Kit capable of simultaneously detecting plantain herb mosaic virus and lily X virus
A technology of mosaic virus and plantain, applied to the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve simple operation, good reproducibility, and stable results
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Embodiment 1
[0037] Embodiment 1, simultaneously detect plantain mosaic virus and lily X virus kit
[0038] 1. A complete set of primer pairs for the detection of plantain mosaic virus and lily X virus
[0039] The invention designs and screens specific primers for detecting the two viruses according to the conserved regions of the plantain mosaic virus and lily X virus genomes.
[0040] The primer pair designed for the plantain mosaic virus in the present invention consists of a P1AMV forward primer and a P1AMV reverse primer, and the size of the target amplified fragment is 338bp. The primer sequences are as follows:
[0041] PlAMV forward primer: 5'-CCTTGCCCTGGTCAACGCAT-3';
[0042] PlAMV reverse primer: 5'-TCTCGTTTGCTGTGACCTCG-3';
[0043] The primer pair designed in the present invention for lily X virus is composed of LVX forward primer and LVX reverse primer, and the size of the target amplified fragment is 439bp. The primer sequences are as follows:
[0044] LVX forward primer...
Embodiment 2
[0062] Embodiment 2: The detection method of simultaneous detection plantain mosaic virus and lily X virus
[0063] 1) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, 1 μL of 10 mmol / L PlAMV reverse primer, 1 μL of 10 mmol / L LVX reverse primer and RNase-free ddH into the PCR reaction tube 2 O 6 μL, 70 °C water bath for 10 min, ice bath for 5 min, and then add the following reagents in sequence: 5 × RT Buffer 5 μL, 10 mmol / L dNTPs 2 μL, 200 U / μL reverse transcriptase 1 μL, 40 U / μL RNase inhibitor 1 μL. After 60 minutes in a water bath at 42°C, cool to room temperature at 70°C for 10 minutes to synthesize cDNA;
[0064] 2) PCR: Add 3 μL of cDNA synthesized by reverse transcription, 12.5 μL of 2×PCR Mix, 1 μL of 10 mmol / L PlAMV forward primer, 1 μL of 10 mmol / L PlAMV reverse primer, and 1 μL of 10 mmol / L LVX forward primer into the PCR reaction tube , 10mmol / LLVX reverse primer 1μL, RNase-free ddH 2 O 5.5 μL; the mixed reaction solution was pre-denature...
Embodiment 3
[0066] Embodiment 3: the primer specificity determination of kit
[0067] 1) Extraction of total RNA from samples: plantain mosaic virus, lily X virus, cucumber mosaic virus (CMV), lily asymptomatic virus (LSV), lily mottle virus (LMoV), strawberry latent ring spot virus ( SLRSV), tobacco mosaic virus (TMV), tomato ringspot virus (ToRSV) samples and negative controls were used as materials, and total RNA was extracted using RNA extraction kits;
[0068] 2) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, 1 μL of 10 mmol / L PlAMV reverse primer, 1 μL of 10 mmol / L LVX reverse primer and RNase-free ddH into the PCR reaction tube 2 O 6 μL, 70 °C water bath for 10 min, ice bath for 5 min, and then add the following reagents in sequence: 5 × RT Buffer 5 μL, 10 mmol / L dNTPs 2 μL, 200 U / μL reverse transcriptase 1 μL, 40 U / μL RNase inhibitor 1 μL. After 60 minutes in a water bath at 42°C, cool to room temperature at 70°C for 10 minutes to synthesize cDNA;
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