A primer probe combination for identifying mixed infection pathogens of cattle and application and kit thereof

Abstract

The present application relates to the technical field of virus detection, in particular to a primer probe combination for identifying mixed infection pathogens of cattle and application and kit thereof. When identifying cattle pathogens, the nucleotide sequence of the upstream primer of the primer probe combination used is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, the nucleotide sequence of the probe is shown in SEQ ID No. 3, and the like scheme. The present application can detect and identify bovine epidemic fever virus, bovine herpes virus 4, bovine rotavirus and clostridium welchii through four groups of primer probes. The primer probe combination provided by the present application can identify and diagnose four common pathogens of cattle through a one-tube reaction system by fluorescence quantitative PCR method, and can also identify mixed infection samples, has the advantages of accurate identification, high sensitivity, simple operation, short time consumption and the like, and can provide effective technical means for the diagnosis of cattle infectious disease pathogens and disease prevention in China.

Description

TECHNICAL FIELD

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

[0002] Cattle, as an important economic animal, plays an important role in global agriculture and animal husbandry. However, the occurrence and spread of cattle infectious diseases have caused serious economic losses to the breeding industry and pose a potential threat to public health safety. Among the numerous cattle infectious diseases, Bovine Ephemeral Fever Virus (BEFV), Bovine Herpesvirus 4 (BoHV4), Bovine Rotavirus (BRV) and Clostridium perfringens (CP) are four common pathogens, and their infection can cause different types of diseases, including respiratory diseases, digestive diseases and systemic infections.

[0003] BEFV is an RNA virus that mainly causes bovine ephemeral 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 significant decline in production performance of cattle herd. BoHV4 is a DNA virus associated with various cattle diseases, including reproductive system diseases, mastitis and immune suppression, and its infection is often occult and prone to mixed infection with other pathogens, leading to complex disease conditions. BRV is a double-stranded RNA virus belonging to the Reoviridae family, and 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 herds, with high morbidity and mortality. CP is a gram-positive anaerobic bacteria that widely exists in the environment and in the intestinal tract of cattle, and certain virulence factors (such as toxin A and toxin B) can cause acute enterotoxemia or necrotic enteritis, which is commonly seen in intensive breeding farms and causes mass mortality.

[0004] Co-infection of BRV and CP often leads to severe diarrhea and even death in calves, and the synergistic effect of BEFV and BoHV4 may exacerbate respiratory symptoms. BEFV and BoHV4 have increased risk of transmission during the peak period of mosquito activity in summer and autumn, while BRV and CP are more likely to outbreak under closed feeding conditions in winter. Due to the prevalence of the above-mentioned mixed infections in cattle herds, whether vaccination, drug treatment or environmental management, the prevention and control measures for the four pathogens emphasize early detection, early isolation and early intervention. Therefore, it is particularly important to develop a technology that can simultaneously detect the above-mentioned pathogens.

[0005] Quantitative PCR (qPCR) has been widely used in pathogen detection due to its high sensitivity, high specificity and rapidity. Up to four targets can be detected simultaneously in one reaction system. Therefore, the establishment of a multiplex qPCR method for simultaneous detection of the four important pathogens of cattle can not only fill the gap in the detection of common important pathogens of cattle, shorten the diagnosis cycle and improve the prevention and control efficiency, but also provide a new technical means for the precise prevention and control of cattle infectious diseases and strong technical support for large-scale farms. SUMMARY

[0006] In order to solve the above problems, the present application provides a primer probe combination for identifying mixed infection pathogens of cattle and its application and kit, the primer probe combination designed by the present application can identify four common pathogens of cattle simultaneously, the primer probe combination is mixed to form a primer pool, and a multiplex real-time fluorescent quantitative PCR kit for identifying four common pathogens of cattle is provided.

[0007] In order to achieve the above purpose, the present application provides the following technical scheme:

[0008] The present application provides a primer probe combination for identifying mixed infection pathogens of cattle, wherein the mixed infection pathogens of cattle include bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii,

[0009] When the bovine epidemic fever virus is identified, the nucleotide sequence of the upstream primer of the primer probe combination 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 the bovine herpes virus is identified, the nucleotide sequence of the upstream primer of the primer probe combination 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 the bovine rotavirus is identified, the nucleotide sequence of the upstream primer of the primer probe combination 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 the Clostridium welchii is identified, the nucleotide sequence of the upstream primer of the primer probe combination 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 for identifying the bovine epidemic fever virus is modified with a ROX group and the 3' end is modified with MGB;

[0015] The 5' end of the probe used for identifying 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 for identifying the bovine rotavirus is modified with a FAM group and the 3' end is modified with MGB;

[0017] The 5' end of the probe used for identifying the Clostridium welchii is modified with a VIC group and the 3' end is modified with MGB.

[0018] Preferably, the target gene for detecting the bovine epidemic fever virus is G;

[0019] The target gene for detecting the bovine herpes virus is gB;

[0020] The target gene for detecting the bovine rotavirus is VP6;

[0021] The target gene for detecting the Clostridium welchii is cpa.

[0022] The present application also provides the use of the primer probe combination in the preparation of a reagent for identifying bovine mixed infection pathogens.

[0023] Preferably, the bovine mixed infection pathogens include bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii.

[0024] The present application also provides a kit for identifying bovine mixed infection pathogens, comprising a primer pool, wherein the primer pool comprises the primer probes for identifying bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii in the primer probe combination.

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

[0026] The concentrations of the upstream and downstream primers for identifying the bovine herpes virus are both 100nM, and the concentration of the probe is 250nM;

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

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

[0029] The application further provides application of the kit in preparation of a product for identifying mixed infection pathogens of cattle.

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

[0031] Advantages of the application:

[0032] The application can detect and identify bovine epidemic fever virus (BEFV), bovine herpes virus 4 (BoHV4), bovine rotavirus (BRV) and Clostridium perfringens (CP) through four groups of primers and probes. The four common pathogens of cattle can be differentially diagnosed through one tube reaction system by using the primer and probe combination provided by the application through the fluorescent quantitative PCR method, and mixed infection samples can also be identified. The application has the advantages of accurate identification, high sensitivity, simple operation, short time consumption, etc., and can provide effective technical means for diagnosis of bovine infectious disease pathogens and disease prevention in China. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0034] Figure 1 Optimization results of the concentration of the primer pool primers and probes;

[0035] Figure 2 Optimization results of the annealing temperature;

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

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

[0038] Figure 5 Amplification curves for four target plasmids (each 8 repeats) at a concentration of 5 copies / μl;

[0039] Figure 6 Amplification curves for four target plasmids (each 16 repeats) at a concentration of 10 5 copies / μl;

[0040] Figure 7 Amplification curves for four target plasmids (each 16 repeats) at a concentration of 10 2 copies / μl. DETAILED DESCRIPTION

[0041] The application provides a primer probe combination for identifying mixed infection pathogens of cattle, the mixed infection pathogens of cattle including bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii, when the bovine epidemic fever virus is identified, the nucleotide sequence of the upstream primer of the primer probe group 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 the bovine herpes virus is identified, the nucleotide sequence of the upstream primer of the primer probe group 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 the bovine rotavirus is identified, the nucleotide sequence of the upstream primer of the primer probe group 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 the Clostridium welchii is identified, the nucleotide sequence of the upstream primer of the primer probe group 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.

[0042] In the application, the 5' end of the probe used for identifying the bovine epidemic fever virus is preferably modified by using a ROX group, and the 3' end is preferably modified by using MGB; the 5' end of the probe used for identifying the bovine herpes virus is preferably modified by using a Cy5 group, and the 3' end is preferably modified by using BHQ2; the 5' end of the probe used for identifying the bovine rotavirus is preferably modified by using a FAM group, and the 3' end is preferably modified by using MGB; and the 5' end of the probe used for identifying the Clostridium welchii is preferably modified by using a VIC group, and the 3' end is preferably modified by using MGB.

[0043] In the application, the detection target gene of the bovine epidemic fever virus is preferably G; the detection target gene of the bovine herpes virus is preferably gB; the detection target gene of the bovine rotavirus is preferably VP6; and the detection target gene of the Clostridium welchii is preferably cpa.

[0044] The application also provides application of the primer probe combination in the preparation of a reagent for identifying mixed infection pathogens of cattle.

[0045] The application further provides a kit for identifying mixed infection pathogens of cattle, comprising a primer pool, wherein the primer pool comprises primers and probes for identifying bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium perfringens in the primer probe combination.

[0046] The application further provides application of the kit in preparation of a product for identifying mixed infection pathogens of cattle.

[0047] In order to further illustrate the application, the application is described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

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

[0049] Example 1

[0050] 1. Design of specific primers and probes. The nucleic acid sequences of the conserved genes or virulence genes of four pathogens prevalent in China in recent years were downloaded from Genbank. Sequence alignment software was used for multiple sequence alignment. Then, specific primers and probes were designed for regions conserved in each strain of the pathogen but specific to other common pathogens of cattle. Specifically, the above primers and probes are shown in Table 1, and the primers and probes were synthesized by Shanghai Jerui Biological Engineering Co., Ltd.

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

[0052]

[0053]

[0054] 2. Synthesis of standard plasmids. According to the designed four pairs of primers and probes, the corresponding target fragments were synthesized and cloned into pUC57 plasmid vectors to obtain four standard plasmids, and the standard plasmids were synthesized by Suzhou Hongxun Biological Technology Co., Ltd.

[0055] 3. Nucleic acid extraction and amplification kit. The kit for nucleic acid extraction was purchased from Suzhou Youyilan Di Biological Technology Co., Ltd: UE body fluid virus DNA / RNA small amount preparation kit; the kit for real-time fluorescent quantitative PCR was purchased from Yixing Biological Technology Co., Ltd: ⅢOne Step RT-qPCR Probe Kit. The specific extraction and amplification steps were carried out according to the kit instructions.

[0056] 4. Verification of effectiveness and specificity of primers and probes. Four pairs of primers and probes were mixed to form a primer pool, and the primer pool mixing conditions are shown in Table 2.

[0057] Table 2 qPCR primer pool for detection of 4 common bovine 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 synthesized four standard plasmids were diluted to 10 5 copies / μl as templates, and other bovine infectious pathogen nucleic acids preserved in the laboratory were used as templates, 8 pairs of primers and probes were used, and qPCR detection was performed using ⅢOne Step RT-qPCR Probe Kit, and the qPCR system is shown in Table 3.

[0060] Table 3 qPCR system

[0061]

[0062] SLAN-96S fluorescent quantitative PCR instrument of Shanghai Hongshi Medical Technology Co., Ltd. was used for RT-qPCR amplification, and the RT-qPCR amplification program was: 50℃ reverse transcription for 15min; 95℃ pre-denaturation for 30s; 95℃ denaturation for 30s, 60℃ extension for 30s, and amplification for 45 cycles. FAM, VIC, ROX, and Cy5 channels were collected for fluorescence signal during extension.

[0063] Results: When the nucleic acid containing the target fragment was added, there were amplification curves, and the CT value was between 19-22. When the nucleic acid without the target fragment was added, there was no amplification, and the results are shown in Table 4, indicating that the primer pool has good effectiveness and specificity.

[0064] Table 4 Verification results of effectiveness and specificity of primer pool

[0065]

[0066]

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

[0068] 5. Optimization of the optimal working concentration of primer probe

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

[0070] Prepare the primer mixture containing BEFV-F / BEFV-R with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. Fix the concentrations of other primers and probes, use the plasmid containing the target fragment as the template, amplify the plasmid containing the target fragment, and detect 3 repeats for each concentration. The qPCR reaction system is shown in Table 5.

[0071] Table 5. Optimization of BEFV-F / BEFV-R primer concentration

[0072]

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

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

[0075] Prepare each primer pair with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively, in the same way as the BEFV-F / BEFV-R primer concentration optimization method, and then amplify the plasmid containing the target fragment according to the method of fixing the concentrations of other primers and probes.

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

[0077] Similarly, prepare the probes with working concentrations of 50 nM, 100 nM, 150 nM, 200 nM, and 250 nM, respectively, and then amplify the plasmid containing the target fragment according to the method of fixing the concentrations of other primers and probes.

[0078] (4) Optimization results of primer and probe concentration

[0079] Each sample will obtain the amplification curve CT value and fluorescence signal intensity Rn after qPCR detection. Take the average of 3 replicate wells to statistically summarize the above primer and probe concentration optimization results, as shown in Table 6, the method for screening the optimal primer or probe concentration is to preferentially select the primer or probe concentration with the smallest CT value, and if the CT values are the same, select the primer or probe concentration with the largest Rn value. Finally, the optimal working concentrations of the optimized primers and probes are as follows: Figure 1

[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 the two primer pools according to the optimal primer and probe concentrations optimized in step 5, as shown in Table 6.

[0086] Table 6 Primer cell preparation after concentration optimization.

[0087]

[0088] 10 containing the target fragment 5 The standard plasmid was mixed in copies / μl and used as a template. qPCR was performed using a SLAN-96S instrument. Triple replicates were performed 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 Optimization of Annealing Temperature and Reaction Procedure

[0090]

[0091] Results: The average CT values ​​of the three replicates after the experiment were used to statistically summarize the results of the annealing temperature optimization. Figure 2 As shown, the annealing temperature with the lowest CT value and the second highest Rn value was selected as the optimal annealing temperature. Finally, the optimal annealing temperature was determined to be 62℃.

[0092] Based on the optimization results of primer and probe concentrations and annealing temperature, the final reaction system and reaction procedure for the detection kit of four common bovine pathogens were determined. The primer pool was prepared as shown in step 6, the reaction system was the same as in step 4, and the reaction procedure was the same as described in step 6. The annealing temperature was selected as 62℃. In addition, a mixed standard plasmid (each plasmid concentration was approximately 10) was selected. 5 (copies / μl) served as a positive control for this kit.

[0093] 7. Establishment of the standard curve

[0094] The four standard plasmids with known copy numbers were diluted by 10-fold gradient, and qPCR was performed using the kit prepared by the present application and the SLAN-96S instrument. Each concentration of plasmid was detected in 3 replicates. The standard curve was plotted using the amplification curve CT value as the vertical coordinate and the logarithm of the template copy number as the horizontal coordinate by Graphpad Prism 8 software. The standard curve is shown in Figure 3-4 . It can be seen that 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, and the amplification efficiencies thereof are 89.51%, 98.80%, 94.88% and 93.29%, respectively, and the R 2 values are all greater than 0.999, indicating that the primer pool has high amplification efficiency.

[0095] 8. Sensitivity experiment

[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 mixed plasmids. The mixed plasmids were used as templates, and each concentration was detected in 8 replicates to determine the detection limit of the kit prepared by the present application for each pathogen. The lowest detection repeat experiment with a detection rate of 100% was identified as the detection limit of the pathogen. The detection results of each concentration of plasmid are shown in Table 8, indicating that the detection limit of the kit prepared by the present application 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 5 .

[0097] Table 8 Sensitivity experiment results

[0098]

[0099] 9. Reproducibility experiment

[0100] As shown in Figure 6-7 , the standard plasmid was diluted to 10 5 copies / μl and 10 2 copies / μl, respectively, and then mixed to obtain mixed plasmids. The mixed plasmids were used as templates, and each concentration was detected in 16 replicates to evaluate the reproducibility of the kit prepared by the present application. After detection, the amplification CT value of each sample was obtained, and the average CT, standard deviation (SD) and coefficient of variation (CV) were calculated. The reproducibility experiment results (Table 9) show that for the template of 10 5 copies / μl, the amplification CT value is between 19 and 22, and the coefficient of variation is 0.13%-2.26%, and for the template of 10 2The amplification CT value is between 30-33, and the coefficient of variation is 0.87%-2.18%, which indicates that the kit prepared by the application has high repeatability.

[0101] Table 9 repeatability test results

[0102]

[0103] 10. Clinical sample detection

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

[0105] The results are shown in Table 10. One case of Clostridium welchii infection was detected in cattle farm A. In cattle farm B, one case of bovine herpes virus 4 virus infection, five cases of bovine rotavirus infection, and three cases of Clostridium welchii infection were detected. Compared with cattle farm A, the total number of infections in cattle farm B was more. In addition, one sample of mixed infection of bovine rotavirus and bovine herpes virus 4 was detected in cattle farm B, and one sample of mixed infection of bovine rotavirus and Clostridium welchii was detected. The detection effect of the kit prepared by the application on clinical samples was successfully verified, and mixed infection of clinical samples could be further detected, which could provide timely and effective reference for the prevention and control of bovine diseases in clinic.

[0106] Table 10 clinical sample detection results

[0107]

[0108] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which belong to the protection scope of the application.

Claims

1. A kit for the identification of mixed infection pathogens in cattle, characterized in that, The primer pool comprises primer probes for identifying bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii in a primer probe combination; When identifying the bovine epidemic 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 herpes virus, 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 welchii, 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; The 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group; The 5' end of the probe used for identifying the bovine epidemic fever virus is modified with a ROX group, and the 3' end is modified with MGB; The 5' end of the probe used for identifying 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 for identifying the bovine rotavirus is modified with a FAM group, and the 3' end is modified with MGB; The 5' end of the probe used for identifying the Clostridium welchii is modified with a VIC group, and the 3' end is modified with MGB; The detection target gene of the bovine epidemic 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 the Clostridium welchii is cpa; The concentrations of the upstream and downstream primers for identifying the bovine epidemic fever virus in the primer pool are both 300 nM, and the concentration of the probe is 250 nM; The concentrations of the upstream and downstream primers for identifying the bovine herpes virus are both 100 nM, and the concentration of the probe is 250 nM; The concentrations of the upstream and downstream primers for identifying the bovine rotavirus are both 300 nM, and the concentration of the probe is 250 nM; The concentrations of the upstream and downstream primers for identifying the Clostridium welchii are both 100 nM, and the concentration of the probe is 50 nM.

2. Use of the kit of claim 1 in the preparation of a product for identifying mixed infection pathogens of cattle; The mixed infection pathogens of cattle are bovine epidemic fever virus, bovine herpes virus, bovine rotavirus and Clostridium welchii.

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