Identification of a Morinda officinalis variola virus and its ribosomal rna gene and application
A leaf virus and ribosome technology, applied in the identification of Morinda officinalis variola leaf virus and its ribosomal RNA gene and application fields, can solve the problems of detection method limitations and other problems, and achieve accurate and reliable detection results, rapid detection, and wide promotion The effect of applying the foreground
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Embodiment 1
[0037] The acquisition of the ribosomal RNA gene of embodiment 1 Morinda officinalis mosaic virus
[0038] 1. Experimental method
[0039] The acquisition of the ribosomal RNA gene of Morinda officinalis small mosaic virus, concrete steps are as follows:
[0040] S1. Collect Morinda officinalis diseased leaves;
[0041] S2. extracting the total RNA of Morinda officinalis diseased leaves;
[0042] S3. Ribosomal RNA library construction: according to the high-throughput sequencing library construction process, the above total RNA was constructed into a paired-end high-throughput sequencing library with a fragment size of 500bp;
[0043] S4. High-throughput sequencing;
[0044] S5. Sequence tag assembly: SPAdes v.3.5.0 software was used for sequence tag assembly. According to the overlap (overlapping region) relationship between short sequencing fragments, the sequencing fragments were assembled into contig sequences, and based on the contig sequences, combined The distance r...
Embodiment 2
[0052] Embodiment 2 detects the method for Morinda officinalis small mosaic virus
[0053] 1. Experimental method
[0054] (1) PCR amplification reaction
[0055] Extract the total RNA of Morinda officinalis variola virus obtained in the example and store the extracted total RNA at -80°C, and reverse transcribe the total RNA into a cDNA library. Design specific primers (F1 / R1 primer set, F2 / R2 primer set and F3 / R3 primer set), the nucleotide sequences of the primers are respectively shown in SEQ ID NO.2~SEQ ID NO.7 in Table 1; The cDNA library was used as a template for PCR detection. The PCR amplification system was shown in Table 2. The amplification reaction products were subjected to agarose electrophoresis and recovered.
[0056] Table 1 Specific primers used in PCR detection
[0057]
[0058] Table 2 PCR amplification system
[0059]
[0060] The PCR amplification program is: 94°C pre-denaturation for 2 minutes; 94°C denaturation for 30s; , it takes 1 min to a...
Embodiment 3
[0065] The detection of embodiment 3 Morinda officinalis smallpox
[0066] The leaves of Morinda officinalis with typical mosaic symptoms were randomly found in the field and collected; the lesion area in the leaves of Morinda officinalis was cut, and the cut lesion material was fully ground with liquid nitrogen, and the total amount of diseased leaves of Morinda officinalis was extracted. RNA; the extracted total RNA was stored at -80°C, and the total RNA was reverse-transcribed into a cDNA library; the specific amplification primers were the same as in Example 2.
[0067] The electropherogram of the detection result is the same as figure 2 , the results showed that the three sets of primers could amplify the corresponding target bands.
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