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Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1

A calicivirus, primer probe technology, applied in the field of rapid detection of target RNA fragments, can solve problems such as hindering disease early warning and prevention

Active Publication Date: 2021-07-30
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peeled catfish calicivirus-1 has not been reported at home and abroad, and there is no detection kit and detection method report for this virus, which seriously hinders the early warning and prevention of diseases caused by the virus. Therefore, the virus detection reagent The development of cartridges and the research of detection technology are imminent

Method used

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  • Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1
  • Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1
  • Specific primer, probe and rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A primer-probe mixture for detecting yellow catfish calicivirus-1, which consists of primer set A and Taqman fluorescent probe B, the 5' end of probe B is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0029] The primer set A includes primer YCCV-1-q183F and primer YCCV-1-q183R;

[0030] The primer YCCV-1-q183F sequence is shown in SEQ ID NO: 1;

[0031] The primer YCCV-1-q183R sequence is shown in SEQ ID NO: 2;

[0032] The nucleotide sequence of the probe B is shown in SEQ ID NO:3.

[0033] Wherein, the fluorescent reporter group is selected from 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxyl-X-rhodamine, 6-carboxytetramethyl Rhodamine, sulforhodamine, 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein succinimide ester, cyanine 3, cyanine 3.5, cyanine 5 and one or more of cyanine 5.5; the fluorescence quenching group is selected fro...

Embodiment 2

[0039] Yellow catfish calicivirus-1 fluorescence quantitative detection kit, including individually packaged viral nucleic acid extraction solution, one-step RT-qPCR reaction solution, positive quality control material, negative quality control material and primer-probe mixture.

[0040]The primer-probe mixture is composed of primer set A and Taqman fluorescent probe B, wherein the final concentration of upstream and downstream primers of primer set A is 0.15 μM, and the final concentration of probe is 0.1 μM.

[0041] Taqman fluorescent probe B, its nucleotide sequence is labeled with HEX at the 5' end and BHQ1 at the 3' end;

[0042] The negative quality control product is sterile saline without RNase; the positive quality control product uses the purified yellow catfish calicivirus-1 genome as the template, and is amplified by RT-PCR with primer set A, and the amplified product is connected to the vector , the pseudovirus obtained by packaging after being confirmed to be co...

Embodiment 3

[0046] Yellow catfish calicivirus-1 fluorescence quantitative detection kit was applied, take 0.05g of the sample to be tested and add 1mL of virus nucleic acid extract to fully grind, centrifuge at 12000r / min for 10min, take 800uL of supernatant, add 400uL of ethanol, and after 5min at room temperature , centrifuge at 12000r / min for 5min, wash the precipitate once with 75% ethanol, dry at room temperature for 5min, add 50uL DEPC water to dissolve the RNA, take 5uL into the reaction tube, add 2uL of primer-probe mixture, and then add one-step RT - 10uL of qPCR reaction solution, add 3uL of DEPC water, mix well and perform fluorescence quantitative PCR. The reaction conditions are 42°C for 20 minutes, 95°C for 5-10 minutes; then 95°C for 5-15 seconds, 60°C for 20 minutes -45 seconds, a total of 45 cycles; the fluorescence signal collection is set to HEX, and the fluorescence signal collection is set to 60 °C;

[0047] Result judgment:

[0048] If there is no S-shaped amplifica...

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Abstract

The invention discloses a specific primer, a probe and a rapid detection kit for detecting pelteobagrus fulvidraco calicivirus-1, and the fluorescent quantitative detection kit for the pelteobagrus fulvidraco calicivirus-1 comprises a specific primer group A and a Taqman fluorescent probe B, according to the primer group and the kit for detecting the pelteobagrus fulvidraco calicivirus-1, the pelteobagrus fulvidraco calicivirus-1 is detected by detecting capsid protein genes of the pelteobagrus fulvidraco calicivirus-1 by adopting a fluorescent quantitative technology, and the primer group and the kit for detecting the pelteobagrus fulvidraco calicivirus-1 are developed for the first time at home and abroad at present. The development of the kit lays a foundation for monitoring and preventing the pelteobagrus fulvidraco calicivirus-1 disease.

Description

technical field [0001] The invention belongs to the field of rapid detection of target RNA fragments, in particular to a specific primer, probe and rapid detection kit for detecting yellow catfish calicivirus-1. Background technique [0002] Yellow catfish Calicivirus-1 (YCCV-1) is a potential pathogenic pathogen of yellow catfish. It has a certain pathogenicity to yellow catfish, often causing hemorrhage and enteritis on the body surface of yellow catfish, and a few diseased fish have bleeding spots in the liver. The diseases caused by it do great harm to the yellow catfish aquaculture industry. YCCV-1 is a single-stranded RNA virus belonging to the family Caliciviridae. The taxonomic status of the genus has not been determined. Through genome comparison, it is found that the virus with the highest genome homology to the virus is Conger japonicus Calicivirus. The homology was 38%, indicating that yellow catfish calicivirus-1 is a completely new virus, unlike any other kno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/701C12Q1/6851C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/113C12Q2521/107Y02A50/30
Inventor 潘晓艺蔺凌云沈锦玉姚嘉赟尹文林黄雷袁雪梅
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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