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Specific primer group, kit comprising the primer group, use method and detection method

A technology of specificity and primer set, applied in the fields of virology and molecular biology, can solve the problem of rapid detection of red sea bream iridescent virus that has not been seen yet

Inactive Publication Date: 2012-07-11
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to the search, there is no report on the rapid detection of red sea bream iridescent virus using the loop-mediated isothermal amplification technique

Method used

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  • Specific primer group, kit comprising the primer group, use method and detection method
  • Specific primer group, kit comprising the primer group, use method and detection method
  • Specific primer group, kit comprising the primer group, use method and detection method

Examples

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Effect test

Embodiment 1

[0220] Carry out 10-fold serial gradient dilution of the whole gene recombinant plasmid containing the main capsid protein of red sea bream iridescent virus preserved in this laboratory, adopt the present invention to detect it, determine the detection limit of the present invention, and the primer sequences adopted are as follows:

[0221] RSIV-F3: 5'-GTCACACCCGCAGACAAT-3'

[0222] RSIV-B3: 5'-ACCTCGGACAGGGGATTG-3'

[0223] RSIV-FIP:

[0224] 5'-TGCGGTGGGTGACGTTCTTTAC-TTTT-CTTGGTGCATCTGGACCTC-3'

[0225] RSIV-BIP:

[0226] 5'-ACGTGCAAAGCAATTACACCGC-TTTT-GCAAAGGCAGATTGACCTTG-3'

[0227] RSIV-LF: 5'-TTGACGGGATGACTGAACCT-3'

[0228] RSIV-LB: 5'-GGCCAGTCCCGTGTATGTCAAC-3'

[0229] Primers were synthesized by Dalian Bao Biological Company.

[0230] Carry out the red sea bream iridescent virus ring-mediated isothermal amplification reaction according to the following procedure:

[0231] Take a 0.2mL eppendorf tube and add the following reagents: 2.5μL 10×Thermoplol buffer, 2μL ...

Embodiment 2

[0236] The specificity of the ring-mediated isothermal amplification detection method of red snapper iridescent virus was analyzed; as a control, other viruses that were closely related to red snapper iridescent virus were used for detection by the ring-mediated isothermal amplification detection method, and these viruses belonged to Iridoviridae, they are epidemic hematopoietic necrosis virus (EHNV), lymphocyst virus (LCDV), frog virus 3 (FV 3), soft shell turtle iridescent virus (STIV); detection step is the same as embodiment 1, just will treat The DNA of the test sample was replaced with the DNA of epidemic hematopoietic organ necrosis virus (EHNV), lymphocyst virus (LCDV), frog virus 3 (FV 3), and soft shell turtle iridescent virus (STIV); the test results showed that red sea bream iridescent virus The primers of the loop-mediated isothermal amplification detection method only amplify red sea bream iridescent virus, and have no amplification reaction to EHNV, LCDV, FV3, an...

Embodiment 3

[0238] A total of 120 batches of 120 batches of seabass, rock butterfly, and turbot were collected from farms in Shandong sea area, with 30 fish in each batch, and were tested for red sea bream iridescent virus. The samples taken had no typical clinical symptoms.

[0239] Follow the procedure below:

[0240] 1. Extraction of sample DNA

[0241] For each batch of 30 samples, the brain, spleen, and kidney tissues were collected, homogenized, and the DNA of the samples was extracted using a DNA extraction kit (DV811A, TAKARA) according to the instructions.

[0242] 2. Loop-mediated isothermal amplification reaction of red snapper iridescent virus

[0243] Take a 0.2 mL polypropylene plastic tube, add 1.0 μL of the sample DNA extracted in step (1), and then add other reaction components and the steps are the same as in Example 1.

[0244] Place the above plastic tube in a constant temperature water bath at 63°C for 60 minutes to carry out the loop-mediated isothermal amplificati...

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Abstract

The invention provides a detection kit for red sea bream iridovirus, comprising a specific primer group which is designed for detecting the red sea bream iridovirus. The specific primer group is composed of the following 6 specific primers: a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), a backward inner primer (BIP), a loop forward primer (LF) and a loop backward primer (LB). The kit provided by the invention adopts the loop-mediated isothermal amplification (LAMP) technology and designs 6 primers based on the full gene sequence of the main capsid protein (MCP) of the red sea bream iridovirus, so that the target fragment is subjected to circulatory displacement and amplification in an isothermal environment, the amplification of a target sequence is realized, and further, the detection purpose is achieved. The kit for detecting the red sea bream iridovirus is rapid and efficient, can meet the onsite and timely detection requirements and provides technical support for the quick detection of the red sea bream iridovirus in aquaculture industry of Chinese and international fish trade.

Description

technical field [0001] The invention belongs to the fields of virology and molecular biology, and in particular relates to a red sea bream iridescent virus detection kit. Background technique [0002] Red sea bream iridovirus (RSIV) belongs to the family Iridoviridae and belongs to the genus Megalocytivirus. Japan, subsequently, broke out in East Asia and Southeast Asia one after another. When the disease breaks out in nature, most of them are very sudden, and the fatality rate is high. It is currently one of the most serious diseases for mariculture in various countries. At the same time, the disease also seriously affects the import and export trade of economic fish. The disease is listed as a disease that must be reported to the World Organization for Animal Health (OIE), and the Ministry of Agriculture of my country lists it as a second-class disease, and monitors the disease in my country's coastal waters twice a year. [0003] Existing detection methods for red sea b...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 赵玉然李八方岳志芹马亚梁成珠徐彪
Owner OCEAN UNIV OF CHINA
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