Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I
A real-time fluorescence quantitative, reovirus technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome operation, long cycle, unable to quantitative analysis, etc., to achieve detection sensitivity high effect
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Embodiment 1
[0034] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;
[0035] 2. Primer design:
[0036] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:
[0037] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'
[0038] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';
[0039] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:
[0040] Take 1 μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5...
Embodiment 2
[0051] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;
[0052] 2. Primer design:
[0053] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:
[0054] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'
[0055] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';
[0056] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:
[0057] Take 5μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5 ...
Embodiment 3
[0068] 1. Treat the extracted grass carp reovirus total RNA with lysate and RNA extractant;
[0069] 2. Primer design:
[0070] According to the grass carp hemorrhagic disease-873 outer capsid protein (VP7) gene sequence (AF403396) in GenBank, two pairs of specific primers were designed, and the length of the amplified fragment was 98bp. The designed primers were as follows:
[0071] Upstream primer: 5'-GGAATTCACCACGATGCCACTTCAC-3'
[0072] Downstream primer: 5'-CGGGATCCCGGTGCTTAATCGGATGG-3';
[0073] 3. RT-PCR amplification of grass carp reovirus outer capsid protein (VP7) gene:
[0074] Take 10μg total virus RNA, add 20pmol upstream primer, follow Improm-Ⅱ TM ReverseTranscription System instructions for reverse transcription. Take 8 microliters of reverse transcription product cDNA, add 5 microliters of 10×Taq reaction buffer, 4 microliters of 2.5 mmol / L dNTPs, 50 micromol 1 / liter of dNTPs, 1 microliter of upstream and downstream, sterilized double distilled water 30.5 ...
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