African swine fever virus (ASFV) detection kit and application thereof
A technology of African swine fever virus and kit, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of poor sensitivity and achieve the effect of high sensitivity and high practical value
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Embodiment 1
[0058] Embodiment 1: Preparation and application of African swine fever virus PCR detection kit
[0059] One, the preparation of African swine fever virus PCR detection kit
[0060] 1. Synthesis of Primers
[0061] The following primers were artificially synthesized:
[0062] Upstream primer P72F3: 5'-GTTTCGGTACGCATTCTTTGTG-3';
[0063] Downstream primer P72R3: 5'-CGGATATGACTGGGACAACCA-3';
[0064] 2. Preparation of Positive Control
[0065] According to the African swine fever virus Georgia 2007 / 1 strain genome sequence published by Genbank (serial number: FR682468), its 103590 to 105530 nucleotide sequence was intercepted, namely the B646L gene sequence, which was artificially synthesized and inserted into the plasmid, such as pUC- 57 plasmids. The obtained plasmid was transformed into Escherichia coli, the plasmid was extracted, the concentration was determined and converted into copy number. Obtaining a concentration of 5.7 x 10 8 Copy / μL African swine fever virus B...
Embodiment 2
[0080] Embodiment 2. Preparation and application of African swine fever virus Taqman fluorescent PCR detection kit
[0081] One, the preparation of African swine fever virus Taqman fluorescent PCR detection kit
[0082] 1. Synthesis of Primers and Probes
[0083] The following primers and probes were artificially synthesized:
[0084] Primer P72F3: 5'-GTTTCGGTACGCATTCTTTGTG-3';
[0085] Primer P72R3: 5'-CGGATATGACTGGGACAACCA-3';
[0086] Probe P72P3: 5'-ATCTACAAGCGTGTAAACGGCGCCC-3'
[0087] The 5'-end of the probe P72P3 is labeled with the fluorescent reporter group FAM, and the 3'-end is labeled with the fluorescent quencher group BHQ-1.
[0088] 2. Preparation of Positive Control
[0089] With embodiment 1.
[0090] 3. Prepare fluorescent PCR reaction buffer
[0091] Prepare 2× fluorescent PCR reaction buffer, containing 0.4mM each of dNTPs, MgSO4 2.8-4mM, DNA polymerase 1U / μL, the primer P72F3 0.4-1.2μM, the primer P72R3 0.4-1.2μM, the fluorescent probe P72P3 0.2~0....
Embodiment 3
[0104] Embodiment 3. Preparation and application of African swine fever virus Taqman fluorescent PCR detection kit
[0105] One, the preparation of African swine fever virus Taqman fluorescent PCR detection kit
[0106] 1. Synthesis of Primers and Probes
[0107] The following primers and probes were artificially synthesized:
[0108] Primer P72F3: 5'-GTTTCGGTACGCATTCTTTGTG-3';
[0109] Primer P72R3: 5'-CGGATATGACTGGGACAACCA-3';
[0110] Probe P72cP3: 5'-GGGCGCCGTTTACACGCTTGTAGAT-3'
[0111] The 5'-end of the probe P72cP3 was labeled with the fluorescent reporter group FAM, and the 3'-end was labeled with the fluorescent quencher group BHQ-1.
[0112] 2. Preparation of Positive Control
[0113] With embodiment 1.
[0114] 3. Prepare fluorescent PCR reaction buffer
[0115] Prepare 2× fluorescent PCR reaction buffer, containing 0.4mM each of dNTPs, MgSO4 2.8-4mM, DNA polymerase 1U / μL, the primer P72F3 0.4-1.2μM, the primer P72R3 0.4-1.2μM, the fluorescent probe P72cP3 0.2...
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