Peste des petits ruminants virus universal/virulent double-fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection reagent, kit and method
A PPR, dual fluorescence technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve high extraction efficiency and high specificity
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Embodiment 1
[0061] The design of embodiment 1 primer, probe
[0062] According to the published N gene sequence of PPR virus (Genbank accession number EU267273.1) and the virulent H gene sequence of PPR virus (Genbank accession number AJ849636.2), the general primers and probes of PPR virus were designed and Peste des petits ruminants virulent primers and probe sequences. The sizes of the expected amplified fragments were 111bp and 120bp respectively. The 5' end of the general probe sequence of Peste des petits ruminants virus is labeled with the reporter fluorescent group FAM (6-carboxy-fluorescein), and the 3' end is labeled with the quencher group TAMRA; the 5' end of the virulent probe sequence for Peste des petits ruminants is labeled The reporter fluorophore HEX (5-hexachloro-fluorescein) is labeled with the quencher fluorophore TAMRA at the 3' end. Through analytical experiments, the following primers were obtained:
[0063] Universal-upstream primer: 5'GCCAACYGCTYCYGGARATCA3'...
Embodiment 2
[0069] Example 2 Column type extraction operation method extracts RNA
[0070] 1. Take a sterilized 1.5mL centrifuge tube, add 500mL of BufferA and 200mL of each sample, mix well, and place at room temperature for 10 minutes;
[0071] 2. Take the same amount of RNase-Free adsorption column as the centrifuge tube; transfer the solution and flocculent precipitate in the centrifuge tube to the RNase-Free adsorption column, and cover the adsorption column with a collection tube (to avoid clogging the adsorption column, try not to absorb the suspension Impurities); centrifuge at room temperature for 30 seconds at 13,000 rpm; discard the liquid in the collection tube, and put the adsorption column back into the collection tube;
[0072] 3. Add 600uL BufferB to the adsorption column, centrifuge at 13,000 rpm for 30 seconds; discard the liquid in the collection tube, and put the adsorption column back into the collection tube;
[0073] 4. Repeat step 3; centrifuge the empty column ...
Embodiment 3
[0080] Embodiment 3 configuration PCR amplification reaction system
[0081] Add 15 μL fluorescent RT-PCR reaction solution and 1.0 μL enzyme mixture into a small tube of appropriate volume, mix well, take 15 μL as the detection reagent; put the detection reagent into the PCR tube, then add 10ul template RNA, and finally make 25ul reaction system (1 part).
[0082] composition Volume (uL) Upstream primer 10μmol / L-universal 1 Downstream primer 10μmol / L-universal 1 Probe 10μmol / L—general purpose 0.5 Upstream primer 10μmol / L-very toxic 1.25 Downstream primer 10μmol / L-very toxic 1.25 Probe 10μmol / L-very toxic 0.75 5xRT 3 10×PCR 1.5 MgCl 2 (25mmol / L) 1.5 dNTPs (2.5mmol / L) 2 DEPC water 1.25
[0083] ; 10×PCR buffer, 25mmol / LMgCl2 was purchased from Promega; 5×RT-buffer, 2.5mmol / LdNTP was purchased from Dalian Bao Biological Engineering Co., Ltd. Primers and probes were entrusted to Dalian Bao Biologi...
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