Molecular probe for detecting African swine fever virus, kit and application thereof
An African swine fever virus and detection kit technology, applied in biological testing, measuring devices, analytical materials, etc., can solve the problems of poor reliability and practicability of detection technology, and achieve advantages of application and promotion, high sensitivity, and repeatability. good effect
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Embodiment 1
[0060] Embodiment 1: Design a kind of molecular probe for detecting ASFV
[0061] According to the 54 strains of African swine fever virus VP72 genes on GeneBank, a conserved sequence with a nucleotide sequence size of 258 bp at the 3' end of the VP72 gene was screened out, as shown in SEQ ID NO.1:
[0062] CGTATCCGATCACATTACCTATTATTAAAAACATTTCCGTAACTGCTCATGGTATCAATCTTATCGATAAATTTCCATCAAAGTTCTGCAGCTCTACATACCCTTCCACTACGGAGGCAATGCGATTAAAACCCCCGATGATCCGGGTGCGATGATGATTACCTTTGCTTTGAAGCCACGGGAGGAATACCAACCCAGTGTCATATTAACGTATCCAGAGCAAGATTGAATG.
[0063] Taking the conserved sequence as the target gene, considering various factors and combining years of practical experience, a molecular probe was designed, as shown in SEQ ID NO.2:
[0064] 5'-[6-carboxy-fluorescein (FAM)]-CCAACCCAGTGGTCATATTAACGTATCC-3'-[6-carboxy-tetramethyl-rhodamine (TAM-RA)].
[0065] The fluorescent reporter group labeled at the 5' end of the probe is 6-carboxy-fluorescein (FAM), and the fluorescent quencher grou...
Embodiment 2
[0066] Embodiment 2: design a kind of specific primer for detecting ASFV
[0067] Taking the conserved sequence shown in SEQ ID NO.1 as the target gene, a pair of specific primers were designed by comprehensive consideration of various factors and years of practical experience, as shown in SEQ ID NO.3 and SEQ ID NO.4:
[0068] ASFV-For: 5'-CGTATCCGATCACATTACC-3'
[0069] ASFV-Back: 5'-CGTAATCCGTGTCCCAAC-3'
Embodiment 3
[0070] Embodiment 3: extract ASFV total DNA
[0071] (1) Reagent preparation:
[0072] Separation buffer: 10mmoL / L Tris-Cl pH 7.4, 10mmoL / L NaCl, 25mmoL / L EDTA;
[0073] Other reagents: 10% SDS, protein kinase K (20mg / mL or powder), diethyl ether, phenol: chloroform: isoamyl alcohol (25:24:1), absolute ethanol and 70% ethanol, 5 mol / L NaCl, 3 mol / L NaAc, TE;
[0074] (2) Cut off about 5g of tissue, remove the connective tissue, absorb the blood with absorbent paper, cut it into pieces (the more broken the better), and put it in a mortar;
[0075] (3) Pour into liquid nitrogen, grind into powder, add 10mL separation buffer;
[0076] (4) Add 10 mL 10% SDS, mix well, and the sample becomes very viscous at this time;
[0077] (5) Add 50 μL or 1 mg of protein kinase K and incubate at 37 °C for 1-2 h to guide the complete dissociation of the tissue;
[0078] (6) Add 1 mL 5mol / L NaCl, mix well, and centrifuge at 5000 rpm for a few seconds;
[0079] (7) Take the supernatant in a...
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