Narcissus latent virus detection kit and detection method thereof
A detection kit, latent virus technology, applied in microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as molecular detection kits that have not yet been seen, and achieve good practical application value The effect of fast detection time and simple production method
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Embodiment 1
[0027] Example 1: Configuration of Narcissus Latent Virus Detection Kit (10 tests)
[0028] 1) Upstream primer: 10 μmol / L, 1 tube (30 μL);
[0029] 2) Downstream primer: 10 μmol / L, 1 tube (30 μL);
[0030] 3) RT Buffer: 5×, 1 tube (30μL);
[0031] 4) RNase inhibitor: 40U / μL, 1 tube (5μL);
[0032] 5) Reverse transcriptase: 200U / μL, 1 tube (5μL);
[0033] 6) dNTPs: 10mmol / L, 1 tube (30μL);
[0034] 7) PCR Buffer: 10×, 1 tube (30μL);
[0035] 8) Mgcl 2 : 25mmol / L, 1 tube (30μL);
[0036] 9) Taq DNA polymerase: 5U / μL, 1 tube (5μL);
[0037] 10) Positive control sample of narcissus latent virus, 1 tube (20 μL);
[0038] 11) Negative control sample without narcissus latent virus, 1 tube (20 μL);
[0039] 12) RNase-free ddH 2 O, 1 tube (1 mL).
Embodiment 2
[0040] Example 2: Detection method of Narcissus latent virus detection kit
[0041] The detection method of above-mentioned Narcissus latent virus detection kit may further comprise the steps:
[0042] 1) Reverse transcription reaction: Add 2 μL of total RNA of the sample to be tested, 1 μL of downstream primers with a concentration of 10 μmol / L and RNase-free ddH in a PCR tube 2 O 5 μL, 70 ° C water bath for 10 min, rapid ice bath for 5 min, then add the following reagents: 5 × RT Buffer 2.5 μL, the concentration is 10mmol / L dNTPs 1 μL, the concentration is 200U / μL reverse transcriptase 0.5μL, the concentration is 40U / μL RNase inhibitor 0.5 μL. 42°C water bath for 60 minutes, 70°C water bath for 10 minutes, and then naturally cool to room temperature to synthesize cDNA;
[0043] 2) PCR reaction: Take 3 μL of cDNA synthesized in step 1), add 0.5 μL of Taq DNA polymerase at a concentration of 5 U / μL, 0.5 μL of dNTPs at a concentration of 10 mmol / L, and 2.5 μL of 10×PCR Buffer...
Embodiment 3
[0045] Example 3: Specific Validation of Narcissus Latent Virus Detection Kit
[0046] 1) Extraction of total RNA from narcissus samples: Narcissus latent virus (NLV), narcissus mosaic virus ( Narcissus mosaic virus , NMV), Narcissus Yellow Streak Virus ( Narcissus yellow stripe virus , NYSV), Narcissus late season yellowing virus ( Narcissus late season yellow virus , NLSYV), Arabidopsis mosaic virus ( Arabis mosaic virus , ArMV) narcissus samples as materials, put 0.1g each in a mortar, add 1mL PBST buffer to grind, centrifuge at 10,000g for 5min at 4°C, take the supernatant and transfer it to a sterilized 1.5mL centrifuge Add 1mL TrizoL reagent to the tube, shake vigorously, let stand at room temperature for 5min; centrifuge at 12,000g for 10min at 4°C, and take the supernatant; add 300μL of chloroform, shake vigorously for 15s, let stand at room temperature for 5min, centrifuge at 12,000g for 15min at 4°C, and take the upper layer Aqueous phase; add an equal volum...
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