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Primer group, kit and detection method for detecting reovirus and bicistronic virus of scylla serrata

A reovirus and bicistronic technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms, can solve unseen problems, achieve high practical value, and improve scientific management efficiency , the effect of avoiding the spread of the virus

Inactive Publication Date: 2013-05-01
SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no effective treatment for viral diseases. Preventing the infection and prevalence of viruses is the main measure that can be taken. Early and rapid diagnosis is an effective way to reduce losses. Therefore, a simple, rapid, specific and sensitive diagnosis The kit and its detection method are of great significance to the prevention of diseases
[0003] The current popular polymerase chain reaction (polymerase chain reaction, PCR technology for short) is a technology for amplifying specific DNA fragments in vitro. It can detect multiple pathogens at the same time and improve work efficiency, but there is no report on double RT-PCR for simultaneous detection of Scylla serrata reovirus disease and Scylla serrata bicistrovirus

Method used

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  • Primer group, kit and detection method for detecting reovirus and bicistronic virus of scylla serrata
  • Primer group, kit and detection method for detecting reovirus and bicistronic virus of scylla serrata
  • Primer group, kit and detection method for detecting reovirus and bicistronic virus of scylla serrata

Examples

Experimental program
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Effect test

Embodiment 1

[0051]The primer set used for detecting Scylla serrata reovirus and dicistronic virus provided in this embodiment includes a primer set of Scylla serrata reovirus and a primer set of bicistronic virus, wherein Scylla serrata reovirus primer set The crab reovirus primer set includes the reovirus forward primer ReoF and the reverse primer ReoR, the Scylla serrata dicistronic virus primer set includes the dicistrovirus forward primer DicF and the reverse primer DicR, each The primers are as follows:

[0052] ReoF: 5- ACTCATAGAGCAGTCATGGG-3

[0053] ReoR: 5-ATATCGTCAGAATGTCGTTC-3

[0054] DicF: 5-GGATACTATGGATGATGTTTC-3

[0055] DicR: 5-ACAAAATACCAGATAAAGCAA-3.

Embodiment 2

[0057] The Scylla serrata reovirus and dicistrovirus detection kit containing the above primer set provided in this example is composed of the following parts (10 samples):

[0058] (1). RNA extraction solution (solution A), 2 tubes, 5mL tube, filled with Trizol solution, used as lysis solution;

[0059] (2). Liquid B, 1 tube, filled with chloroform (you can also bring your own), 2 mL in total, used for RNA extraction;

[0060] (3). Solution C, 1 tube, filled with isopropanol (you can also bring your own), 5mL in total, used to precipitate RNA;

[0061] (4). Solution D, 1 tube, containing 70% ethanol by volume (you can also prepare your own), 10mL in total, for washing RNA;

[0062] (5). Solution E, 1 tube, 1mL / tube, filled with DEPC (diethylpyrocarbonate) water, used to dissolve RNA;

[0063] (6). RT reaction solution (solution F), 1 tube, 0.1mL / tube, containing RT reaction solution, RT reaction solution includes buffer Buffer, deoxyribonucleoside triphosphate dNTP, D...

Embodiment 3

[0091] The double RT-PCR detection method for Scylla serrata reovirus and dicistronic virus provided in this example uses the primer set in Example 1 and the kit in Example 2, specifically comprising the following steps:

[0092] (1). Take one diseased Scylla serrata that may be infected with reovirus, bicistronic virus, or both viruses at the same time, and one that has not been infected by these two viruses. Add 1mL of solution A to 0.1g of gill tissue, homogenize in an ice bath in a homogenizer, and let it stand at room temperature for 3-5 minutes. The amount of gill tissue of each crab can be within the range of 0.05-0.1g. 0.5-1mL is acceptable;

[0093] (2). Add 200 μL of B solution to the above homogenate, mix it upside down, and let it stand for 5 minutes. The amount of B solution can be within the range of 100-200 μL;

[0094] (3). Centrifuge at 12000r / min for 5-15min at 4°C, where the centrifugation temperature is 3-5°C, the speed is 10000-12000r / min, the time is ...

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Abstract

The invention discloses a primer group for detecting the reovirus and bicistronic virus of scylla serrata, comprising a reovirus forward primer ReoF, a reovirus reverse primer ReoR, a bicistronic virus forward primer DicF and a bicistronic virus reverse primer DicR, wherein each primer is specifically as follows: ReoF: 5-ACTCATAGAGCAGTCATGGG-3; ReoR: 5-ATATCGTCAGAATGTCGTTC-3; DicF: 5-GGATACTATGGATGATGTTTC-3; and DicR: 5-ACAAAATACCAGATAAAGCAA-3. The invention further discloses a kit containing the primer group and a detection method. The primer group, the kit and the detection method are shortin detection time, strong in specificity, high in detection sensitivity, and capable of simultaneously detecting the reovirus and bicistronic virus of scylla serrata.

Description

technical field [0001] The invention relates to a primer set, a kit and a double RT-PCR detection method for detecting Scylla serrata reovirus and bicistrovirus. Background technique [0002] Scylla serrata has delicious meat and rich nutrition. It is an important aquatic economic animal in my country. It is widely cultivated in coastal areas of Guangdong, Fujian, Hainan, Zhejiang and other provinces. However, with the increase of breeding scale and density, the disease becomes more and more serious. Since the spring of 2004, the cultured mud crabs in various regions of Guangdong Province have continued to die, which has caused huge economic losses to the farmers and severely affected the development of the industry. Investigations have found that Scylla serrata reovirus and Scylla serrata dicistrovirus are the main causes of mass mortalities, and both viruses are often co-detected in diseased Scylla serrata arrive. So far, there is no effective treatment for viral diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 郭志勋冯娟苏友禄张迪吴开畅
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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