Primer for detecting freesia mosaic virus (FreMV) and detection method of freesia mosaic virus
A technology of virus and freesia, which is applied in the detection field of freesia virus primers and its detection, can solve the problems of low detection sensitivity, high price of antiserum, false positive reaction, etc., and achieve high detection efficiency, high detection sensitivity, low cost effect
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Embodiment 1
[0027] Concrete operation steps of the present invention are as follows:
[0028] Step 100: Utilize Primer5.0 software to design three pairs of specificity according to the coat protein gene nucleotide sequences of the three viruses of CMV (cucumber mosaic virus), FreMV (freesia mosaic virus) and BYMV (bean yellow mosaic virus) Primers, and then use DNAman software to correct the three pairs of specific primers designed to confirm that there is no self-matching and competitive amplification among the three pairs of primers, so as to ensure that the above three viruses can amplify the specific fragments with obvious differences. The above three pairs of specific primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0029] Step 200: 0.2 g of healthy leaves and 0.2 g of freesia leaves co-infected by CMV, FreMV and BYMV were subjected to RNA extraction respectively, and healthy leaves were used as negative controls. RNA was extracted using the Trizol method: 0.2g...
Embodiment 2
[0039] This part differs from Example 1 in that:
[0040] Step 200: Randomly select 6 samples of freesia leaves with compound infection symptoms in the germplasm resource garden of the Flower Research Center of Fujian Academy of Agricultural Sciences for RNA extraction, and the 6 freesia leaf samples are all 0.2g;
[0041] Step 400: In this embodiment, multiple PCR amplification reactions are carried out. The specific content of the reaction system and reaction procedure of the PCR amplification reaction is as follows: the multiple PCR reaction system includes a total volume of 50 μL of the reaction system, and contains 0.3 μL of 5U / μL Taq enzyme , 5 μL of 10×Taq enzyme buffer, 4 μL of 2.5 mmol / L dNTPs, 1 μL of template, 0.38 μmol / L of CMV forward primer, 0.38 μmol / L of CMV reverse primer, 0.04 μmol / L of FreMV forward primer, 0.04 μmol / L of FreMV reverse primer, 0.07 μmol / L of BYMV forward primer, 0.07 μmol / L of BYMV reverse primer, and the remaining components are water; 30s...
Embodiment 3
[0044] This part differs from Embodiment 2 in that:
[0045] Step 200: Randomly select 0.2 g of a freesia leaf sample with compound infection symptoms in the germplasm resource garden of the Flower Research Center of Fujian Academy of Agricultural Sciences for RNA extraction;
[0046] Step 400: The reaction system and reaction procedure of the PCR amplification reaction are as follows: the multiplex PCR reaction system includes a reaction system with a total volume of 50 μL, containing 0.3 μL 5U / μL Taq enzyme, 5 μL 10×Taq enzyme buffer, 4 μL 2.5 mmol / L dNTPs, 1 μL template, 0.34 μmol / L CMV forward primer, 0.34 μmol / L CMV reverse primer, 0.03 μmol / L FreMV forward primer, 0.03 μmol / L FreMV reverse primer, 0.05 μmol / L BYMV forward primer, 0.05μmol / L BYMV reverse primer, and water as the rest; the PCR reaction program is A. Pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute , cycled 35 ...
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