Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

CyHV-2 (Cyprinid herpesvirus II) LAMP (loop-mediated isothermal amplification) detection kit and detection method

A technology of carp herpes virus and detection kit, which is applied in the field of fish virus detection, can solve the problems of high mortality and rising incidence, and achieve the effect of simple identification, low detection cost and good specificity

Inactive Publication Date: 2013-04-17
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In the main breeding areas of crucian carp in my country, viral hemorrhagic disease of crucian carp sporadically occurred in 2009. The incidence has been increasing year by year, and the mortality rate is high. At present, there are no effective control measures. Preliminary research shows that the pathogen is carp herpes virus type Ⅱ ( Cyprinid herpesvirus II, CyHV-2), initially named the disease as Crucian Carp Hematopoietic Necrosis

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
  • CyHV-2 (Cyprinid herpesvirus II) LAMP (loop-mediated isothermal amplification) detection kit and detection method
  • CyHV-2 (Cyprinid herpesvirus II) LAMP (loop-mediated isothermal amplification) detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A detection kit for carp herpesvirus type 2 LAMP, comprising the following components:

[0044] The kit includes the following components: 10×ThermoPol Reaction Buffer; Bst DNA polymerase (NEB); dNTPs; outer primers F3 and B3, inner primers FIP and BIP, Betaine (Sigma), MgCl 2 , 1000 x SYBR GreenI (Invitrogen).

[0045] F3: 5'-TTGGATCTGAACGCTTCGG-3';

[0046] B3: 5'-CGTTGGTCTGTATGGGAGC-3';

[0047]FIP: 5'-GCGATGTAAGCCCTGTGAGACTTTTTACGAGACGTGGTTCCTAGC-3';

[0048] BIP: 5'-AACGCACGAGTGCGAGTCTCTTTTGCTGTGGATCGTCCATCC-3'.

Embodiment 2

[0050] Carp herpesvirus type 2 LAMP detection kit with different concentrations of Mg 2+ Optimization

[0051] 1. Take the sample to be tested and extract the virus DNA:

[0052] Cultivate Koi fin ray cell line (Koi-Fin) to a confluent monolayer, suck out the culture medium, and inoculate 1 mL of carp herpesvirus type 2 tissue toxic material ( Doszpoly, A., M. Benko, Gy. Csaba, A. Dan, M. Lang, B. Harrach. 2011. Introduction of the family Alloherpesviridae: the first molecular detection of herpesviruses of cyprinid fish in Hungary. Magyar Allatorvosok Lapja133 (3 ):174-181.), add 10 μL of Polybrene (final concentration 10 μg / ml), and place in a 26°C incubator for 1 hour to adsorb the virus to the cells. During the adsorption process, gently shake the culture bottle once every 20 minutes to make the virus liquid Fully and evenly contact with the cell monolayer, after 1 hour of adsorption, discard the virus liquid, add 5mL of 2% fetal bovine serum (V / V) culture solution, and c...

Embodiment 3

[0061] Optimization of the reaction temperature of LAMP detection method for carp herpesvirus type 2:

[0062] 1. Take the sample to be tested and extract the virus DNA:

[0063] Harvest the Koi-Fin cells infected with carp herpesvirus type 2 after 90% of the lesions appear (the preparation method is the same as in Example 2), freeze and thaw 3 times at -80°C to room temperature, centrifuge at 5000r / min for 30min, and take 250 μl of the supernatant. DNA was extracted using the Viral DNA Kit kit according to the instructions, and finally dissolved in 50 μl of sterilized water, and stored at -20°C for future use.

[0064] 2. The reaction system of LAMP amplification:

[0065] A 25 μl reaction system was used, including: 0.8 μM each of the inner primers FIP and BIP, 0.1 μM each of the outer primers F3 and B3, dNTPs1 mM, Betaine0.5M, MgCl 2 8mM, Bst DNA polymerase 8U, template DNA 5μl, 10×ThermoPolReaction Buffer 2.5μl, deionized water to make up the balance.

[0066] 3. Reacti...

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 discloses a CyHV-2 (Cyprinid herpesvirus II) LAMP (loop-mediated isothermal amplification) detection kit and detection method. The CyHV-2 LAMP detection kit comprises the following components: 10*ThermoPol Reaction buffer solutions, Bst DNA polymerase, dNTPs, outer primers F3 and B3, inner primers FIP and BIP, Betaine, MgCl2 and 1000*SYBR Green I. The invention has the characteristics of convenience, quickness and high specificity and sensitivity; and the CyHV-2 in a sample can be accurately detected within 2 hours by means of a water bath or metal bath alone, CyHV-2 infected sick fish tissues can be detected, and CyHV-2 infected cells can be detected, thereby ensuring that the invention is very applicable to on-site quick CyHV-2 detection.

Description

technical field [0001] The invention belongs to the technical field of fish virus detection, in particular to a carp herpes virus type 2 LAMP detection kit, and also relates to a method for detecting carp herpes virus type 2 LAMP detection kit. Background technique [0002] In the main breeding areas of crucian carp in my country, viral hemorrhagic disease of crucian carp sporadically occurred in 2009. The incidence has been increasing year by year, and the mortality rate is high. At present, there are no effective control measures. Preliminary research shows that the pathogen is carp herpes virus type Ⅱ ( Cyprinid herpesvirus II, CyHV-2), initially named the disease as Crucian Carp Hematopoietic Necrosis. Cases of CyHV-2 infection were also found in gibel carp cultured in Hungary in 2011. [0003] The proliferation of CyHV-2 in several fish cell lines can not exceed 4 generations. It is necessary to establish a rapid, sensitive, specific and suitable method for on-site diag...

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): C12Q1/70C12Q1/68
Inventor 曾令兵范玉顶张辉周勇徐进
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com