR detection was performed using the kit prepared by the present invention and the SLAN-96S instrument.

[0105] The results are shown in Table 10. One case of Clostridium perfringens infection was detected in Farm A. One case of bovine herpesvirus type 4 infection, five cases of bovine rotavirus infection, and three cases of Clostridium perfringens infection were detected in Farm B. The overall number of infections in Farm B was higher than that in Farm A. In addition, one sample of mixed infection with bovine rotavirus and bovine herpesvirus type 4, and one sample of mixed infection with bovine rotavirus and Clostridium perfringens were detected in Farm B. The detection effect of the test kit prepared by the present invention on clinical samples was successfully verified, and it can further detect clinical samples with mixed infections, which can provide timely and effective reference for the prevention and control of cattle diseases in clinical practice.

[0106] Table 10 Clinical sample test results

[0107]

[0108] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A primer-probe combination for identifying mixed pathogens of cattle infection, wherein the mixed pathogens of cattle infection include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens, characterized in that: When identifying the bovine fever virus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the probe is shown in SEQ ID No. 3; When identifying the bovine herpesvirus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the probe is shown in SEQ ID No. 6; When identifying the bovine rotavirus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 7, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 8, and the nucleotide sequence of the probe is shown in SEQ ID No. 9; When identifying the Clostridium perfringens, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 10, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 11, and the nucleotide sequence of the probe is shown in SEQ ID No.

12.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quencher group.

3. The primer-probe combination according to claim 2, characterized in that The 5' end of the probe used to identify the bovine fever virus is modified with a ROX group and the 3' end is modified with MGB; The 5' end of the probe used to identify the bovine herpes virus is modified with a Cy5 group and the 3' end is modified with BHQ2; The 5' end of the probe used to identify the bovine rotavirus is modified with a FAM group and the 3' end is modified with an MGB group; The 5' end of the probe used to identify Clostridium perfringens was modified with a VIC group, and the 3' end was modified with an MGB group.

4. The primer-probe combination according to claim 1, characterized in that The detection target gene of the bovine fever virus is G; The detection target gene of the bovine herpes virus is gB; The detection target gene of the bovine rotavirus is VP6; The detection target gene of Clostridium perfringens is cpa.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a reagent for identifying mixed pathogens of cattle infections.

6. The use according to claim 5, characterized in that The cattle mixed infection pathogens include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

7. A kit for identifying pathogens of mixed infections in cattle, characterized in that: The invention comprises a primer pool, wherein the primer pool comprises primer probes for identifying bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens in the primer-probe combination according to claim 1.

8. The kit according to claim 7, characterized in that The concentrations of the upstream and downstream primers for identifying bovine fever virus in the primer pool are both 300 nM, and the concentration of the probe is 250 nM; The concentrations of upstream and downstream primers for identifying bovine herpesvirus were both 100 nM, and the concentration of the probe was 250 nM; The concentrations of upstream and downstream primers for identifying bovine rotavirus were both 300 nM, and the concentration of the probe was 250 nM; The concentrations of the upstream and downstream primers for identifying Clostridium welchii were both 100 nM, and the concentration of the probe was 50 nM.

9. Use of the kit according to claim 7 or 8 in the preparation of a product for identifying cattle pathogens.

10. The use according to claim 9, characterized in that The cattle mixed infection pathogens include bovine typhoid virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens.

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