Primer and method for detecting pathogen of CSVd (Chrysanthemum Stunt Viroid)
A detection device and technology of Compositae plants, applied in the field of plant disease diagnosis, can solve problems such as no effective detection method for new virus CVR
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Embodiment 1
[0111] Embodiment 1. Discovery of new pathogen of chrysanthemum dwarf disease
[0112] During the investigation of chrysanthemum virus disease in Beijing area, it was found that chrysanthemum dwarf disease is an important disease that endangers the production of chrysanthemum. The application took 4 chrysanthemum dwarf samples in Yanqing District, Beijing, and used the RT-PCR method to detect chrysanthemum common virus chrysanthemum B virus (Chrysanthemum virus B, CVB), tomato infertility virus (Tomato aspermy virus, TAV) and chrysanthemum stunt viroid (Chrysanthemum stunt viroid, CSVd). However, in sample No. 2, the above-mentioned virus was not detected, so we speculate that the dwarfing of this strain of chrysanthemum is caused by the infection of a new pathogen (for example, it is most likely a virus).
Embodiment 2
[0113] Example 2. Obtaining, Sequencing and Database Searching of Genomic RNA of New Pathogens
[0114] This application uses the Illumina small RNA high-throughput sequencing method to identify the pathogen in sample No. 2. The specific method is as follows:
[0115] 1. Take 0.1 g of chrysanthemum leaves and grind them fully in liquid nitrogen, and extract the total RNA of chrysanthemum leaves by TRNzol method (this application refers to the extraction of plant total RNA by TRNzol method, but is not limited to TRNzol method).
[0116] 2. This application uses the total RNA sample as a template to construct a small RNA library, and performs Illumina small RNA high-throughput sequencing on the library, and the number of sequencing reads is not less than 20M.
[0117] 3. The analysis of the raw data generated by sequencing mainly includes: using the Velet algorithm to remove low-quality sequencing reads and joints in the reads, and assembling the processed reads into contigs; u...
Embodiment 3
[0135] Example 3. Establishment of a method for detecting chrysanthemum R virus (CVR)
[0136] 1. Design of primers:
[0137] Detection primers for CVRs are allowed to be designed based on any fragment at any position in the nucleotide sequence of CVRs encoding replicase, three-gene box and coat protein (ie ORF1 to 5). Primer design principles are:
[0138] 1) The primer specifically binds to any fragment of the CVR genome;
[0139] 2) The primer length is 15bp to 30bp;
[0140] 3) G+C content between 40% and 60%;
[0141] 4) There can be no consecutive 4 base complementarity within and between primers;
[0142] 5) The Tm value of the primer is 55±5°C;
[0143] 6) The length of the amplified product is 300bp to 1200bp.
[0144] The detection primers designed according to the above design principles are, for example:
[0145] Table 2. The designed detection primers
[0146]
[0147] 2. The detection method is as follows:
[0148] Using the above-mentioned Chrysanthe...
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