Primer probe set, reaction system and application of BRV, BCoV and BVDV TaqMan multiplex fluorescent quantitative PCR detection

Abstract

The application belongs to the technical field of molecular biology detection, and particularly relates to a primer probe set, a reaction system and application for BRV, BCoV and BVDV TaqMan multiplex fluorescent quantitative PCR detection, wherein the primer probe set comprises three pairs of specific primers and three TaqMan probes designed respectively for BRV VP1 gene, BCoV N gene and BVDV 5'-UTR gene, the three probes are respectively marked with FAM, HEX and CY5 fluorescent groups, the primer probe set can simultaneously detect three pathogens of bovine rotavirus, bovine coronavirus and bovine viral diarrhea virus in the same reaction system, has the advantages of fast detection speed, high sensitivity, strong specificity and good repeatability, and is suitable for epidemiological investigation, disease monitoring, clinical rapid diagnosis and disease prevention and control of bovine diarrhea cases caused by main viruses.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically relating to a primer and probe set, reaction system, and application for TaqMan multiplex quantitative PCR detection of BRV, BCoV, and BVDV. Background Technology

[0002] Bovine diarrhea is a common outbreak disease in the cattle industry, especially harmful to calves. It can lead to stunted growth, decreased productivity, and even death in severe cases, causing significant economic losses to the cattle industry. Bovine rotavirus (BRV), bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV) are not only involved in independent infections clinically, but more often occur as mixed infections of two or more viruses with similar symptoms, making diagnosis difficult based on clinical signs alone. Single-sample real-time quantitative PCR testing is inefficient and costly, failing to meet the needs of rapid diagnosis and disease control. Summary of the Invention

[0003] This invention aims to provide a primer and probe set, reaction system, and application for TaqMan multiplex quantitative PCR detection of BRV, BCoV, and BVDV. The goal is to establish a TaqMan multiplex quantitative PCR method capable of simultaneously detecting BRV, BCoV, and BVDV, thereby providing effective technical support for the rapid diagnosis and epidemiological investigation of bovine diarrhea.

[0004] Because single-item real-time quantitative PCR detection is inefficient, costly, and unable to meet the needs of rapid diagnosis and epidemic prevention and control, TaqMan multiplex quantitative PCR technology, by adding multiple pairs of specific primers and probes labeled with different fluorescent groups to a single reaction system, can simultaneously perform qualitative or quantitative detection of multiple target nucleic acids in the same tube, offering advantages such as high throughput, speed, specificity, and low risk of contamination. Based on this, to achieve the above objectives, the present invention adopts the following technical solution: A primer and probe set for TaqMan multiplex quantitative PCR detection of BRV, BCoV, and BVDV is provided, the primer and probe set comprising: (1) Specific primer pairs and probes targeting the BRV VP1 gene: The upstream primer BRV-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer BRV-R has the nucleotide sequence shown in SEQ ID NO:2; The probe BRV-Probe has the nucleotide sequence shown in SEQ ID NO:3; (2) Specific primer pairs and probes targeting the BCoV N gene: Upstream primer BCoV-F: Its nucleotide sequence is shown in SEQ ID NO:4; Downstream primer BCoV-R: Its nucleotide sequence is shown in SEQ ID NO:5; The probe BCoV-Probe has the following nucleotide sequence as shown in SEQ ID NO:6; (3) Specific primer pairs and probes targeting the BVDV 5'-UTR gene: Upstream primer BVDV-F: Its nucleotide sequence is shown in SEQ ID NO:7; Downstream primer BVDV-R: Its nucleotide sequence is shown in SEQ ID NO:8; The probe BVDV-Probe has the following nucleotide sequence as shown in SEQ ID NO:9.

[0005] Preferably, the fluorescent reporter groups of the probes BRV-Probe, BCoV-Probe, and BVDV-Probe are of different types.

[0006] The present invention also provides a reaction system for the simultaneous detection of BRV, BCoV and BVDV, comprising the primer and probe set described in any of the preceding claims.

[0007] This invention also provides the application of the aforementioned primer-probe set in the preparation of bovine diarrhea virus detection reagents or reaction systems.

[0008] Compared with existing technologies, the advantages of this invention are as follows: This invention designs specific primers and probes for the VP1 gene of BRV, the N gene of BCoV, and the 5'-UTR gene of BVDV. The three probes are labeled with three different fluorescent groups, FAM, HEX, and CY5, respectively, enabling the simultaneous detection of the three viruses in the same reaction tube without interference, greatly improving detection efficiency and reducing detection costs. The reaction system of this primer and probe set has high detection specificity and no cross-reactivity with other bovine viruses such as bovine astrovirus (BAstV), bovine norovirus (BNoV), bovine herpesvirus type 4 (BoHV-4), bovine infectious rhinotracheitis virus (IBRV), bovine parvovirus (BPV), and bovine topovirus (BToV); moreover, this primer and probe set has high detection sensitivity and good repeatability, which can meet the needs of rapid detection and diagnosis. Attached Figure Description

[0009] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a graph showing the results of RT-PCR detection of recombinant plasmids; Figure 2 The figure shows the optimization results of annealing temperature for TaqMan single-fluorescent quantitative PCR for each pathogen, where A represents BRV, BCoV, and C represents BVDV. Figure 3 The figure shows the optimization results of TaqMan single fluorescent quantitative PCR probes and primer concentrations for three pathogens, where A represents BRV, BCoV, and C represents BVDV. Figure 4 This is a standard curve for BRV single-particle quantitative PCR. Figure 5 Standard curve for BCoV singleton quantitative PCR; Figure 6 This is a standard curve for BVDV single-cell quantitative PCR. Figure 7 The images show the results of TaqMan single-fluorescent quantitative PCR specificity assays for each pathogen, where A represents BRV, BCoV, and C represents BVDV. Figure 8 The image shows the results of TaqMan single-fluorescent quantitative PCR sensitivity assays for each pathogen, where A represents BRV, BCoV, and C represents BVDV. Figure 9 Figure showing the optimization results of TaqMan multiplex quantitative PCR for three pathogens; Figure 10 Figure 1 shows the optimization results of the best primers and probe concentrations for TaqMan multiplex quantitative PCR of various pathogens. Figure 11 Figure showing the results of a TaqMan multiplex quantitative PCR cross-validation assay; Figure 12 Figure 1 shows the results of a TaqMan multiplex quantitative PCR specificity assay. Figure 13 Figure 1 shows the results of a TaqMan multiplex quantitative PCR sensitivity assay. Figure 14 This is a BRV standard curve diagram for multiplex PCR. Figure 15 A standard curve of BCoV in multiplex PCR; Figure 16 This is a standard curve of BVDV in multiplex PCR. Detailed Implementation

[0010] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual, or as recommended by the manufacturer.

[0012] The materials used in each embodiment include: 1. Viral positive nucleic acids and samples: Positive nucleic acids for viruses such as bovine astrovirus (BAstV), bovine norovirus (BNoV), bovine herpesvirus type 4 (BoHV-4), bovine infectious rhinotracheitis virus (IBRV), bovine rotavirus (BRV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine parvovirus (BPV), and bovine topovirus (BToV) were all identified by our laboratory and preserved at low temperatures.

[0013] 2. Test instruments: The instruments and equipment used in each embodiment and their manufacturers are shown in Table 1-1.

[0014] Table 1-1 Instrument and Equipment Information

[0015] 3. Main reagents: The main reagents used and their manufacturers are listed in Table 1-2.

[0016] Table 1-2 Reagent Information

[0017] Based on the above experimental materials, the following are examples of related experiments: Specific primer design and probe synthesis Published specific or conserved gene sequences of BRV, BCoV, and BVDV were screened from the GenBank database. The VP1 gene of BRV, the N gene of BCoV, and the 5'-UTR gene of BVDV were selected as amplification target genes. Specific primers and probes were designed using Primer-BLAST from NCBI. After BLAST validation, primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Primer and probe information is shown in Tables 1-3. The primer-probe set includes three pairs of specific primers and three TaqMan probes designed for the BRV VP1 gene, BCoV N gene, and BVDV 5'-UTR gene, respectively. The three probes are labeled with FAM, HEX, and CY5 fluorescent groups, respectively.

[0018] Table 1-3 Primer and Probe Information

[0019] Preparation of positive standard plasmids Using BRV cDNA, BCoV cDNA, and BVDV cDNA as templates, and employing 2×TaqPlus Master Mix reagents, the templates were amplified using primers designed in Table 1-3 and the corresponding annealing temperatures. The reaction system and procedure are detailed in Tables 1-4 and 1-5.

[0020] Table 1-4 PCR Reaction System

[0021] Table 1-5 PCR reaction procedures

[0022] The PCR products were detected using a 1% agarose gel electrophoresis system at a constant voltage of 120 V for 40 min. The position and size of the target band were then observed and confirmed using a gel imaging system to ensure they matched the primer design. The target band was then excised using a UV gel cutter, and the PCR products were recovered and purified using the OMEGA Gel Extraction Kit. The specific steps are as follows: (1) Place the cut gel block containing the target fragment into a sterile 1.5 mL centrifuge tube, weigh it, add an equal amount of XP2Binding Buffer, and incubate in a 55℃ water bath until the gel is completely dissolved. You can use a vortex mixer to shake it.

[0023] (2) Transfer all of the above solution to a HiBind® DNA Mini binding column equipped with a 2 mL collection tube, centrifuge at 10000×g for 1 min, and discard the filtrate in the collection tube; (3) Put the binding column back into the collection tube, add 300 μL XP2 Binding Buffer into the column, centrifuge at the maximum speed for 1 min, and discard the filtrate; (4) Keep the binding column and the collection tube in the assembled state, add 700 μL LSPW Buffer, centrifuge at 10000×g for 1 min and discard the filtrate; (5) Repeat the washing operation in step 4; (6) Put the binding column back into the collection tube again, centrifuge at 10000×g for 2min to completely remove residual buffer; (7) Transfer the treated binding column to a new sterile 1.5 mL centrifuge tube, add 30 μL ddH2O, let stand at room temperature for 5 min, and centrifuge at 13,000 × g for 1 min to complete the elution of DNA; (8) After measuring the concentration of the PCR purified product obtained by elution, store it at -20℃ for later use.

[0024] (9) The purified PCR product was ligated with the pMD18-T cloning vector of Baori Biotechnology (Beijing) Co., Ltd. The specific ligation system is shown in Table 1-6.

[0025] Table 1-6 Connecting Carrier Systems

[0026] Invert the mixture to mix thoroughly, then incubate in an ice box for 12-16 hours. Subsequently, proceed with the transformation according to the DH5α competent cell instructions from Baori Biotechnology (Beijing) Co., Ltd. The steps are as follows: (1) Thaw DH5α competent cells in advance, take 50 μL of thawed competent cells and add each ligation product, mix thoroughly, and incubate on ice for 30 min. (2) Place the above mixture in a 42°C water bath for 30-60 seconds, and then transfer it to ice to cool for 2 minutes. (3) Add 1 ml of sterile antibiotic-free LB broth medium to each tube of reaction solution, gently invert and mix, then place in an intelligent precision shaker with the temperature set to 37°C and shake at 200 rpm for 1 h to complete the bacterial culture. (4) Take 200 μL of the cultured bacterial solution and spread it evenly on the surface of LB solid agar medium containing ampicillin (Amp). After the bacterial solution is completely absorbed by the medium, invert the plate and place it in a 37°C constant temperature incubator for overnight culture. (5) Prepare 15 mL sterile centrifuge tubes in advance and add 7 mL of LB broth medium containing Amp into the tubes; (6) After white colonies grow on the plate, use a sterile pipette to pick up individual colonies and inoculate them into the above-mentioned LB broth medium containing antibiotics. Repeat the pipetting several times to ensure that the colonies are fully dispersed. (7) Place the inoculated centrifuge tubes in a 37°C air bath constant temperature shaker and shake for 12–16 h; (8) After observing that the bacterial solution is turbid and that sediment appears at the bottom of the tube, take a portion of the bacterial solution and mix it with 50% glycerol solution at a ratio of 1:1, and store it at -80℃ for later use.

[0027] The remaining bacterial culture was used for plasmid extraction using an OMEGA plasmid mini-prep kit. The specific steps are as follows: (1) Collection of bacterial cells: Take an appropriate amount of cultured bacterial solution, transfer it to a 1.5 mL sterile centrifuge tube, centrifuge at 8000×g for 90 s at room temperature, discard the supernatant, and collect the bacterial cell precipitate.

[0028] (2) Resuspension: Add 250 µL of a mixture of Solution I and RNase A to a centrifuge tube and use a vortex mixer or pipette to disperse the bacterial pellet completely, ensuring that there are no visible cell clumps.

[0029] (3) Lysis: Add 250µL Solution II to the tube and immediately gently invert the centrifuge tube 4-6 times to mix the contents thoroughly (avoid vigorous shaking). The solution should become clear and viscous at this point. Let it stand at room temperature for 2-3 minutes to ensure complete lysis of the bacteria.

[0030] (4) Neutralization: Then add 350 µL Solution III and immediately gently invert the centrifuge tube several times until a large amount of white flocculent precipitate (genomic DNA, protein and SDS complex) is observed to form. Place the centrifuge tube in a centrifuge and centrifuge at 13000×g at room temperature for 10 min.

[0031] (5) Binding: Carefully aspirate the supernatant (approximately 850 µL) after centrifugation and transfer it to a HiBind® DNA binding column fitted with a collection tube, taking care to avoid aspirating the white precipitate. Centrifuge at 10000 ×g for 60 s at room temperature to allow the plasmid DNA to adsorb onto the membrane of the collection column wells, and discard the filtrate in the collection tube.

[0032] (6) Washing: Add 500 µL of HBC Buffer to the binding column, centrifuge at 10000 × g for 60 s, and discard the filtrate (this step is to remove impurities such as proteins). Add 700 µL of DNA Wash Buffer (with anhydrous ethanol added according to the instructions) to the binding column, centrifuge at 10000 × g for 60 s, and discard the filtrate. Repeat the above steps of adding 700 µL of DNA Wash Buffer and centrifuging to wash once.

[0033] (7) Drying: Place the binding column back into the empty collection tube and centrifuge again at the maximum speed (≥13000 ×g) for 2 min to completely remove residual ethanol and prevent it from affecting subsequent enzymatic reactions.

[0034] (8) Elution: Transfer the binding column to a new 1.5 mL sterile centrifuge tube, and add 50 µL of Elution Buffer or sterile DEPC water to the center of the membrane well of the binding column. After standing at room temperature for 2 min, centrifuge at 13000 × g for 90 s. The liquid in the collection tube is the purified plasmid DNA solution, which should be stored at -20℃ for later use.

[0035] (9) The prepared positive recombinant plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmids that were verified by sequencing results were named pMD18-T-BRV, pMD18-T-BCoV and pMD18-T-BVDV, respectively. The plasmid concentration was detected by an enzyme-linked immunosorbent assay (ELISA) reader and the copy number was calculated to construct recombinant plasmid standards.

[0036] Establishment and optimization of TaqMan single-fluorescent quantitative PCR detection method for three pathogens Dilute the standards of each pathogen-positive recombinant plasmid to 10 μL. 8 Using copies / µL as the template for optimization, the 2×M5 GoldStar TaqMan Mixture kit from Polymer Biotech was used to initially construct and optimize the BRV single-fluorescent quantitative PCR reaction system. The total reaction volume was 25 µL. The basic reaction mixture was prepared according to the kit instructions. Specific sample loading procedures are shown in Table 1-7, and thermal cycling parameters are set in Table 1-8.

[0037] To obtain optimal reaction performance, a checkerboard method was used to systematically optimize key reaction conditions. First, an annealing temperature gradient (53℃~63℃) was established to determine the optimal annealing temperature. Simultaneously, the final concentrations of primers and probes were screened using a gradient approach, setting six concentration levels (0.15 µmol / L, 0.20 µmol / L, 0.25 µmol / L, 0.30 µmol / L, 0.35 µmol / L, and 0.40 µmol / L). Matrix experiments with different concentration combinations were conducted to investigate the impact of each condition combination on amplification efficiency. The fluorescence intensity, peak time (Ct value), and overall curve shape of the amplification curve were used as evaluation indicators. The optimal reaction conditions for single-pathogen quantitative PCR were selected based on the highest fluorescence signal gain, the lowest Ct value, and the parabolic amplification curve.

[0038] Table 1-7 TaqMan single-fluorescent quantitative PCR reaction systems for three pathogens

[0039] Table 1-8 TaqMan Single / Multiplex Real-Time Quantitative PCR Reaction Procedures

[0040] Establishment and optimization of TaqMan multiplex quantitative PCR detection method Based on the optimized singlet PCR, a TaqMan multiplex real-time PCR detection method was established. Three positive recombinant plasmids mixed in the same proportion were used as positive templates, three pairs of primers were mixed in equal proportions as primer mixtures, and three probes were mixed in equal proportions as probe mixtures. The reaction system and procedure are shown in Tables 1-9 and 1-8. The optimal reaction conditions were determined based on the amplification curves by optimizing the annealing temperature, primer and probe concentrations.

[0041] Table 1-9 TaqMan multiplex quantitative PCR reaction systems for three pathogens

[0042] Cross-test To verify the specificity and effectiveness of this method, specific primers and TaqMan probes for bovine rotavirus (BRV), bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV) were designed and amplified under optimized conditions using their corresponding positive recombinant plasmids as templates.

[0043] Specificity test An optimized single-particle and TaqMan multiplex quantitative PCR detection system was used, with three positive recombinant plasmids and their equal-volume mixtures as positive controls, and DEPC water as a negative control, to detect the nucleic acids of bovine rotavirus (BRV), bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV). The specificity of the detection method was validated by observing the real-time quantitative PCR curve results.

[0044] Sensitivity test To verify the sensitivity of this method, three positive recombinant plasmids and a mixture of the three plasmids in equal proportions were serially diluted 10-fold (a total of 7 gradients, with a concentration range of 10). 7 ~10 1 The diluted plasmid (copies / μL) was used as a template, and the optimized TaqMan single and multiplex real-time PCR methods were used for detection.

[0045] Repeatability test With a concentration of 10 after dilution 6 copies / uL, 10 5copies / uL and 10 4 Using three positive recombinant plasmids with copies / uL as templates, repeatability tests were performed using optimized TaqMan single and triple quantitative PCR. Three replicates were set for each concentration, and the method repeatability was evaluated by standard deviation and coefficient of variation.

[0046] Application test The constructed TaqMan single and triple quantitative PCR technologies were used to detect clinical serum samples, and the results were compared with the quantitative PCR methods for BRV, BVDV, and BCoV reported in the domestic AKAV industry standard and literature to evaluate their compliance rate.

[0047] The results of the above experiments are as follows: Positive recombinant plasmid standard test results Sequencing analysis of the constructed recombinant plasmid and alignment with the NCBI BLAST tool showed that it was consistent with the target sequence. Further RT-PCR detection revealed specific bands of the expected design size for all bands: 93 bp for BRV, 98 bp for BCoV, and 102 bp for BVDV (e.g., 102 bp for BVDV). Figure 1 The reaction system is shown in the figure. Note: In Figure 1 In the RT-PCR detection results of the recombinant plasmids, M is the DL500 DNA Marker; 1-3 are BRV, BCoV, and BVDV, respectively; 4. Negative control. These results indicate that the three recombinant plasmids were successfully constructed and named pMD18-T-BRV, pMD18-T-BCoV, and pMD18-T-BVDV, respectively. The concentrations of the three plasmids were determined by microplate spectrophotometry, and were 165.32, 158.54, and 256.72 mg / L, respectively. The copy numbers were converted to 5.01 × 10⁻⁶, respectively. 10 4.80×10 10 and 7.78×10 10 The plasmid was copied / µL and then stored at -80℃ for later use.

[0048] Determination of Optimal Annealing Temperature for TaqMan Single-Fluorescent Quantitative PCR To determine the optimal annealing temperature, eight temperature gradients (53℃, 53.7℃, 55.0℃, 56.9℃, 59.4℃, 61.3℃, 62.5℃, and 63℃) were used for amplification, while other reaction conditions remained consistent. The amplification results showed that all three pathogens produced specific amplification curves within the experimental temperature range (e.g., [missing information]). Figure 2 As shown, note: in Figure 2In the TaqMan single-fluorescence quantitative PCR annealing temperatures for each pathogen (A represents BRV, B represents BCoV, and C represents BVDV), the Ct values ​​obtained under each temperature condition were compared, and the temperature corresponding to the highest amplification efficiency was selected as the optimal annealing temperature. After optimization, the optimal annealing temperatures for pMD18-T-BRV, pMD18-T-BCoV, and pMD18-T-BVDV were finally determined to be 55.0℃, 56.9℃, and 55.0℃, respectively.

[0049] Determination of optimal primer and probe concentrations for TaqMan single-color quantitative PCR To optimize primer and probe concentrations, six final concentration gradients were set: 0.15 µmol / L, 0.20 µmol / L, 0.25 µmol / L, 0.30 µmol / L, 0.35 µmol / L, and 0.40 µmol / L. TaqMan singlet quantitative PCR amplification was performed using the checkerboard method at the optimal annealing temperatures for each of the three pathogens, while other reaction parameters remained constant. Experimental results showed that typical amplification curves (e.g., [missing information]) could be obtained for all three pathogens within the tested primer and probe concentration ranges. Figure 3 As shown, note: Figure 3 Among the three pathogens identified by TaqMan single-probe quantitative PCR, A represents BRV, B represents BCoV, and C represents BVDV. By comprehensively analyzing the Ct values ​​obtained under various concentration combinations, and using the principle of maximizing amplification efficiency while minimizing primer and probe usage, the optimal primer and probe concentrations for each recombinant plasmid were determined as follows: pMD18-T-BRV: 0.35 µmol / L, pMD18-T-BCoV: 0.35 µmol / L, and pMD18-T-BVDV: 0.3 µmol / L.

[0050] Establishment of standard curve To evaluate the amplification efficiency and linearity of the established TagMan singlet real-time PCR method, three plasmid standards, pMD18-T-BRV, pMD18-T-BCoV, and pMD18-T-BVDV, were serially diluted 10-fold to obtain concentrations of 10... 7 copies / uL, 10 6 copies / uL, 10 5 copies / uL, 10 4 copies / uL and 10 3A series of templates in copies / µL were amplified using an optimized singlet real-time PCR method. Linear regression analysis was performed on the obtained Ct values ​​and logarithmic values ​​of each template concentration using the Bio-Rad CFX Manager real-time PCR instrument software and Excel to construct standard curves for the three pathogens. The results showed that the regression equation for the standard curve of pMD18-T-BRV was y = -2.7754x + 30.904, with a coefficient of determination R² = 0.9909 (see...). Figure 4 The standard curve regression equation for pMD18-T-BCoV is y = -3.5716x + 37.056, R² = 0.9948 (see...). Figure 5 The standard curve regression equation for pMD18-T-BVDV is y = -4.1616x + 42.726, R² = 0.9948 (see...). Figure 6 The above results show that the standard curves established by the three methods all have good linearity and meet the requirements for quantitative detection.

[0051] TaqMan single-fluorescent quantitative PCR specificity assay results The established TaqMan single-fluorescent quantitative PCR method was used to amplify nucleic acid samples of three virus-positive recombinant plasmids: pMD18-T-BVDV, pMD18-T-BRV, pMD18-T-BCoV, as well as BoHV-4, IBRV, BAstV, and BToV. The results showed that only the target virus exhibited an S-type specific amplification curve, while no amplification curve was observed for the other viruses. Figure 7 This indicates that the constructed TaqMan single-fluorescent quantitative PCR detection method has good specificity.

[0052] TaqMan single-fluorescence quantitative PCR sensitivity test results Recombinant plasmid standards for BVDV, BCoV, and BRV viruses were serially diluted 10-fold, resulting in a total of 8 dilution gradients and plasmid concentrations ranging from 10 to 10. 1 ~10 8 copies / µL. Amplification was performed using recombinant plasmids of various dilutions as templates. The lowest plasmid concentration that consistently produced a specific S-shaped amplification curve was used to determine the limit of detection (LOD) of the TaqMan single-color quantitative PCR method. Results showed ( Figure 8 Within the Ct value range of less than 35, the lowest detection limit of pMD18-T-BRV is 10. 1 The lowest detection limit for pMD18-T-BCoV is 10 copies / μL. 1 The limit of detection for pMD18-T-BVDV is 10 copies / μL.3 copies / μL. Results of optimal annealing temperature for TaqMan multiplex quantitative PCR Based on the optimal annealing temperature for TaqMan singlet quantitative PCR, similar annealing gradients were selected for screening to obtain the optimal annealing temperature for TaqMan multiplex quantitative PCR. The temperature gradients were set to 53.7℃, 55.0℃, 56.9℃, and 59.4℃, with other reaction conditions remaining unchanged. The results are shown below ( Figure 9 As shown in the figure, the annealing temperature of 55.0℃, which has the highest amplification efficiency, was finally selected as the optimal annealing temperature.

[0053] Determination of optimal primer and probe concentrations for TaqMan multiplex quantitative PCR To optimize the working concentrations of primers and probes, six concentration levels were set in the range of 0.15–0.40 µmol / L with a gradient of 0.05 µmol / L. Using the checkerboard method, with the annealing temperature fixed at 55.0℃ and other conditions unchanged, the concentrations showing the most pronounced amplification curves and the lowest primer and probe usage were selected as the optimal working concentrations. The optimal primer and probe concentrations for this method were ultimately determined to be 0.35 µmol / L. The results are shown in (…). Figure 10 ).

[0054] TaqMan multiplex quantitative PCR cross-test results pMD18-T-BRV, pMD18-T-BCoV, and pMD18-T-BVDV reacted only with their respective primers and probes. Results are shown below. Figure 11 As shown in the figure, this indicates that the primers and probes are highly specific and have no cross-reactivity.

[0055] TaqMan multiplex quantitative PCR specificity assay results The established TaqMan multiplex quantitative PCR method was used to amplify nucleic acid samples of three virus-positive recombinant plasmids: pMD18-T-BVDV, pMD18-T-BRV, pMD18-T-BCoV, as well as BoHV-4, IBRV, BAstV, and BToV, with DEPC water used as a negative control. The experimental results showed ( Figure 12 Only the target virus showed an S-type specific amplification curve, while other viruses and the negative control did not show this curve, indicating that the method has good specificity.

[0056] TaqMan multiplex quantitative PCR sensitivity test results Recombinant plasmid standards for BVDV, BCoV, and BRV viruses were serially diluted 10-fold, resulting in a total of 7 dilution gradients and plasmid concentrations ranging from 10 to 10. 7~10 1 copies / µL. Recombinant plasmids of each dilution were mixed in equal proportions and used as templates. Sensitivity tests were performed using the optimized TaqMan multiplex quantitative PCR reaction system. Results (e.g.) Figure 13 As shown in the figure. Within the Ct value range of less than 35, the limit of detection for plasmid pMD18-T-BRV is 10. 2 copies / uL, the standard curve is y=-3.6216x+41.206, R2=0.9923 (see Figure 14 The lowest detection limit for pMD18-T-BCoV is 10. 2 The standard curve is y = -3.5216x + 36.906, with R² = 0.9947 (see [reference]). Figure 15 The lowest detection limit for pMD18-T-BVDV is 10. 2 copies / uL, the standard curve is y=-4.5316x+44.256, R2=0.9991 (see) Figure 16 ).

[0057] TaqMan multiplex quantitative PCR repeatability test results The standard template, a mixture of pMD18-T-BVDV, pMD18-T-BRV, and pMD18-T-BCoV plasmids, was diluted 10-fold. 6 copies / uL, 10 5 copies / uL and 10 4 The TaqMan multiplex quantitative PCR detection method was tested for intra- and inter-group reproducibility using a mixed standard plasmid template with copies / L. The results are shown in Table 1-10. The coefficients of variation for the mixed standard plasmid template were all less than 4%, indicating that the method has good reproducibility.

[0058] Table 1-10 Results of TaqMan Multiplex Quantitative PCR Repeatability Tests

[0059] TaqMan multiplex quantitative PCR application test results Ninety-six fecal samples from calves with diarrhea, collected from large-scale cattle farms in Ningxia, were tested using TaqMan multiplex quantitative PCR. Validation experiments were also conducted using PCR detection methods according to relevant Chinese industry standards. The specific results of the two methods are detailed in Table 1-11. Single and mixed infections were found.

[0060] Single infection: Multiplex quantitative PCR was used to test 96 clinical samples. The results showed that 17 samples were positive for BRV, with a positive rate of 17.71% (17 / 96); 10 samples were positive for BCoV, with a positive rate of 10.41% (10 / 96); and 14 samples were positive for BVDV, with a positive rate of 14.58% (14 / 96). The above detection results were compared with industry standard methods one by one: BRV detection results were compared with the standard NY / T 4742-2025 "Diagnostic Techniques for Bovine Rotavirus Infection" (this standard uses the VP7 gene as the target gene, while this method uses the VP1 gene); BCoV detection results were compared with the standard T / CVMA 203—2024 "Diagnostic Techniques for Bovine Coronavirus Infection" (this technique is consistent with this paper, both using the N gene as the target gene); and BVDV detection results were compared with the standard GB / T 18637-2018 "Diagnostic Technical Specifications for Bovine Viral Diarrhea / Mucosal Disease" (this technique is consistent with this paper, both using the 5'-UTR gene as the target gene). The overall concordance rate of the two methods in the detection of the three pathogens was over 93%. This result indicates that the primers and probes designed in this embodiment have good specificity and sensitivity, and the detection results are highly consistent with industry standard methods, fully demonstrating the accuracy of this method.

[0061] Mixed Infections: Multiplex detection further revealed complex multi-pathogen mixed infections in calf diarrhea samples from Ningxia. The detected mixed infection patterns were as follows: 6 samples with BRV and BCoV co-infection (6.25%, 6 / 96); 8 samples with BRV and BVDV co-infection (8.34%, 8 / 96); 4 samples with BCoV and BVDV co-infection (4.17%, 4 / 96); and 4 samples with BRV, BCoV, and BVDV tri-infection (4.17%, 4 / 96). All mixed infection samples detected by this method were compared and verified with the results of industry standard methods. The results showed that the detection results for all mixed infection patterns were completely consistent, with a concordance rate of 100%. This result demonstrates that the multiplex quantitative PCR method established in this embodiment can accurately identify mixed infections of multiple pathogens in complex clinical samples, providing technical support for clinical diagnosis and disease control.

[0062] In summary, the TaqMan multiplex quantitative PCR method constructed in this embodiment of the invention showed extremely high consistency with the Chinese industry standard PCR detection method in the detection of 96 clinical samples, regardless of whether the infection was caused by a single pathogen or a mixed infection. The overall concordance rate was over 93%, and the detection rate for mixed infections was 100%. This result indicates that the multiplex RT-PCR detection method has good clinical applicability and high reliability, and its detection results are stable and accurate, meeting the needs for rapid clinical detection and diagnosis of calf diarrhea-related viruses.

[0063] Table 1-11 Sample Test Results

[0064] Bovine diarrhea is a significant disease hindering the healthy development of beef and dairy cattle farming in Ningxia. Its pathogens are complex, often exhibiting mixed infections involving viruses, bacteria, and parasites, making single detection methods insufficient for rapid and accurate clinical diagnosis. This invention, based on the epidemiological characteristics of bovine diarrhea in Ningxia, establishes a triple TaqMan quantitative PCR detection method capable of simultaneously detecting BRV, BCoV, and BVDV. Ninety-six clinical diarrheal calf samples were collected for applied testing, aiming to clarify the prevalence patterns of major pathogens causing bovine diarrhea in the region and provide technical support for disease prevention and control.

[0065] The multiplex quantitative PCR method established in this invention exhibits significant advantages in detection efficiency and avoids false positives caused by environmental contamination in conventional PCR, meeting the practical needs of rapid clinical pathogen screening. Compared to traditional PCR and pathogen isolation and identification methods, this method can simultaneously achieve qualitative and quantitative detection of multiple diarrheal pathogens, significantly shortening the detection cycle. It also boasts high sensitivity and specificity, effectively avoiding missed detections and false positives caused by environmental contamination in conventional PCR. Related studies have shown that multiplex real-time quantitative PCR technology, with its high throughput and precise quantification, has become the mainstream method for detecting pathogens causing calf diarrhea. Its detection limit for target pathogens can reach 10¹~10² copies / µL, far superior to conventional PCR methods. The method constructed in the experiments of this invention, after experimental optimization and verification, shows good sensitivity for detecting common pathogens causing calf diarrhea in Ningxia, with no cross-amplification, indicating good reliability. It is suitable for rapid detection of large-scale clinical samples, providing technical support for the simultaneous quantitative detection of multiple pathogens causing calf diarrhea in Ningxia and other major dairy and beef cattle farming areas in China.

[0066] Ninety-six clinically diagnosed calf samples were tested using a constructed multiplex quantitative PCR method. The results showed that the positive rates for BRV, BCoV, and BVDV were 18.75%, 11.46%, and 14.58%, respectively. The positive rates for mixed BRV and BCoV infection were 6.25%, 8.34%, 4.17%, and 4.17% for mixed BCoV and BVDV infection, and 4.17% for mixed BRV, BCoV, and BVDV infection. These results indicate that mixed-pathogen bovine diarrhea is prevalent in Ningxia. This method provides solid technical and theoretical support for the timely development of scientific and effective prevention and control strategies based on mixed infection situations.

[0067] In the experiments described above, TaqMan single-cell quantitative PCR methods for BRV, BCoV, and BVDV were established. After optimization of annealing temperature, primer and probe concentrations, the method demonstrated good specificity, sensitivity, and reproducibility. Results showed that this method can accurately detect each single pathogen. Furthermore, based on the TaqMan single-cell quantitative PCR methods for each pathogen, a TaqMan multiplex PCR detection method for the simultaneous detection of BRV, BCoV, and BVDV was established. Annealing temperature, specificity, and sensitivity were optimized, and the reproducibility of the method was verified. Clinical sample application trials showed that this method can effectively identify and detect the three pathogens.

[0068] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0069] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A primer and probe set for TaqMan multiplex real-time PCR detection of BRV, BCoV, and BVDV, characterized in that, The primer-probe set includes: (1) Specific primer pairs and probes targeting the BRV VP1 gene: The upstream primer BRV-F has the nucleotide sequence shown in SEQ ID NO:1; The downstream primer BRV-R has the nucleotide sequence shown in SEQ ID NO:2; The probe BRV-Probe has the nucleotide sequence shown in SEQ ID NO:3; (2) Specific primer pairs and probes targeting the BCoV N gene: Upstream primer BCoV-F: Its nucleotide sequence is shown in SEQ ID NO:4; Downstream primer BCoV-R: Its nucleotide sequence is shown in SEQ ID NO:5; The probe BCoV-Probe has the following nucleotide sequence as shown in SEQ ID NO:6; (3) Specific primer pairs and probes targeting the BVDV 5'-UTR gene: Upstream primer BVDV-F: Its nucleotide sequence is shown in SEQ ID NO:7; Downstream primer BVDV-R: Its nucleotide sequence is shown in SEQ ID NO:8; Probe BVDV-Probe: Its nucleotide sequence is shown in SEQ ID NO:

9.

2. The primer and probe set for BRV, BCoV, and BVDV TaqMan multiplex real-time PCR detection according to claim 1, characterized in that: The fluorescent reporter groups of the probes BRV-Probe, BCoV-Probe, and BVDV-Probe are of different types.

3. A reaction system for simultaneous detection of BRV, BCoV, and BVDV, characterized in that, It comprises the primer and probe set according to any one of claims 1-2.

4. The application of the primer-probe set according to claim 1 or 2 in the preparation of bovine diarrhea virus detection reagents or reaction systems.

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