Narcissus retrovirus nested RT-PCR detection kit and its detection method
A Narcissus degenerated virus, RT-PCR technology, applied in biochemical equipment and methods, methods based on microorganisms, measurement/testing of microorganisms, etc., can solve problems such as nested RT-PCR detection kits for rapid detection of viruses that have not yet been seen , to achieve the effect of high sensitivity, strong specificity and short cycle
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Embodiment 1
[0032] Embodiment 1: the configuration of narcissus retrovirus nested RT-PCR detection kit (10 detections)
[0033] 1) Outer forward primer: 10 μmol / L, 1 tube (30 μL);
[0034] 2) Outer reverse primer: 10 μmol / L, 1 tube (30 μL);
[0035] 3) Inner forward primer: 10 μmol / L, 1 tube (30 μL);
[0036] 4) Inner reverse primer: 10 μmol / L, 1 tube (30 μL);
[0037] 5) RTBuffer: 5×, 1 tube (30 μL);
[0038] 6) RNase inhibitor: 40U / μL, 1 tube (5μL);
[0039] 7) Reverse transcriptase: 200U / μL, 1 tube (5μL);
[0040] 8) dNTPs: 10mmol / L, 1 tube (30μL);
[0041] 9) PCRBuffer: 10×, 1 tube (60 μL);
[0042] 10) Mgcl 2 : 25mmol / L, 1 tube (50μL);
[0043] 11) TaqDNA polymerase: 5U / μL, 1 tube (10μL);
[0044] 12) Positive control sample of Narcissus retrograde virus, 1 tube (50 μL);
[0045] 13) Negative control sample without narcissus retrovirus, 1 tube (50 μL);
[0046] 14) RNase-freeddH 2 0, 1 tube (1 mL).
Embodiment 2
[0047] Embodiment 2: the detection method of Narcissus retrograde virus nested RT-PCR detection kit
[0048] The detection method of above-mentioned narcissus retrovirus nested RT-PCR detection kit comprises the following steps:
[0049] 1) Reverse transcription reaction: Add 3 μL of the total RNA of the sample to be tested, 1 μL of the outer reverse primer with a concentration of 10 μmol / L and RNase-freedH in the PCR tube 2 O7μL, 70℃ water bath for 10min, rapid ice bath for 5min, then add the following reagents: 5×RTBuffer 2.5μL, concentration 10mmol / LdNTPs 1μL, concentration 200U / μL reverse transcriptase 0.5μL, concentration 40U / μL RNase inhibitor 0.5 μL. 42°C water bath for 60 minutes, 70°C water bath for 10 minutes, synthesize cDNA;
[0050] 2) The first round of PCR reaction: take 3 μL of cDNA synthesized in step 1), add 0.5 μL of TaqDNA polymerase at a concentration of 5 U / μL, 0.5 μL of 10 mmol / LdNTPs, 2.5 μL of 10×PCRBuffer, and a concentration of 25 mmol / LMgCl into e...
Embodiment 3
[0053] Example 3: Specificity determination of Narcissus retrovirus nested RT-PCR detection kit
[0054] 1) Extraction of total RNA from Narcissus samples: Narcissus degenerate virus (NDV), Narcissus late season yellowing virus (NLSYV), Narcissus mosaic virus (NMV), Narcissus yellow stripe virus (Narcissus yellowstripevirus, NYSV), Narcissus latent virus (Narcissus latent virus, NLV) and Arabismosaic virus (Arabismosaicvirus, ArMV) narcissus samples were used as materials, each 0.1g was placed in a mortar, 1mL of LPBST buffer was added to grind, 4°C, centrifuged at 10000g for 5min, and the Supernatant and quickly transfer the supernatant to a sterilized 1.5mL centrifuge tube, add 1mL TrizoL reagent, shake vigorously, let stand at room temperature for 5min; centrifuge at 12000g at 4°C for 10min, take the supernatant; add 300μL chloroform, shake vigorously for 15s , let stand at room temperature for 5min, centrifuge at 12000g at 4°C for 15min, take the upper aqueous phase; add a...
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