Visualized rapid detection kit for carp dropsy virus and detection method thereof
A technology for detecting kits and viruses, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the lack of high-sensitivity detection kits for carp edema virus, which cannot meet the needs of disease quarantine and prevention work and other problems, to overcome the long detection time, reduce background influence, and easy identification
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Embodiment 1
[0047] Example 1 Visual rapid detection kit for carp edema virus
[0048] Carp edema virus visual rapid detection kit includes virus DNA extraction reagents and reaction reagents. The kit only needs a conventional centrifuge and a water bath to complete nucleic acid extraction and detection, and the entire detection process only takes 2 hours, and the detection product is closed for observation. It will not pollute the environment. In addition, the test result can be judged by observing the color change with the naked eye, which is very practical.
[0049] Wherein, the viral DNA extraction reagent comprises the following components: 80mM Tris, 50mM EDTA, 500mM NaCl, 1.5% by mass SDS, 0.01% by volume lysate A of pH 8.0 of β-mercaptoethanol; Tris saturated phenol, pH8.0 ; sodium acetate aqueous solution, concentration 3mol / L; absolute ethanol; lotion A containing 75% absolute ethanol configured with DEPC water; DEPC water.
[0050] The reaction reagent contains a nucleic acid p...
Embodiment 2
[0057] Example 2 The method for detection using the carp edema virus visualization rapid detection kit described in Example 1
[0058] (1) DNA extraction:
[0059] Take carp spleen or heart tissue 30-80mg in a 2mL centrifuge tube, grind it with a grinding rod on ice, add 600μL lysate A, continue grinding, add 600μL Tris saturated phenol with a pH value of 8.0, shake vigorously, and centrifuge at 11000g for 10min. Take the supernatant and repeat the phenol extraction; take the supernatant, add 0.1 times the volume of sodium acetate aqueous solution with a concentration of 3mol / L, mix well, add two times the volume of ice-cold absolute ethanol, mix well and let stand at low temperature Centrifuge at 15,000 g for 5 min for 10 min, discard the supernatant, wash the pellet twice with washing solution A containing 75% absolute ethanol prepared in DEPC water, dry at room temperature for 5-10 min, resuspend in 30 μL DEPC water, and store at -80°C for future use. , Lysis solution A co...
Embodiment 3
[0065] Example 3 Kit Specific Detection
[0066] (1) DNA extraction:
[0067] Take carp edema virus-positive carp tissue samples of 30-80 mg in a 2 mL centrifuge tube, grind on ice with a grinding rod, add 600 μL of lysate A, continue to grind fully, add 600 μL of Tris saturated phenol with a pH value of 8.0, shake vigorously, and centrifuge at 11,000 g 10min, take the supernatant, and repeat the phenol extraction; take the supernatant, add 0.1 times the volume of sodium acetate aqueous solution with a concentration of 3mol / L, mix well, add two times the volume of ice-cold absolute ethanol, mix well and then lower the temperature Let stand for 10 minutes, centrifuge at 15,000 g for 5 minutes, discard the supernatant, wash the precipitate twice with washing solution A containing 75% absolute ethanol prepared in DEPC water, dry at room temperature for 5-10 minutes, resuspend in 30 μL DEPC water, and store at -80°C for later use.
[0068] (2) Rapid amplification of carp edema vi...
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