Canna yellow mottle virus nested PCR detection kit and its detection method
A technology for detecting kits and mottled viruses, which is applied in the direction of biochemical equipment and methods, and the measurement/inspection of microorganisms, can solve the problems of unseen nested PCR detection kits and nested PCR detection methods, and achieve detection The effect of fast time, short detection time and high sensitivity
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Embodiment 1
[0026] Example 1: Configuration of Canna Yellow Mottle Virus Nested PCR Detection Kit (10 detections)
[0027] 1) Outer upstream primer: 10 μmol / L, 1 tube (30 μL);
[0028] 2) Outer downstream primer: 10 μmol / L, 1 tube (30 μL);
[0029] 3) Inner upstream primer: 10 μmol / L, 1 tube (30 μL);
[0030] 4) Inner downstream primer: 10 μmol / L, 1 tube (30 μL);
[0031] 5) dNTPs: 10mmol / L, 1 tube (20μL);
[0032] 6) PCR Buffer: 10×, 1 tube (60μL);
[0033] 7) Mgcl 2 : 25mmol / L, 1 tube (50μL);
[0034] 8) Taq DNA polymerase: 2.5U / μL, 1 tube (10μL);
[0035] 9) Canna yellow mottle virus positive control sample, 1 tube (50 μL);
[0036] 10) Negative control sample without canna yellow mottle virus, 1 tube (50 μL);
[0037] 11) ddH 2 O, 1 tube (1 mL).
Embodiment 2
[0038] Embodiment 2: the detection method of canna yellow mottle virus nested PCR detection kit
[0039] The detection method of above-mentioned canna yellow mottle virus nested PCR detection kit comprises the following steps:
[0040] 1) The first round of PCR reaction: Add 4 μL of the sample DNA to be tested in the PCR tube, add 0.5 μL of Taq DNA polymerase at a concentration of 2.5 U / μL, 0.5 μL of dNTPs at a concentration of 10 mmol / L, and 2.5 μL of 10×PCR Buffer for each tube , the concentration is 25mmol / L MgCl 2 2 μL, concentration of 10 μmol / L outer upstream primer 2 μL, concentration of 10 μmol / L outer downstream primer 2 μL, ddH 2 O11.5 μL, so that the total reaction volume is 25 μL; the mixed reaction solution is pre-denatured at 94°C for 3 minutes, then denatured at 94°C for 30 s, annealed at 53°C for 45 s, and extended at 72°C for 1 min, so a total of 35 cycles, after the end of the last cycle Continue to extend at 72°C for 10 minutes, and the reaction ends;
[00...
Embodiment 3
[0043] Example 3: Specificity determination of canna yellow mottle virus nested PCR detection kit
[0044] 1) DNA extraction: Canna yellow mosaic virus (CaYMV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), tomato infertility virus (Tomato aspermy virus) Virus, TAV) samples as materials, take 50-100mg of each sample and thoroughly grind it with liquid nitrogen, transfer to a 1.5mL centrifuge tube, add 500μL TES, 50-100μg proteinase K, incubate at 56°C for 0.5-1h, and shake gently during the Mix well; add a certain volume of 10mol / L NaCl (to make the final concentration 1.4mol / L), 1 / 10 times the volume of 10% CTAB, and incubate at 65°C for 10 minutes; add 1 times the volume of chloroform / isoamyl alcohol (24:1 ), shake gently to mix, put on ice for 30min, centrifuge at 12000r / min at 4℃ for 10min; take the supernatant, add 225μL NH 4 AC (5mol / L), mix well, place on ice for 1h, centrifuge at 12000r / min at 4°C for 10min; take the supernatant, add 3μL RNase, incubat...
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