Specific primers, probes and rapid detection kits for detecting yellow catfish calicivirus-1

A technology of calicivirus and yellow catfish, applied in biochemical equipment and methods, measurement/testing of microorganisms, and resistance to vector-borne diseases, etc., can solve problems that hinder early warning and prevention of diseases, and achieve low cost and high detection sensitivity High and efficient effect

Active Publication Date: 2022-05-20
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • 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 primers, probes and rapid detection kits for detecting yellow catfish calicivirus-1
  • Specific primers, probes and rapid detection kits for detecting yellow catfish calicivirus-1
  • Specific primers, probes and rapid detection kits for detecting yellow catfish calicivirus-1

Examples

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 fluorescence quencher group.

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

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

[0031] The sequence of the primer YCCV-1-q183R 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 reporting group is selected from 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethyl Rhodamine, sulforhodamine, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and one or more of cyanine 5.5; the fluorescence quenching group ...

Embodiment 2

[0039] Yellow catfish calicivirus-1 fluorescent quantitative detection kit, including individually packaged virus nucleic acid extraction solution, one-step RT-qPCR reaction solution, positive quality control, negative quality control 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, whose nucleotide sequence is marked with HEX at the 5' end and BHQ1 at the 3' end;

[0042] The negative quality control product is sterilized normal saline without RNase; the positive quality control product is based on the purified yellow catfish calicivirus-1 genome as a template, and the primer set A is used for RT-PCR amplification, and the amplified product is connected to the carrier , the pseudovirus that is packaged after being confirmed to be ...

Embodiment 3

[0046] For the application of the yellow catfish calicivirus-1 fluorescent quantitative detection kit, take 0.05g of the sample to be tested and add 1mL of virus nucleic acid extraction solution to fully grind, centrifuge at 12000r / min for 10min, take 800uL of the supernatant, add 400uL of ethanol, and wait for 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 RNA, take 5uL and add it to the reaction tube, then 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 fluorescent quantitative PCR. The reaction conditions are 42°C for 20 minutes, 95°C for 5-10 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-type amplification curve...

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Abstract

The invention discloses a specific primer, a probe and a rapid detection kit for detecting yellow catfish calicivirus-1, wherein the fluorescent quantitative detection kit for yellow catfish calicivirus-1 includes specific primer group A and Taqman fluorescent probe B; the present invention uses fluorescence quantitative technology to detect the capsid protein gene of yellow catfish calicivirus-1 to detect yellow catfish calicivirus-1, which is the first development and detection of yellow catfish calicivirus-1 at home and abroad Primer sets and kits for Fish Calicivirus‑1. The development of the kit laid the foundation for the monitoring and prevention of yellow catfish calicivirus-1 disease.

Description

technical field [0001] The invention belongs to the field of rapid detection of target RNA fragments, and in particular relates to a specific primer, a probe and a rapid detection kit for detecting yellow catfish calicivirus-1. Background technique [0002] Yellow catfish Calicivirus-1 (Yellow catfish Calicivirus, YCCV-1) is a potential pathogenic pathogen of yellow catfish. It has a certain pathogenicity to yellow catfish, and often causes surface hemorrhage and enteritis of yellow catfish, and a few diseased fish have bleeding spots in the liver. The diseases caused by it are very harmful to the aquaculture industry of yellow catfish. 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 was found that the virus with the highest homology to the virus genome is Conger japonicus Calicivirus. The homology was 38%, indicating that the yellow catfish calicivirus-1 i...

Claims

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Application Information

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Patent Type & Authority Patents(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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