Kit for diagnosing reovirus genes of grass carps and application thereof

A technology for reovirus and gene diagnosis, which is applied to a grass carp reovirus gene diagnosis kit and its application field, and can solve problems such as failure to amplify

Active Publication Date: 2010-12-08
ZHEJIANG INST OF FRESH WATER FISHERIES
View PDF1 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the GCRV RT-PCR method has also been reported in the literature, only the original strain virus used to design primers is PCR positive, and the corresponding fragments of other GCRVs cannot be amplified. This test is based on

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Kit for diagnosing reovirus genes of grass carps and application thereof
  • Kit for diagnosing reovirus genes of grass carps and application thereof
  • Kit for diagnosing reovirus genes of grass carps and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Genetic diagnostic kit for grass carp reovirus

[0053] The kit consists of the following parts (10 samples of virus in tissues and cells are extracted):

[0054] 1. One tube each of virus RNA extraction solutions A, B, C and D in tissues, A is 0.5mL proteinase K (stored in a refrigerator at 4°C), B is 0.5mL 1% SDS, and C is 8mL phenol / chloroform / isoamyl Alcohol mixed solution, D is 8mL chloroform / isoamyl alcohol mixed solution.

[0055] 2. One tube each of virus RNA extraction solution a and b in cells, a is 10mL Trizol solution, and b is 2.5mL chloroform (you can also prepare it yourself).

[0056] 3. Liquid E, 1 tube, filled with isopropanol (you can also bring your own), 10mL in total.

[0057] 4. Solution F, 2 tubes, 10mL / tube, filled with 70% ethanol (you can also bring your own).

[0058] 5. Liquid G, 2 tubes, 5mL / tube, filled with DEPC water.

[0059] 6. 1 tube each of RT reaction solution H and I, containing RT reaction solution (25 μL system), o...

Embodiment 2

[0079] Example 2: Detection of GCRV in liver, spleen and kidney tissues of grass carp

[0080] (1) Extraction of sample nucleic acid

[0081] Add 100 mg of diseased fish tissue to 500 μL DEPC water, mash and homogenate, centrifuge at 10,000 rpm for 10 minutes, take the supernatant, add 40 μL proteinase K and 40 μL 1% SDS, and incubate at 37°C for 30 minutes; then add 600 μL phenol / chloroform / isoamyl alcohol, mix thoroughly Homogenize for 30S, centrifuge at 12,000rpm for 5min, take the upper aqueous phase; then add an equal volume of chloroform / isoamyl alcohol mixture, mix thoroughly for 30S, and centrifuge at 12,000rpm for 5min, take the upper aqueous phase; add an equal volume of isopropanol, mix After homogenization, the nucleic acid was precipitated at -20°C for more than 1h, centrifuged at 12,000rpm for 10min, the supernatant was discarded, washed with 70% ethanol, dried, and an appropriate amount of DEPC water was added to dissolve it, and set aside.

[0082] (2) RT-PCR ...

Embodiment 3

[0112] Example 3: Detection of Grass Carp Kidney Cell Separation GCRV

[0113] (1) Extraction of RNA of CIK cell infection virus

[0114] The infected CIK cell solution was repeatedly frozen and thawed three times. Take 250 μL virus suspension, add 750 μL Trizol to mix at high speed, room temperature for 5 minutes, then add 200 μL chloroform for vigorous shaking, room temperature for 5 minutes; Precipitate with isopropanol at room temperature for 15-20 minutes, centrifuge at 12,000 rpm for 15 minutes, wash with 70% ethanol, and blow dry. Then add an appropriate amount of DEPC water to dissolve and set aside.

[0115] (2) RT-PCR amplification

[0116] RT reaction: a total of 25 μL of reaction system

[0117] RNA template 13.5 μL

[0118] Rnasin (RI) 0.5μL

[0119] 1 μL of the reverse primer R1

[0120] Place in a 72°C water bath for 10 minutes and ice bath for 5 minutes.

[0121] Then add in order

[0122] 5×AMV Buffer 5.0μL

[0123] dNTP 4μL

[0124] AMV reverse tran...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a kit for diagnosing the reovirus genes of grass carps, comprising an RNA (Ribonucleic Acid) extraction reagent for viruses in visceral tissues, an RAN extraction reagent for cell infecting viruses, an RT (Reverse Transcriptase) reaction reagent and a PCR (Polymerase Chain Reaction) reaction reagent, wherein the RNA extraction reagent for the viruses in the visceral tissues comprises the components of DEPC (Diethyl Pyrocarbonate) water, protease K (1 mg/mL, pH is 8.0), SDS (Sodium Dodecyl Sulfate) (1%), phenol/chloroform/isoamylol (25/24/1), chloroform/isoamylol (24/1), isopropanol and 70% ethanol; the RAN extraction reagent for the cell infecting viruses comprises the components of Trizol, chloroform, isopropanol, 70% ethanol and the DEPC water; the RT reaction reagent comprises the components of 5*AMV Buffer, dNTP, the DEPC water, a reverse primer R1, an RNA enzyme inhibitor RNAsin (R1) and a reverse transcriptase AMV; and the PCR reaction reagent comprises the components of 10*PCR buffer (containing Mg<2+>) and DNTP Mixture (which are respectively 2.5 mmol/L), a specific oligonucleotide primer F1, the primer R1, the DEPC water and Taq E (5U/muL), the forward primer F1:5'-ATCCCGTATATCTATGGCTT-3', and the reverse primer R1:5'TTGGAGACGAACATAGACGC-3. The kit is used for diagnosing the reovirus genes of the grass carps.

Description

technical field [0001] The diagnostic kit and application of the aquatic animal virus of the present invention are mainly a diagnostic kit and a detection method for the conserved part of the sixth gene segment of Grass carp reovirus (GCRV, Grass carp reovirus). Background technique [0002] Grass Carp Hemorrhage Virus (GCHV) belongs to aquatic reovirus, the virus particles are spherical particles with icosahedral symmetry, the diameter of the mature virus is 75nm, it has a double-layer capsid, no envelope, and its genome Consisting of 11 dsRNAs, it is very stable in the range of pH3-11 and resistant to chloroform and ether. The fifth meeting of the International Committee on Taxonomy of Viruses decided to formally create a new genus of aquatic reovirus (Aquareovirus, ARV) in the family Reoviridae, and to include grass carp hemorrhagic disease virus into this genus, named Grass carp reovirus (Grass carp reovirus). reovirus, GCRV), but it is different from the representative...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 郝贵杰沈锦玉潘晓艺徐洋姚嘉赟尹文林
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap