Porcine reproductive and respiratory syndrome virus European type, American type double droplet digital PCR absolute quantitative detection kit
A technology for respiratory syndrome and pig reproduction, which is applied in the measurement/inspection of microorganisms, microorganisms, recombinant DNA technology, etc. It can solve the problem of inability to accurately quantify viral nucleic acids, unable to very well reflect the core concept of digital PCR, and long time required for the test. And other issues
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Embodiment 1
[0073] Embodiment 1, design and screening of primers and probes
[0074] 1. Design and screening of primers and probes for detection of European strains of porcine reproductive and respiratory syndrome virus
[0075] Through a large number of sequence acquisition, analysis, comparison and preliminary experiments, four primers and two probes were designed and preliminarily screened. The nucleotide sequences are as follows:
[0076] LV-F1 (sequence 1 of the sequence listing): 5'-GCTGCMGGTTGCYCATACA-3';
[0077] LV-R1 (sequence 2 of the sequence listing): 5'-CAATCGHGGCCATTCAC-3';
[0078] LV-P1 (SEQ ID NO: 3 of the Sequence Listing): 5'-TGCGBCTGATTCGCGTDACTTC-3';.
[0079] LV-F2: 5'-TGCTGCMGGTTGCTCATAC-3';
[0080] LV-R2: 5'-TCAATCGCGKCCATTCA-3';
[0081] LV-P2: 5'-CGCMTGATTCGCGTGACTTCTAVATCC-3'.
[0082] In the above nucleotide sequence, M stands for A or C, Y stands for C or T, H stands for A, T or C, B stands for G, T or C, D stands for G, A or T, M stands for A or C, K ...
Embodiment 2
[0108] Embodiment 2, optimization of relevant reaction parameters in digital PCR
[0109] 1. Optimization of annealing temperature
[0110] 1. Take the European strain of porcine reproductive and respiratory syndrome virus and extract total RNA.
[0111] 2. Take the American strain of porcine reproductive and respiratory syndrome virus, and extract the total RNA.
[0112] 3. Mix the total RNA obtained in step 1 with the total RNA obtained in step 2 to obtain mixed RNA.
[0113] 4. Using the mixed RNA obtained in step 3 as a template, carry out digital RT-PCR with a primer combination consisting of primer probe set LV-F1R1P1 and primer probe set VR2332-F1R1P1.
[0114] (1) Prepare the following system (20μL): 10μL 2×One-step RT-ddPCR Supermix for probes, 1μL LV-F1, 1μL LV-R1, 0.5μL LV-P1, 1μL VR2332-F1, 1μL VR2332-R1, 0.5 μL VR2332-P1, 2 μL template, 3 μL RNase Free dH 2 O. In the system, the concentration of the upstream primer is 0.5 μM, the concentration of the downstre...
Embodiment 3
[0140] Embodiment 3, the preparation of kit
[0141] 1. Preparation of each reagent
[0142] Solution A is the one-step ddPCR probe master mix. The composition of each 900 μL solution A is as follows: 500 μL 2×One-step RT-ddPCR Supermix for probes, 90 μL LV-F1 solution (the concentration of LV-F1 in the LV-F1 solution is 10 μM), 90 μL VR2332-F1 solution (VR2332-F1 The concentration of VR2332-F1 in the solution is 10 μM), 90 μL of LV-R1 solution (the concentration of LV-R1 in the LV-R1 solution is 10 μM), 90 μL of VR2332-R1 solution (the concentration of VR2332-R1 in the VR2332-R1 solution is 10 μM) , 20 μL LV-P1 solution (the concentration of LV-P1 in the LV-P1 solution is 10 μM), 20 μL VR2332-P1 solution (the concentration of VR2332-P1 in the VR2332-P1 solution is 10 μM).
[0143] Solution B is droplet generating oil.
[0144] Solution C is a positive control. The preparation method of solution C: extract the total RNA of the European type of porcine reproductive and resp...
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