Method for detecting pigeon Newcastle disease virus based on fluorescence quantitative PCR technology
A fluorescent quantitative technology for Newcastle disease virus, applied in the field of detection of pigeon Newcastle disease virus type VI based on fluorescent quantitative PCR technology, can solve the problem of distinguishing chicken-derived NDV and pigeon-derived NDV, which takes a long time and easily delays clinical diagnosis, etc. problem, to achieve the effect of improving the detection speed and detection cycle
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Embodiment 1
[0048] Example 1 Primer Design
[0049] According to the isolated pigeon Newcastle disease virus CQE3 strain, use Primer Primer5 software to design 1 pair of primers and 1 probe, specifically including:
[0050] The sequence of the upstream primer (pF1) is: 5'-CAGAGAGTAATGGCAAAC-3';
[0051] The sequence of the downstream primer (pR1) is 5'-ACGGTTAGAGCGATATAG-3';
[0052] The fluorescent probe (probe) sequence is:
[0053] 5'FAM-TCGGACTAATCAACACATCTGCTCT-TEMRA3'.
[0054] The above primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and diluted to 20 μmol / L with DEPC-treated water. Primers pF1 and pR1 were used for fluorescent quantitative amplification, and the target fragment was 82 bp in length.
Embodiment 2
[0055] Example 2 Pigeon Newcastle Disease Virus Total RNA Extraction and Reverse Transcription
[0056] Use the Trizol reagent method to extract total virus RNA, take 0.2 mL of the allantoic fluid inoculated with Newcastle disease virus from pigeons, add 1 mL of Trizol, make it fully mixed and let it stand for 5 minutes, add 0.2 mL of chloroform, shake vigorously for 15 seconds, and let it stand at room temperature for 2-3 minutes , followed by centrifugation at 12,000×g (4°C) for 15 minutes, absorbing the supernatant aqueous phase, transferring it to a new EP tube, adding an equal volume of pre-cooled isopropanol, standing at room temperature for 10 minutes, centrifuging at 12,000×g (4°C) for 15 minutes, Discard the supernatant, and add 1 mL of 70% pre-cooled ethanol to the pellet to wash the RNA pellet, centrifuge at 7,500×g (4°C) for 5 min, dry at room temperature for 10 min, and add 14 μL of DEPC water to fully dissolve the RNA for later use.
[0057] Add 1 μL of random pr...
Embodiment 3
[0058] Example 3 pMD18T-F plasmid construction
[0059] The following primers were designed to amplify the NDV F gene using the reverse transcription cDNA as a template:
[0060] The sequence of the upstream primer (pF3) is: 5'-CAggATgggCTCCAgACCTT-3';
[0061] The downstream primer (pR1) sequence is 5'-ACCTTCgTTCCTCATCTgTgTT-3'.
[0062] The target fragment is 1687bp in length, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The PCR amplified product was connected to the pMD18-T vector according to conventional methods, and transformed into DH5α competent cells. The 1687bp target fragment can be obtained from the positive plasmid identified by PCR, and the positive clone is sent to the sample for sequencing. The PCR results are shown in figure 1 shown.
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