Primer probe combination for cattle respiratory disease syndrome 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 bovine respiratory disease syndrome pathogen identification and application and a kit thereof. When the bovine viral diarrhea virus is identified, the nucleotide sequence of the upstream primer of the used primer probe group is as shown in SEQ ID No.1, the nucleotide sequence of the 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 primer probe combination provided by the invention can be used for identifying and diagnosing eight common pathogens capable of causing the bovine respiratory disease syndrome at one time through a two-tube reaction system by using a fluorescent quantitative PCR method, can also be used for identifying a mixed infected sample, has the advantages of accurate identification, high sensitivity, simplicity in operation, short time consumption and the like, and can be used for detecting the bovine respiratory disease syndrome. And an effective technical means can be provided for pathogen diagnosis and disease prevention of the bovine respiratory disease syndrome in China.

Description

Technical Field

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

[0002] Bovine respiratory disease complex (BRDC) is a general term for respiratory diseases such as pneumonia and bronchitis in cattle caused by the interaction of multiple bacteria, viruses and stress factors. The disease is distributed worldwide and can lead to decreased production performance, increased treatment and prevention costs, and even death, causing harm to my country's cattle industry.

[0003] The etiology of BRDC is complex, and various pathogenic microorganisms and non-infectious factors can cause the disease. Common infectious pathogens that can cause BRDC include: Bovine viral diarrhea virus (BVDV), Bovine parainfluenza virus type 3 (BPIV3), Bovine respiratory syncytial virus (BRSV), Bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella bovis (PM), Mannheimia hemolytica (MH), Bovine infectious rhinotracheitis virus (IBRV), influenza virus type D, Haemophilus somnus, Cryptobacterium pyogenes, Dictyocaulus elegans, etc. Since there are many pathogens that can cause BRDC, and the clinical symptoms of these pathogens are similar to the lesions in the autopsy, it is of great significance to establish a high-throughput method that can quickly and accurately identify and diagnose the pathogens that cause BRDC for targeted treatment and prevention and control of BRDC.

[0004] Real-time fluorescence quantitative PCR (qPCR) has the advantages of accuracy, sensitivity, and rapidity. It is widely used in the clinical diagnosis of pathogens. At present, many studies have reported qPCR methods that can simultaneously detect multiple pathogens of BRDC, but only a few pathogens can be detected. There are also studies that have established high-throughput qPCR detection methods for detecting more than 10 BRDC pathogens. However, the increase in the number of pathogens detected also increases the detection cost, detection time, and operation difficulty, and it cannot be generally applied to the rapid detection of a large number of clinical samples in farms. Therefore, the establishment of a multiplex qPCR method that can identify multiple common pathogens of BRDC, which is simple to operate, short in time, highly sensitive, and low in cost, can provide a very practical tool for the diagnosis of clinical BRDC, and can provide great assistance for the prevention and control of BRDC, thereby effectively helping the cattle industry reduce economic losses. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a primer-probe combination for identifying the pathogen of bovine respiratory disease syndrome, and its application and kit. The primer-probe combination provided by the present invention can be used to perform differential diagnosis on 8 common pathogens that can cause bovine respiratory disease syndrome at one time through a two-tube reaction system through a fluorescent quantitative PCR method, and can also identify mixed-infected samples. It has the advantages of accurate identification, high sensitivity, simple operation, short time consumption, etc., and can provide an effective technical means for the pathogen diagnosis and disease prevention of bovine respiratory disease syndrome in my country.

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

[0007] The present invention provides a primer-probe combination for identifying pathogens of bovine respiratory disease syndrome, wherein the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus. When identifying the bovine viral diarrhea virus, the nucleotide sequence of the upstream primer of the primer-probe combination 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;

[0008] When identifying the bovine parainfluenza virus type 3, 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;

[0009] When identifying the bovine respiratory syncytial virus, 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;

[0010] When identifying the bovine coronavirus, 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;

[0011] When identifying the bovine Mycoplasma, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 13, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 14, and the nucleotide sequence of the probe is shown in SEQ ID No. 15;

[0012] When identifying the bovine Pasteurella, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 16, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 17, and the nucleotide sequence of the probe is shown in SEQ ID No. 18;

[0013] When identifying the bovine hemolytic Mannheimia, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 19, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 20, and the nucleotide sequence of the probe is shown in SEQ ID No. 21;

[0014] When identifying the infectious bovine rhinotracheitis virus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No.22, the nucleotide sequence of the downstream primer is shown in SEQ ID No.23, and the nucleotide sequence of the probe is shown in SEQ ID No.24.

[0015] Preferably, the 5' end of the probe is modified with a fluorescent group, and the 3' end is modified with a quenching group.

[0016] Preferably, the 5' end of the probe used to identify the bovine viral diarrhea virus 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 the bovine parainfluenza virus type 3 is modified with a VIC group and the 3' end is modified with an MGB group;

[0018] The 5' end of the probe used to identify the bovine respiratory syncytial virus is modified with a ROX group, and the 3' end is modified with MGB;

[0019] The 5' end of the probe used to identify the bovine coronavirus is modified with a Cy5 group, and the 3' end is modified with BHQ2;

[0020] The 5' end of the probe used to identify the bovine Mycoplasma is modified with a FAM group, and the 3' end is modified with an MGB group;

[0021] The 5' end of the probe used to identify the bovine Pasteurella is modified with a VIC group, and the 3' end is modified with MGB;

[0022] The 5' end of the probe used to identify the bovine hemolytic Mannheimia is modified with a ROX group, and the 3' end is modified with MGB;

[0023] The 5' end of the probe used to identify the infectious bovine rhinotracheitis virus is modified with a Cy5 group, and the 3' end is modified with BHQ2.

[0024] Preferably, the detection target gene of the bovine viral diarrhea virus is 5'UTR;

[0025] The detection target gene of bovine parainfluenza virus type 3 is N;

[0026] The detection target gene of the bovine respiratory syncytial virus is N;

[0027] The detection target gene of the bovine coronavirus is N;

[0028] The detection target gene of bovine Mycoplasma is oppD;

[0029] The detection target gene of the bovine Pasteurella is kmt1;

[0030] The detection target gene of the bovine hemolytic Mannheimia is lktA;

[0031] The detection target gene of the bovine infectious rhinotracheitis virus is gB.

[0032] The present invention also provides the use of the primer-probe combination described in the above technical solution in the preparation of a reagent for identifying the pathogen of bovine respiratory disease syndrome.

[0033] Preferably, the bovine respiratory disease syndrome pathogens include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

[0034] The present invention also provides a kit for identifying pathogens of bovine respiratory disease syndrome, comprising a primer pool A and a primer pool B;

[0035] The primer pool A includes primer probes for identifying bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus and bovine coronavirus in the primer-probe combination described in the above technical solution;

[0036] The primer pool B includes primer probes for identifying Mycoplasma bovis, Pasteurella bovis, Mannheimia bovis and infectious bovine rhinotracheitis virus in the primer-probe combination of the above technical solution.

[0037] Preferably, the concentrations of the upstream and downstream primers for identifying bovine viral diarrhea virus in the primer pool A are both 300 nM, and the concentration of the probe is 250 nM;

[0038] The concentrations of the upstream and downstream primers for identifying bovine parainfluenza virus type 3 were both 100 nM, and the concentration of the probe was 100 nM;

[0039] The concentrations of the upstream and downstream primers for identifying bovine respiratory syncytial virus were both 300 nM, and the concentration of the probe was 250 nM;

[0040] The concentrations of the upstream and downstream primers for identifying bovine coronavirus were both 200 nM, and the concentration of the probe was 250 nM.

[0041] Preferably, the concentrations of the upstream and downstream primers for identifying Mycoplasma bovis in the primer pool B are both 300 nM, and the concentration of the probe is 250 nM;

[0042] The concentrations of the upstream and downstream primers for identifying Pasteurella bovis were both 100 nM, and the concentration of the probe was 50 nM;

[0043] The concentrations of the upstream and downstream primers for identifying Mannheimia bovis were both 100 nM, and the concentration of the probe was 250 nM;

[0044] The concentrations of the upstream and downstream primers for identifying bovine infectious rhinotracheitis virus were both 300 nM, and the concentration of the probe was 250 nM.

[0045] The present invention also provides the use of the kit described in the above technical scheme in the preparation of a product for identifying the pathogens of bovine respiratory disease syndrome; the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

[0046] Beneficial effects of the present invention:

[0047] By using the primer-probe combination provided by the present invention, a fluorescent quantitative PCR method can be used to perform differential diagnosis on eight common pathogens that can cause bovine respiratory disease syndrome at one time through a two-tube reaction system, and mixed-infected samples can also be identified. The method has the advantages of accurate identification, high sensitivity, simple operation, and short time consumption, and can provide an effective technical means for the pathogen diagnosis and disease prevention of bovine respiratory disease syndrome in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0049] Figure 1 The results of primer probe concentration optimization for primer pool A in Example 5 are as follows;

[0050] Figure 2 The results of primer probe concentration optimization for primer pool B in Example 5;

[0051] Figure 3 The annealing temperature optimization result of Example 6;

[0052] Figure 4 The standard curve drawn for the amplification of 4 target plasmids by primer pool A in Example 7;

[0053] Figure 5 The amplification curve corresponding to the standard curve of the four target plasmids amplified by primer pool A in Example 7;

[0054] Figure 6 The standard curve drawn for the amplification of 4 target plasmids by primer pool B in Example 7;

[0055] Figure 7 The amplification curve corresponding to the standard curve of the four target plasmids amplified by primer pool B in Example 7;

[0056] Figure 8 The amplification curves of primer pool A in Example 8 for four target plasmids (eight replicates each) at a concentration of 5 copies / μl;

[0057] Fig. 9 The amplification curves of primer pool B in Example 8 for four target plasmids (eight replicates each) at a concentration of 5 copies / μl;

[0058] Fig.10 The primer pool A in Example 9 was used for 4 target plasmids (16 replicates each) at 10 5 Amplification curve at the concentration of copies / μl;

[0059] Fig.11 The primer pool A in Example 9 was used for 4 target plasmids (16 replicates each) at 10 2 Amplification curve at the concentration of copies / μl;

[0060] Fig.12 The primer pool B in Example 9 was used for 4 target plasmids (16 replicates each) at 10 5 Amplification curve at the concentration of copies / μl;

[0061] Fig.13 The primer pool B in Example 9 was used for 4 target plasmids (16 replicates each) at 10 2 Amplification curve at the concentration of copies / μl;

[0062] Fig.14 These are the statistical results of multiple infections of clinical samples in Example 10. DETAILED DESCRIPTION

[0063] The present invention provides a primer-probe combination for identifying pathogens of bovine respiratory disease syndrome, wherein the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

[0064] When identifying the bovine viral diarrhea 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;

[0065] When identifying the bovine parainfluenza virus type 3, 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;

[0066] When identifying the bovine respiratory syncytial virus, 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;

[0067] When identifying the bovine coronavirus, 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;

[0068] When identifying the bovine Mycoplasma, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 13, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 14, and the nucleotide sequence of the probe is shown in SEQ ID No. 15;

[0069] When identifying the bovine Pasteurella, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 16, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 17, and the nucleotide sequence of the probe is shown in SEQ ID No. 18;

[0070] When identifying the bovine hemolytic Mannheimia, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 19, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 20, and the nucleotide sequence of the probe is shown in SEQ ID No. 21;

[0071] When identifying the infectious bovine rhinotracheitis virus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No.22, the nucleotide sequence of the downstream primer is shown in SEQ ID No.23, and the nucleotide sequence of the probe is shown in SEQ ID No.24.

[0072] The present invention preferably modifies the 5' end of the probe with a fluorescent group and the 3' end with a quenching group. In the present invention, the 5' end of the probe used to identify the bovine viral diarrhea virus 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 the bovine parainfluenza virus type 3 is preferably modified with a VIC group and the 3' end is preferably modified with MGB; the 5' end of the probe used to identify the bovine respiratory syncytial 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 coronavirus is preferably modified with a Cy5 group and the 3' end is preferably modified with a BH Q2 modification; the 5' end of the probe used to identify the bovine Mycoplasma 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 the bovine Pasteurella is preferably modified with a VIC group, and the 3' end is preferably modified with MGB; the 5' end of the probe used to identify the bovine hemolytic Mannheimia 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 infectious rhinotracheitis virus is preferably modified with a Cy5 group, and the 3' end is preferably modified with BHQ2.

[0073] In the present invention, the detection target gene of bovine viral diarrhea virus is preferably 5'UTR; the detection target gene of bovine parainfluenza virus type 3 is preferably N; the detection target gene of bovine respiratory syncytial virus is preferably N; the detection target gene of bovine coronavirus is preferably N; the detection target gene of bovine Mycoplasma is preferably oppD; the detection target gene of bovine Pasteurella is preferably kmt1; the detection target gene of bovine hemolytic Mannheimia is preferably lktA; the detection target gene of bovine infectious rhinotracheitis virus is preferably gB.

[0074] The present invention also provides the use of the primer-probe combination described in the above technical solution in the preparation of a reagent for identifying the pathogen of bovine respiratory disease syndrome. In the present invention, the pathogen of bovine respiratory disease syndrome preferably includes bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

[0075] The present invention also provides a kit for identifying pathogens of bovine respiratory disease syndrome, comprising a primer pool A and a primer pool B; the primer pool A comprises primer probes for identifying bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus and bovine coronavirus in the primer-probe combination described in the above technical solution; the primer pool B comprises primer probes for identifying bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus in the primer-probe combination described in the above technical solution.

[0076] In the present invention, the concentrations of the upstream and downstream primers for identifying bovine viral diarrhea virus in the primer pool A are preferably 300 nM, and the concentration of the probe is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying bovine parainfluenza virus type 3 are preferably 100 nM, and the concentration of the probe is preferably 100 nM; the concentrations of the upstream and downstream primers for identifying bovine respiratory syncytial virus are preferably 300 nM, and the concentration of the probe is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying bovine coronavirus are preferably 200 nM, and the concentration of the probe is preferably 250 nM.

[0077] In the present invention, the concentrations of the upstream and downstream primers for identifying Mycoplasma bovis in the primer pool B are preferably 300 nM, and the concentration of the probe is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying Pasteurella bovis are preferably 100 nM, and the concentration of the probe is preferably 50 nM; the concentrations of the upstream and downstream primers for identifying Mannheimia hemolytica are preferably 100 nM, and the concentration of the probe is preferably 250 nM; the concentrations of the upstream and downstream primers for identifying bovine infectious rhinotracheitis virus are preferably 300 nM, and the concentration of the probe is preferably 250 nM.

[0078] The present invention also provides the use of the kit described in the above technical scheme in the preparation of a product for identifying the pathogens of bovine respiratory disease syndrome; the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

[0079] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0080] The following example provides a primer-probe combination for pathogen identification of bovine respiratory disease syndrome, its application and kit, which can perform two-tube one-step detection and identification of 8 common pathogens that can cause BRDC: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella bovis (PM), Mannheimia bovis (MH), and infectious bovine rhinotracheitis virus (IBRV).

[0081] Example 1

[0082] 1. Design of specific primer probes. Download the nucleic acid sequences of the conserved genes or virulence genes of 8 BRDC pathogens popular in my country in recent years from Genbank. Use sequence alignment software for multiple sequence alignment. Then select the regions that are conserved in each strain of this pathogen but specific to other common cattle pathogens to design specific primer probes. Specifically, the above primer probes are shown in Table 1, and the primer probes are synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0083] Table 1 Primer and probe sequences for amplifying 8 BRDC pathogens

[0084]

[0085]

[0086] 2. Synthesis of standard plasmids: According to the designed 8 pairs of primer probes, the corresponding target fragments were synthesized and cloned into the pUC57 plasmid vector to obtain 8 standard plasmids. The standard plasmids were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0087] 3. Nucleic acid extraction and amplification kits. The nucleic acid extraction kit was purchased from Suzhou UE Landi Biotechnology Co., Ltd.: UE Body Fluid Virus DNA / RNA Mini-preparation Kit; the real-time fluorescence quantitative PCR kit was purchased from Yisheng Biotechnology Co., Ltd.: ⅢOne Step RT-qPCR Probe Kit. The specific extraction and amplification steps were carried out according to the instructions of the above kit.

[0088] 4. Verification of the effectiveness and specificity of primer probes: Four pairs of primer probes were mixed to form primer pool A, and the remaining three pairs of primer probes were mixed to form primer pool B. The composition of the primer pool is shown in Table 2.

[0089] Table 2 qPCR primer pool for detecting 8BRDC pathogens

[0090]

[0091]

[0092] The 8 synthesized standard plasmids were diluted to 10 5 copies / μl as templates, and other bovine infectious pathogen nucleic acids stored in our laboratory as templates, using 8 pairs of primer probe sets, respectively, using IIIOne StepRT-qPCR Probe Kit uses two primer pools to prepare two reaction tubes for qPCR detection. The qPCR system is shown in Table 3.

[0093] Table 3 qPCR system

[0094]

[0095] RT-qPCR amplification was performed using the SLAN-96S fluorescent quantitative PCR instrument from Shanghai Hongshi Medical Technology Co., Ltd. The RT-qPCR amplification program was as follows: reverse transcription at 50°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 30 s, extension at 60°C for 30 s, and amplification for 45 cycles. During the extension, the fluorescence signals of the four channels of FAM, VIC, ROX, and Cy5 were collected.

[0096] Results: When nucleic acid containing the target fragment was added, both primer pools had amplification curves with CT values ​​between 18-23. When nucleic acid containing no target fragment was added, no amplification occurred. The results are shown in Table 4, indicating that both primer pools have good effectiveness and specificity.

[0097] Table 4 Results of validation of primer pool effectiveness and specificity

[0098]

[0099]

[0100] The value represents the amplification curve CT value -: RT-qPCR has no amplification curve

[0101] 5. Optimization of the optimal working concentration of primers and probes

[0102] 5.1 Optimization of the optimal working concentration of primers and probes in primer pool A

[0103] (1) Optimization of BVDV-F / BVDV-R primer concentration

[0104] Primer mixtures containing BVDV-F / BVDV-R were prepared to have working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM, respectively. The concentrations of 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 for each concentration condition. The qPCR reaction system is shown in Table 5.

[0105] Table 5 Optimization of primer concentrations for BVDV-F / BVDV-R

[0106]

[0107] The reaction conditions are the same as step 4.

[0108] (2) Optimization of primer concentrations for BPIV3-F / BPIV3-R, BRSV-F / BRSV-R, and BcoV-F / BcoV-R

[0109] Similar to the BVDV-F / BVDV-R primer concentration optimization method, primer pairs with working concentrations of 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM were prepared, and the plasmid containing the target fragment was amplified by fixing the concentrations of other primer probes.

[0110] (3) Optimization of BVDV-P, BPIV3-P, BRSV-P, and BcoV-P probe concentrations

[0111] 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 by the method of fixing the concentrations of other primers and probes.

[0112] (4) Primer probe concentration optimization results for primer pool A

[0113] After qPCR testing, each sample will obtain the amplification curve CT value and fluorescence signal intensity Rn. The three replicate well values ​​are averaged and the above primer probe concentration optimization results are statistically summarized, such as Figure 1 As shown, the method for screening the best 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:

[0114] BVDV-F: 300nM, BVDV-R: 300nM, BVDV-P: 250nM;

[0115] BPIV3-F:100nM, BPIV3-R:100nM, BPIV3-P:100nM;

[0116] BRSV-F: 300nM, BRSV-R: 300nM, BRSV-P: 250nM;

[0117] BcoV-F:200nM, BcoV-R:200nM, BcoV-P:250nM;

[0118] 5.2 Optimization of the optimal working concentration of primer probes in primer pool B

[0119] (1) Experimental method: The same as the optimization method for the optimal working concentration of primers and probes in primer pool A.

[0120] (2) Primer probe concentration optimization results for primer pool B

[0121] Similarly, the optimization results of primer and probe concentrations were statistically summarized by taking the average of the values ​​of the three replicate wells after the experiment, such as Figure 2As shown, the primer or probe concentration with the lowest CT value and the highest Rn value is selected as the optimal working concentration. Finally, the optimal working concentrations of each primer and probe after optimization are:

[0122] M.bovis-F:300nM, M.bovis-R:300nM, M.bovis-P:250nM;

[0123] PM-F:100nM, PM-R:100nM, PM-P:50nM;

[0124] MH-F:100nM, MH-R:100nM, MH-P:250nM;

[0125] IBRV-F: 300nM, IBRV-R: 300nM, IBRV-P: 250nM.

[0126] 6. Annealing temperature optimization

[0127] According to the optimal concentration of primer probe optimized in step 5, prepare two primer pools, each with 500 μl, as shown in Table 6.

[0128] Table 6 Primer pool preparation after concentration optimization

[0129]

[0130]

[0131] The 10 5 The standard plasmids with 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 temperatures of primer pool A and primer pool B were screened respectively. The reaction system was the same as in step 4. The reaction procedure is shown in Table 7.

[0132] Table 7 Annealing temperature optimization reaction program

[0133]

[0134] 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 3 As shown, the annealing temperature with the lowest CT value and the highest Rn value is selected as the optimal annealing temperature. Finally, the optimal annealing temperature is 60°C.

[0135] 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 eight common pathogens of bovine respiratory syndrome 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, wherein the annealing temperature was selected to be 60°C, and the mixed standard plasmids (the concentration of each plasmid was about 10 5 copies / μl) was used as the positive control of this kit.

[0136] 7. Establishment of standard curve

[0137] Eight kinds of synthesized standard plasmids with known copy numbers were diluted 10 times in a gradient manner, and qPCR was performed using the kit prepared by the present invention and the SLAN-96S instrument. Each concentration of plasmid was tested in triplicate. The CT value of the amplification curve was used as the ordinate, and the logarithm of the template copy number was used as the abscissa. The standard curve was drawn using Graphpad Prism8 software. The standard curve of primer pool A is shown in FIG. Figure 4-Figure 5 As shown, the standard curve of primer pool B is as follows Figure 6-Figure 7 The slopes of the standard curves of primer pool A for amplifying BVDV, BPIV, BRSV, and BcoV plasmids were -3.468, -3.503, -3.548, and -3.473, respectively, and their amplification efficiencies were 94.25.0%, 92.96%, 91.36%, and 94.06%, respectively. 2 The slopes of the standard curves of primer pool B for amplifying M.bovis, PM, MH, and IBRV plasmids were -3.443, -3.505, -3.535, and -3.426, respectively, and their amplification efficiencies were 95.18%, 92.89%, 91.82%, and 95.83%, respectively. 2 All of them were greater than 0.999, indicating that primer pool B also had a high amplification efficiency.

[0138] 8. Sensitivity test

[0139] The standard plasmid was diluted to 10 copies / μl, 5 copies / μl, 1 copy / 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 8 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 9 shown.

[0140] Table 8 Sensitivity test results

[0141]

[0142] 9. Repeatability Experiment

[0143] like Figure 10-13 As shown, the standard plasmids were diluted to 10 5 copies / μl, 10 2 The mixed plasmids were then mixed to obtain the mixed plasmids. The mixed plasmids were used as templates and 16 replicates were tested at each concentration to evaluate the repeatability of the kit prepared by the present invention. After the test, 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 template with 10 copies / μl was used for the amplification. The CT value was between 19 and 22, and the coefficient of variation was 0.27%-0.61%. 2 The template was 100 copies / μl, the amplified CT value was between 30 and 33, and the coefficient of variation was 0.40%-0.97%, indicating that the kit prepared by the present invention has high repeatability.

[0144] Table 9 Repeatability test results

[0145]

[0146] 10. Clinical sample testing

[0147] In order 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: first, RNA was 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.

[0148] The results are shown in Table 10. Farm A detected four pathogens, BVDV, BcoV, PM and MH, and the positive rates were all high. Farm B detected four pathogens, BcoV, PM, MH and IBRV, among which MH had a higher positive rate. Farm A had a higher overall infection rate than Farm B. The detection effect of the kit prepared by the present invention on clinical samples was successfully verified. In addition, the kit can further detect clinical samples with mixed infections, and it can be found that the pathogens causing BRDC in Farm A are more common with multiple pathogens ( Fig.14 ), which can provide timely and effective reference for the prevention and control of BRDC in clinical practice.

[0149] Table 10 Clinical sample test results

[0150]

[0151] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A primer-probe combination for identifying pathogens of bovine respiratory disease syndrome, wherein the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus, characterized in that: When identifying the bovine viral diarrhea 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 parainfluenza virus type 3, 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 respiratory syncytial virus, 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 bovine coronavirus, 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; When identifying the bovine Mycoplasma, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 13, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 14, and the nucleotide sequence of the probe is shown in SEQ ID No. 15; When identifying the bovine Pasteurella, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 16, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 17, and the nucleotide sequence of the probe is shown in SEQ ID No. 18; When identifying the bovine hemolytic Mannheimia, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No. 19, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 20, and the nucleotide sequence of the probe is shown in SEQ ID No. 21; When identifying the bovine infectious rhinotracheitis virus, the nucleotide sequence of the upstream primer of the primer probe set used is shown in SEQ ID No.22, the nucleotide sequence of the downstream primer is shown in SEQ ID No.23, and the nucleotide sequence of the probe is shown in SEQ ID No.

24.

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 quenching group.

3. The primer-probe combination according to claim 2, characterized in that: The 5' end of the probe used to identify the bovine viral diarrhea virus 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 the bovine parainfluenza virus type 3 is modified with a VIC group and the 3' end is modified with an MGB group; The 5' end of the probe used to identify the bovine respiratory syncytial 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 coronavirus 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 Mycoplasma 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 the bovine Pasteurella is modified with a VIC group, and the 3' end is modified with MGB; The 5' end of the probe used to identify the bovine hemolytic Mannheimia is modified with a ROX group, and the 3' end is modified with MGB; The 5' end of the probe used to identify the infectious bovine rhinotracheitis virus is modified with a Cy5 group, and the 3' end is modified with BHQ2.

4. The primer-probe combination according to claim 1, characterized in that: The detection target gene of the bovine viral diarrhea virus is 5'UTR; The detection target gene of bovine parainfluenza virus type 3 is N; The detection target gene of the bovine respiratory syncytial virus is N; The detection target gene of the bovine coronavirus is N; The detection target gene of bovine Mycoplasma is oppD; The detection target gene of the bovine Pasteurella is kmt1; The detection target gene of the bovine hemolytic Mannheimia is lktA; The detection target gene of the bovine infectious rhinotracheitis virus is gB.

5. Use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of a reagent for identifying the pathogen of bovine respiratory disease syndrome.

6. The use according to claim 5, characterized in that: The pathogens of the bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

7. A kit for identifying the pathogen of bovine respiratory disease syndrome, characterized in that: Including primer pool A and primer pool B; The primer pool A includes primer probes for identifying bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus and bovine coronavirus in the primer-probe combination of claim 1; The primer pool B includes primer probes for identifying Mycoplasma bovis, Pasteurella bovis, Mannheimia hemolytica and bovine infectious rhinotracheitis virus in the primer-probe combination of claim 1.

8. The kit according to claim 7, characterized in that The concentrations of the upstream and downstream primers for identifying bovine viral diarrhea virus in the primer pool A are both 300 nM, and the concentration of the probe is 250 nM; The concentrations of the upstream and downstream primers for identifying bovine parainfluenza virus type 3 were both 100 nM, and the concentration of the probe was 100 nM; The concentrations of the upstream and downstream primers for identifying bovine respiratory syncytial virus were both 300 nM, and the concentration of the probe was 250 nM; The concentrations of the upstream and downstream primers for identifying bovine coronavirus were both 200 nM, and the concentration of the probe was 250 nM.

9. The kit according to claim 7, characterized in that The concentrations of the upstream and downstream primers for identifying Mycoplasma bovis in the primer pool B are both 300 nM, and the concentration of the probe is 250 nM; The concentrations of the upstream and downstream primers for identifying Pasteurella bovis were both 100 nM, and the concentration of the probe was 50 nM; The concentrations of the upstream and downstream primers for identifying Mannheimia bovis were both 100 nM, and the concentration of the probe was 250 nM; The concentrations of the upstream and downstream primers for identifying bovine infectious rhinotracheitis virus were both 300 nM, and the concentration of the probe was 250 nM.

10. Use of the kit according to any one of claims 7 to 9 in the preparation of a product for identifying pathogens of bovine respiratory disease syndrome; the pathogens of bovine respiratory disease syndrome include bovine viral diarrhea virus, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bovine coronavirus, bovine mycoplasma, bovine Pasteurella, bovine hemolytic Mannheimia and bovine infectious rhinotracheitis virus.

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