Specific primer, probe and rapid detection kit for detecting yellow catfish Picornavirus-1

A technology of RNA virus and primer probe, which is applied in the field of rapid detection of target RNA fragments, can solve problems that hinder early warning and prevention of diseases, and achieve the effects of low cost, efficient amplification, and rapid amplification

Pending Publication Date: 2021-08-20
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 picornavirus-1 has not been reported at home and abroad, and there is no detection kit and detection method report for this virus so far, 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 yellow catfish Picornavirus-1
  • Specific primer, probe and rapid detection kit for detecting yellow catfish Picornavirus-1
  • Specific primer, probe and rapid detection kit for detecting yellow catfish Picornavirus-1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A primer-probe mixture for detection of Peeled catfish picornavirus-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 YCPRV-1-q85F and primer YCPRV-1-q85R;

[0030] The sequence of the primer YCPRV-1-q85F is shown in SEQ ID NO: 1;

[0031] The sequence of the primer YCPRV-1-q85R 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 gr...

Embodiment 2

[0039] Yellow catfish picornavirus-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; the positive quality control product is based on the purified yellow catfish picornavirus-1 genome as a template, PCR amplification is carried out by primer set A, the amplified product is connected to the carrier, and the correctness is confirmed by gene sequencing Pseudoviruses obtained by post-...

Embodiment 3

[0046] For the application of the small RNA virus-1 fluorescence quantitative detection kit for peltago catfish, take 0.05g of the sample to be tested and add 1mL of virus nucleic acid extraction solution for full grinding, 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 fluorescence quantitative RT-PCR. The reaction conditions are 42°C for 20 minutes, 95°C for 5-10 minutes; React for 20-40 seconds, a total of 45 cycles; set the fluorescence signal collection to HEX, and set the fluorescence signal collection to 60°C;

[0047] Result judgment:

[0048] If there is no S-t...

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Abstract

The invention discloses a specific primer, a probe and a rapid detection kit for detecting yellow catfish Picornavirus-1. The fluorescent quantitative detection kit for the yellow catfish Picornavirus-1 comprises a specific primer group A and a Taqman fluorescent probe B. The fluorescent quantitative technology is adopted, the yellow catfish Picornavirus-1 is detected by detecting capsid protein genes of the yellow catfish Picornavirus-1, and the primer group and the kit for detecting the yellow catfish Picornavirus-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 yellow catfish Picornavirus-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 picornavirus-1. Background technique [0002] Yellow catfish Picornavirus-1 (Yellow catfish Picornavirus, YCPRV-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. YCPRV-1 is a single-stranded RNA virus belonging to the family Picornaviridae. 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%, which indicated that the Peeled catfish pico...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2561/101C12Q2545/113
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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