TaqMan fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection primer and probe for bovine neovirus and application
A technique for fluorescence quantification and detection of primers, which is applied in the determination/inspection of microorganisms, microorganisms, and methods based on microorganisms, etc. It can solve problems such as the detection of bovine Newburial virus, calf xylose malabsorption, and severe diarrhea. , to achieve the effect of reducing RNA degradation, high sensitivity, and improving sensitivity
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Embodiment 1
[0035] According to the BNebV-RdRp gene sequences of GenBank (MH718886, MG599036, MW036464), homology analysis was performed using the MegAlign program in Lasergener software, and a pair of BNeV primers and probes were designed by selecting conserved sequences. The sequences are shown in Table 1.
[0036] Table 1 Amplification of bovine NZV primers and probe sequence information
[0037]
[0038]
Embodiment 2
[0040] Preparation of standards
[0041]According to the RNeasy Mini Kit kit, the sample RNA was extracted according to the instructions, the target gene was amplified with the upstream and downstream primers in Table 1, the PCR product was recovered and purified by gel, and ligated to the pMD19-T vector (ligation reaction system: pMD19-T Vector 1.0 μL; purified product 4.0 μL) μL; Solution I 5.0 μL; reaction conditions: overnight culture at 4°C) and transformed into Escherichia coli DH5α competent cells, plated, screened out positive clones and enriched for culture (specific steps:
[0042] Convert:
[0043] (1) When the removed DH5α competent cells are thawed to a semi-fog state in ice, immediately add 5 μL of the ligation product to 50 μL of DH5α competent cells, and immediately place in ice for 30 minutes after flicking and mixing.
[0044] (3) Take out the mixed solution and place it in a metal bath at 42°C for 45 sec, then place it in an ice box for 2 min.
[0045] (4)...
Embodiment 3
[0051] Condition optimization of real-time quantitative RT-PCR method
[0052] The template is the positive recombinant plasmid DNA of bovine Newbei virus, and 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1 μL of the upstream and downstream primers were respectively taken to optimize the primer concentration.
[0053] Then take the optimized primer concentration, and absorb 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1 μL of the probe, and the template is the positive recombinant plasmid DNA of bovine Newbull virus, and then optimize the probe concentration.
[0054] In the 20 μL amplification system, the minimum CT value and the highest fluorescence amount were selected as the reference standard. The results showed that the optimal upstream and downstream primer concentrations of bovine Newbuli virus were both 500 nmol / L, and the optimal probe concentration was 400 nmol / L. See optimization curve figure 1 and figure 2 .
[0055] Use optimized primers and probes to carry out fluorescence qu...
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