Bai yang dian virus and duck plague virus double fluorescent quantitation RT-PCR detection reagent kit
A detection kit and duck plague virus technology, which is applied in the determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complexity, high requirements for reagents and primers, and achieve the effect of high detection sensitivity
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Embodiment 1
[0037] Embodiment 1, design and synthesis of primers and Taqman probes
[0038] According to the conserved sequences of duck flavivirus and duck plague virus in GenBank, two pairs of specific primers and two Taqman probes were designed using Primer Express3.0 software (Table 1).
[0039] Table 1 Primers and TaqMan probe sequences (5'-3')
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Embodiment 2
[0041] Embodiment 2, establishment of fluorescent quantitative RT-PCR detection method
[0042] 1. Determination of fluorescent quantitative RT-PCR detection method
[0043] 1. Sample preparation
[0044] According to the instructions of the DNA / RNA extraction kit, extract the DNA of duck plague virus, Muscovy duck parvovirus, duck circovirus and egg drop syndrome virus; simultaneously extract duck yellow virus, duck hepatitis virus, H9 subtype avian influenza virus, The RNA of duck-derived Newcastle disease virus was reverse-transcribed into cDNA according to the reverse transcription instructions, and stored at -30°C for future use.
[0045] 2. Standard product preparation
[0046] PCR amplification was carried out using the cDNA of duck yellow virus obtained above as a template, and the reaction system was 50 μL (containing 25 μL PCRMix, 1 μmol / L primers (BYD(257)-1 and BYD(257)-2, see Table 2), 19 μL ddH 2 (0, 4 μL DNA template), the reaction conditions are: 95°C pre-de...
Embodiment 3
[0077] Embodiment 3, the assembly of detection kit
[0078] According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.
[0079] Solution A: Premix ExTaq 10 μL (Dalian Bao Biological Engineering Co., Ltd., catalog number: DRR390S); BYD (257-62) F, BYD (257-62) R and BYD (257-62) at a final concentration of 0.3 μmol / L ) P; DPV (454-65) F, DPV (454-65) R and DPV (454-65) P at a final concentration of 0.2 μmol / L; add ddH 2 O8.5μL
[0080] Solution B: 1 μL each of BYD+DPV template, as a positive control.
[0081] Solution C: Contains ddH 2 O2μL, as a negative control.
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