A detection method and kit for swine fever virus based on the fluorescence characteristics of g-quadruplex
A swine fever virus and fluorescence detection technology, applied in the field of genetic engineering, can solve problems such as high requirements for operators, obstacles to application, expensive reagents, etc., and achieve the effects of improving detection efficiency, easy operation, and fast detection speed
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Embodiment 1
[0028] The test kit for swine fever virus detection based on G-quadruplex fluorescent properties, which consists of:
[0029] (1) NASBA reaction buffer includes: 40mM Tris-HCl, pH8.0; 70mM KCl; 20mM MgCl 2 ; 1 mM deoxyribonucleoside triphosphates (dNTPs); 1 mM ribonucleoside triphosphates (NTPs); 10 mM dithiothreitol; 10% dimethylsulfoxide (DMSO).
[0030] (2) NASBA forward primer and reverse primer include, 0.5 μM forward primer E2P7f: ACTATGAGCCCAGGGACAGCTACTT; 0.5 μM reverse primer E2P7T7r: GTCGACTAATACGACTCACTATAGGGTTCCCTATCAACACTACCTCACCC T.
[0031] (3) NASBA enzyme reaction solution includes: T7 RNA polymerase 60U; AMV reverse transcriptase 5U; RNase H 0.1U, 2μg of BSA.
[0032] (4) The composition of G-quadruplex fluorescence detection reaction buffer includes: 10mM 4-hydroxyethylpiperazineethanesulfonic acid, 200mM sodium chloride and 20mM potassium chloride; upstream probe 1μM and downstream probe 1 μM. Add sterile double distilled water to make up to 97 μl.
[003...
Embodiment 2
[0040] The test kit for swine fever virus detection based on G-quadruplex fluorescent properties, which consists of:
[0041] (1) NASBA reaction buffer includes: 30mM Tris-HCl, pH8.0; 80mM KCl; 10mM MgCl 2 ; 2mM deoxyribonucleoside triphosphates (dNTPs); 0.5mM ribonucleoside triphosphates (NTPs); 5mM dithiothreitol; 20% dimethylsulfoxide (DMSO).
[0042] (2) NASBA forward primer and reverse primer include, 0.5 μM forward primer E2P7f: ACTATGAGCCCAGGGACAGCTACTT; 0.5 μM reverse primer E2P7T7r: GTCGACTAATACGACTCACTATAGGGTTCCCTATCAACACTACCTCACCC T.
[0043] (3) NASBA enzyme reaction solution includes: 40 U of T7 RNA polymerase; 2 U of AMV reverse transcriptase; 0.15 U of RNase H, 5 μg of BSA.
[0044] (4) The composition of G-quadruplex reaction buffer includes: 10mM 4-hydroxyethylpiperazineethanesulfonic acid, 200mM sodium chloride and 20mM potassium chloride; upstream probe 2μM and downstream probe 2μM, Add sterile double distilled water to make up to 97μl;
[0045] (5) The u...
Embodiment 3
[0052] The test kit for swine fever virus detection based on G-quadruplex fluorescent properties, which consists of:
[0053] (1) NASBA reaction buffer includes: 60mM Tris-HCl, pH8.0; 60mM KCl; 30mM MgCl 2 ; 0.5mM deoxyribonucleoside triphosphates (dNTPs); 2mM ribonucleoside triphosphates (NTPs); 20mM dithiothreitol; 5% dimethylsulfoxide (DMSO).
[0054] (2) NASBA forward primer and reverse primer include, 1 μM forward primer E2P7f: ACTATGAGCCCAGGGACAGCTACTT; 1 μM reverse primer E2P7T7r: GTCGACTAATACGACTCACTATAGGGTTCCTATCAACACTACCTCACCCT.
[0055] (3) NASBA enzyme reaction solution includes: T7 RNA polymerase 60U; AMV reverse transcriptase 5U; RNase H 0.05U, 0.5μg of BSA.
[0056] (4) The composition of G-quadruplex fluorescence detection reaction buffer includes: 10mM 4-hydroxyethylpiperazineethanesulfonic acid, 200mM sodium chloride and 20mM potassium chloride; upstream probe 0.5μM and downstream probe Needle 0.2 μM. Add sterile double distilled water to make up to 97 μl....
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