Reagent assisting in identifying sowbane mosaic virus (SoMV) and application thereof
A grass mosaic virus and mosaic virus technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of cumbersome ELISA operation steps, expensive electron microscopy equipment, and low detection sensitivity. Achieve the effects of avoiding false positive results, wide application range and prospects, and improving sensitivity
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Embodiment 1
[0034] Embodiment 1, the preparation of reagent
[0035] 1. Design of primers and probes
[0036] According to the conserved region of the polyprotein gene (DQ450973) sequence in the quinoa mosaic virus genome in the US NCBI nucleic acid database, an upstream primer (SoMVF), two downstream primers (SoMVR1 and SoMVR2) and a Taqmam probe were designed (So MVP).
[0037] 2. Composition of reagents
[0038] 1. Composition of Reagent A
[0039] Reagent A consists of SoMVF, SoMVR1 and SoMVP (primers and probes were synthesized by Shanghai Sangon).
[0040] SoMVF (upstream primer): 5'-CCGATGGAACACTTATTCAACAGT-3' (sequence 1 of the sequence listing);
[0041] SoMVR1 (downstream primer): 5'-TGGAGTTGGTGGAGGAAGTACA-3' (sequence 2 of the sequence listing);
[0042] SoMVP (probe): 5'(FAM)-TCGCCGGGTGTTATGAAGTCAGGATC-3'(TAMARA); (The nucleotide sequence is sequence 4 of the sequence listing, the 5' end is marked with the reporter fluorescent dye FAM, and the 3' end is marked with the ...
Embodiment 2
[0050] Embodiment 2, the application of reagent
[0051] Get the grape leaves that infect five kinds of viruses (SoMV, TBRV, SLRSV, TBSV and PRMV) respectively, get the healthy grape leaves as a contrast, use reagent A and reagent B prepared in Example 1 to carry out specificity measurement and sensitivity measurement respectively, and carry out Amplification efficiency comparison of reagent A and reagent B.
[0052] 1. Determination of specificity of reagents
[0053] 1. Total RNA extraction and quality control
[0054] Total RNA was extracted from leaves infected with each virus (5 species) and healthy leaves (CK1) with a plant total RNA extraction kit. Use the nucleic acid protein analyzer BioPhotometer to measure its OD value, get its concentration and purity value, and use this to control the quality of nucleic acid.
[0055] 2. Reverse transcription to synthesize cDNA
[0056] The total RNA extracted from the six kinds of leaves in step 1 was reverse-transcribed with...
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