Method for detecting sweet potato leaf curl viruses and special primer set thereof
A technology of sweet potato leafroll virus and primer set, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effect of shortening the reaction time, overcoming the long detection cycle, and easy operation
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Embodiment 1
[0036] Embodiment 1, design and preparation of primers
[0037] Based on a large number of sequence comparisons, primer design, primer screening and effect verification, a set of primers with the best effect for loop-mediated isothermal amplification was obtained. The sequences of each primer in the primer set are as follows:
[0038] SPLCV-F3 (outside upstream primer, sequence 1 of the sequence listing): 5'-GGCTGAACTTCGAGACAG-3';
[0039] SPLCV-B3 (outside downstream primer, sequence 2 of the sequence listing): 5'-AATCCGAGACACAGACAAAC-3';
[0040] SPLCV-FIP (inside upstream primer, sequence 3 of the sequence listing):
[0041]5'-CTCTTCATCCGGACGCCTCCTACACTGGGAATGCTGTC-3';
[0042] SPLCV-BIP (inside downstream primer, sequence 4 of the sequence listing):
[0043] 5'-GGTGACCGCATCCCGAAGTCTTGAACTCATAGTCCTGGA-3';
[0044] SPLCV-LF (loop upstream primer, sequence 5 of the sequence listing): 5'-TAGCTTCGGGCAGCAATT-3';
[0045] SPLCV-LB (loop downstream primer, sequence 6 of the s...
Embodiment 2
[0047] Embodiment 2, specific detection
[0048] Sweet potato leaf curl virus (SPLCV), sweet potato feathery mottle virus (SPFMV), sweet potato asymptomatic virus (SPSMV), sweet potato virus No. 2 (SPV2), sweet potato G virus (SPVG) and sweet potato C virus (SPVC) were selected as the samples to be tested. Virus. Among the above-mentioned viruses, the sweet potato leafroll virus is a DNA virus, and the other viruses are all RNA viruses.
[0049] 1. Extract the genomic DNA of the DNA virus to be tested, or extract the total RNA of the RNA virus to be tested and reverse transcribe it into cDNA.
[0050] 2. Using the genomic DNA or cDNA obtained in step 1 as template DNA, perform loop-mediated isothermal amplification, then observe and take pictures under normal light conditions. During the loop-mediated isothermal amplification process, a real-time fluorescent PCR instrument was used for real-time monitoring. Set up a blank control in which an equal volume of water was used i...
Embodiment 3
[0056] Embodiment 3, sensitivity detection
[0057] 1. Genomic DNA of sweet potato leafroll virus was extracted.
[0058] 2. Using the genomic DNA obtained in step 1 as template DNA, perform loop-mediated isothermal amplification, then observe and take pictures under normal light conditions. During the loop-mediated isothermal amplification process, a real-time fluorescent PCR instrument was used for real-time monitoring. Set up a blank control in which an equal volume of water was used instead of template DNA.
[0059] In the reaction system (25 μl) of loop-mediated isothermal amplification, the concentration of BstDNA polymerase is 0.32 U / μL, the concentration of SPLCV-F3 and SPLCV-B3 is 0.2 μmol / L, and the concentration of SPLCV-FIP and SPLCV-BIP The concentration is 1.6 μmol / L, the concentration of SPLCV-LF and SPLCV-LB is 0.8 μmol / L, the concentration of calcein fluorescent dye is 0.2 μmol / L, the concentration of betaine is 1mol / L, MgSO 4 The concentration of dNTP is 6...
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