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A kind of flounder rhabdovirus detection test paper and preparation method thereof

A technology of virus detection and flounder bomb, which is applied in the field of detection test paper and preparation of flounder rhabdovirus, can solve problems such as limitations

Active Publication Date: 2021-09-14
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, commonly used detection methods for the virus (such as histopathological techniques, electron microscopy techniques, and cell culture techniques) have their own advantages in terms of accuracy, sensitivity, safety, and cost, but they are limited by the need for professionals and complex instruments to varying degrees. limits

Method used

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  • A kind of flounder rhabdovirus detection test paper and preparation method thereof
  • A kind of flounder rhabdovirus detection test paper and preparation method thereof
  • A kind of flounder rhabdovirus detection test paper and preparation method thereof

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Effect test

Embodiment 1

[0046] Embodiment 1: Preparation of anti-flounder rhabdovirus G protein and M protein monoclonal antibody:

[0047] 1. Construction and protein expression of flounder rhabdovirus G and M protein expression plasmids:

[0048] (1) The total RNA of rhabdovirus from flounder was extracted by Trizol method.

[0049] (2) According to the principles of primer design and the published G gene of a flounder rhabdovirus isolate CNPo2015 (GeneBank: KY363350), use the primer design software Primer5.0 to combine with the multiple cloning site of the pET-28(a) plasmid The restriction sites of Bam H I and Xho I were selected as the insertion site of the target gene, and primers for expressing G protein of flounder rhabdovirus were designed.

[0050] Also according to the HIRRV (sequence number: FJ376982.1) and the multiple cloning site sequence of the expression vector pET-32(a) published by GenBank, the specific primers for the M protein of the flounder rhabdovirus were designed and combine...

Embodiment 2

[0142] Embodiment 2: the preparation of flounder rhabdovirus rapid detection test paper:

[0143] 1. Resuscitation and passage of monoclonal antibodies HIRRV-G-4D10, 10HIRRV-M-1F9, and HIRRV-M-4D10, intraperitoneally inject 8- and 12-week-old BALB / C mice with sterile liquid paraffin, inject 0.3 mL per mouse . One week later, use RPMI 1640 to gently blow off the cultured hybridoma cells from the cell culture plate, collect them into a 10 mL sterile centrifuge tube, centrifuge at 1000 rpm for 3 min, and gently resuspend the cell pellet with an appropriate amount of RPMI 1640. Only mice were intraperitoneally injected with 0.5 mL of hybridoma cell fluid. Closely observe the state of the mice. After 7 and 10 days, the abdomen of the mice is obviously enlarged, and the ascites is collected in a sterile centrifuge tube.

[0144] 2. Purification of ascites: Add 33ul of n-octanoic acid per milliliter of ascites, absorb an appropriate amount of n-octanoic acid, and add slowly drop by...

Embodiment 3

[0152] Embodiment 3: the usage method of flounder rhabdovirus detection test paper

[0153] (1) Preparation of test samples: Take the liver, spleen, kidney, heart, etc. of the flounder tissue to be tested, mix them with buffer solution at a ratio of 1:10 (W / V), add an appropriate amount of quartz sand and grind with a mortar, 7000-8000rpm ice homogenate in a bath, and take the supernatant as a test sample.

[0154] (2) Use of flounder rhabdovirus detection test paper: place the test paper flat on the table, drop the sample to be tested in the sample hole, and observe the results for 5-10 minutes.

[0155] (3) Result judgment:

[0156] ① Positive result: Red bands appear on the nitrocellulose membrane detection layer 4, detection line 6 and quality control line 7, which proves that the tested sample has been infected by HIRRV;

[0157] ② Negative result: There is no red band on the test line 6 of the nitrocellulose membrane detection layer 4, and a red band appears on the qua...

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Abstract

The invention discloses a flounder rhabdovirus detection test paper and a preparation method thereof. The detection test paper includes a carrier plate, a sample pad, a gold standard pad, a nitrocellulose membrane detection layer, and an absorbent pad. The sample pad and the absorbent pad are respectively at the two ends of the carrier plate, and one end of the gold standard pad is located below the sample pad. The gold label pad is loaded with colloidal gold-labeled anti-flounder rhabdovirus G protein monoclonal antibody, which is secreted by the hybridoma cell line HIRRV‑G‑4D10 (CCTCC NO: C201869), and the detection line and Quality control line, wherein the detection line is close to the side of the sample pad, and the quality control line is close to the side of the absorbent pad. The control line was coated with goat anti-mouse IgG. The invention can specifically detect the flounder rhabdovirus, has the advantages of rapidity, sensitivity, accuracy, little influence, low cost and the like, and is not restricted by professionals.

Description

technical field [0001] The invention relates to a detection test paper for flounder rhabdovirus (hirame rhabdovirus, HIRRV) and a preparation method thereof, and belongs to the cross technical fields of immunology, virology and diagnostic reagents. Background technique [0002] Flounder rhabdovirus disease is a highly pathogenic disease of flounder, and hiramerhabdovirus (HIRRV) is the causative agent of the disease. Flounder rhabdovirus is a single-stranded negative-sense RNA virus with strong pathogenicity. The virus particles are elastic, about 60-80nm wide and 160-180nm long. Plecoglossus altivelis ) and other muscular tissue organs and visceral hemorrhage, hematopoietic organ necrosis, can be infected from juvenile to adult fish; artificially infected red sea bream ( Pagrosomus major ), black sea bream juveniles are highly pathogenic to rainbow trout ( Oncorhynchus mykiss ) are also pathogenic. The onset seasons are mostly winter and early spring, and the disease m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/558
CPCG01N33/558G01N33/68
Inventor 唐小千战文斌杨秋娟绳秀珍邢婧
Owner OCEAN UNIV OF CHINA
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