A reagent for assisting identification of tobacco ringspot virus and its application
A tobacco ringspot virus and auxiliary identification technology, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problems of ELISA technology such as cumbersome operation steps, prone to false positives, long detection cycle, etc., and achieve broad Application range and prospects, avoiding false positive results, and improving the effect of accuracy
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Embodiment 1
[0037] Embodiment 1, the preparation of reagent
[0038] 1. Design of primers and probes
[0039] According to the conserved region of the coat protein gene (CP) sequence in the TRSV genome in the NCBI nucleic acid database in the United States, two sets of TaqMAN probes and primers were designed respectively. In this study, three primers were designed (two upstream primers TRSVF1, TRSVF2 and one downstream primer TRSVR) and One Taqman probe (TRSVP).
[0040] 2. Composition of reagents
[0041] 1. Composition of Reagent A
[0042] Reagent A consists of TRSVF1, TRSVR and TRSVP (primers and probes were synthesized by Shanghai Sangon).
[0043] TRSVF1 (upstream primer): 5'-CTCCTGATGCACATGTTGGGT-3' (sequence 1 of the sequence listing);
[0044] TRSVR (downstream primer): 5'-GGGCCTGTTTAGACCTTGACC-3' (sequence 3 of the sequence listing);
[0045] TRSVP (probe): 5'(FAM)-AGACGCAACTGTGACGCTCGCATC-3'(TAMARA); (The nucleotide sequence is sequence 4 of the sequence listing, the 5' en...
Embodiment 2
[0053] Embodiment 2, the application of reagent
[0054] Soybean leaves infected with six viruses (TRSV, BPMV, SBMV, PSV, ToRSV, and TSWV) were taken respectively, and healthy soybean leaves were taken as controls, and reagent A and reagent B prepared in Example 1 were used for specificity determination and sensitivity determination respectively. The amplification efficiencies of reagent A and reagent B were compared.
[0055] 1. Determination of specificity of reagents
[0056] 1. Total RNA extraction and quality control
[0057] Total RNA was extracted from leaves infected with each virus (6 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.
[0058] 2. Reverse transcription to synthesize cDNA
[0059] The total RNA extracted from the 7 kinds of leaves in step 1 was reverse-transcri...
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