Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof
A spleen and kidney necrosis virus and gene diagnosis technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of troublesome material preparation, difficulty in popularization, limited application and development, etc., to avoid virus spread and epidemic, The effect of improving scientific management efficiency and high practical value
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Embodiment 1
[0027] Embodiment 1: the genetic diagnosis kit of mandarin fish infectious spleen and kidney necrosis virus
[0028] The kit consists of the following components (10 samples): 1. Release solution (solution A), 2 tubes, 5ml / tube, containing phosphate buffer (1×PBS), pH7.4. 2. Template extract ( Solution B), self-prepared or prepared, phenol / chloroform / isoamyl alcohol, the ratio is 25:24:1. 3. Solution C, 1 tube, 200μl / tube, filled with 5M NaCl. 4. Liquid D, self-prepared, 1ml / part×10parts, 10ml, mainly absolute ethanol. 5. E liquid, self-prepared, mainly 70% ethanol. 6. Solution F, 1 tube, 30μl / part x 10 parts, 300μl / tube, filled with sterilized double distilled water. 7. PCR reaction solution (solution G), 1 tube, 25μl / part × 10 copies, 250μl / tube, containing PCR first amplification reaction solution (25μl system), including ddH 2 O, 10×Buffer (including mg 2+ ), dNTP, outer primer F1, outer primer R1 and TaqE. 8. PCR reaction solution (solution H), 1 tube, 25μl / part × 10...
Embodiment 2
[0051] Embodiment 2: detection method of mandarin fish infectious spleen and kidney necrosis virus
[0052] Use the kit of embodiment 1, carry out according to the following steps: 1. sample 0.1g, add sample diluent (A solution) 1ml to dilute 10 times, homogenize in ice bath in homogenizer. 2. Centrifuge at 4000r / min for 10min. 3. Take 600 μl of supernatant / extract with template extract solution (solution B), invert up and down several times and mix well. 4. Centrifuge at 12000r / min for 10min. 5. Take 500ul supernatant and add 20μl / C solution, then add 1ml D solution, and precipitate at -20℃ for 1 hour. 6. Centrifuge at 12000r / min for 10min. 7. Discard the supernatant, add E solution to wash, centrifuge at 12000r / min for 5min, and wash twice. 8. Discard the supernatant and dry it naturally or on a clean bench. 9. Add 20-30 μl F solution to resuspend and dissolve as template. 10. Take 2 μl of the template and solution I respectively, add them to the PCR reaction solution ...
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