Isolated pathogenic yellow catfish calicivirus and its specific sequence and application
A calicivirus, yellow catfish technology, applied in the direction of viruses, antiviral agents, viruses/phages, etc., can solve the problem of no homology, and achieve the effect of easy analysis and judgment, simplifying the process, and reducing costs
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Embodiment 1
[0035] Discovery of yellow catfish calicivirus and acquisition of specific sequences:
[0036] The applicant collected dying yellow catfish suffering from hemorrhagic disease from different yellow catfish farms for identification of the pathogen. The results showed necrosis and hemorrhage of the spleen and kidney cells of the sick yellow catfish. Ultramicrosection observation results show that there are a large number of spherical non-enveloped virus-like particles in the cells of the visceral tissue of the diseased yellow catfish, with a diameter of about 30-40nm, in the liver, spleen, kidney, heart, gills and other tissues of the yellow catfish. distribution, especially in the spleen and kidney tissues, a large number of viruses multiply. After high-throughput sequencing, it is found that the virus pathogen is a new virus pathogen. The genome sequence is quite different from other members of the Caliciviridae, but the evolutionary relationship It is similar, so it is named ...
Embodiment 2
[0041] Application of the primers designed for the specific sequence of YCCV-NS in the preparation of yellow catfish calicivirus (Yellow catfishcalicivrus, YCCV) detection kit:
[0042] The primers designed for the sequence shown in SEQ ID NO.1 are: YCCV-NS-F: 5'-CGCCTAAAGTTTCCTCCTTGTTGG-3' and YCCV-NS-R: 5'-AAAGTTGGGTGGATTGGTTTGTCAT-3'.
[0043] The amplification system is:
[0044] 1 μl (0.1 μg) of cDNA of the sample to be tested, 1 μl of 10 μM each primer YCCV-NS-F / YCCV-NS-R; 2 μl of 10 mM dNTP Mix; 5 μl of 10×TranStart Taq Buffer (Mg 2+ ) and 1 μl TranStart Taq DNA Polymerase (product of Beijing Quanshijin Biotechnology Co., Ltd.), add ddH2O to make the final volume 50 μl, after mixing, centrifuge for 15s to concentrate the reaction components at the bottom of the tube.
[0045] PCR amplification was performed, and the cycle program was: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min, 32 cycles,...
Embodiment 3
[0050] Sensitivity of primers designed for the specific sequence of YCCV-NS:
[0051] The yellow catfish calicivirus YCCV (Yellow catfish calicivrus, YCCV) genome cDNA was serially diluted 10 times: stock solution 0.1ng / μL, 10 -1 ...to 10 -4 , as template cDNA.
[0052] 2) Carry out RT-PCR amplification according to the method in embodiment 2;
[0053] 4) The detection limit of this primer to YCCV can reach 0.1ng / μL ( Figure 4 ).
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