Primer pairs, method and fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses

A technique for red-spotted grouper and nerve necrosis, which is applied in the field of biological identification of aquatic biological diseases, and achieves the effects of reducing background influence, mild reaction conditions and high detection sensitivity

Inactive Publication Date: 2011-11-09
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

[0006] Due to its own advantages, LAMP has been used in the detection of pathogenic microorganisms and the diagnosis of infectious diseases, such as: detection of severe acute respiratory syndrome coronavirus (SARS-CoV), detection of mycobacteria, adenoviral conjunctivitis detection, fungal detection, etc., there are no relevant reports on the use of LAMP for the detection of red grouper neuronecrosis virus

Method used

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  • Primer pairs, method and fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses
  • Primer pairs, method and fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses
  • Primer pairs, method and fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Primer set for detection of red grouper neuronecrosis virus and effectiveness detection

[0065] In this example, according to the published genome sequence of red-spotted grouper neuronecrosis virus, the specific sequence of red-spotted grouper neuronecrosis virus gene is selected, and then analyzed and designed to be used in LAMP detection technology, which can specifically identify red The specific primer group of grouper neuronecrosis virus, this primer group is made up of following four primers:

[0066] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0067] Internal primer BIP, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0068] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0069] The nucleotide sequence of the outer primer B3 is shown in SEQ ID NO:4.

[0070] The present embodiment selects 8 positive fishes and 2 healthy fishes detected by RT-PCR (such as figure 1 show...

Embodiment 2

[0072] The preparation of embodiment 2 rapid diagnostic kits

[0073] The rapid diagnostic kit of this embodiment can be used for the rapid diagnosis of whether red-spotted grouper neuronecrosis virus is contained in the sample by the loop-mediated isothermal amplification technique. Composition of reaction solution, Bst DNA polymerase, positive control substance, chromogenic solution and foam plate, wherein:

[0074] (1) Primer set for detection of red grouper neuronecrosis virus: DNA synthesizer, 1 tube, inner primer F3, outer primer B3, inner primer FIP and inner primer BIP, inner primer FIP, its nucleotide sequence As shown in SEQ ID NO: 1; inner primer BIP, its nucleotide sequence is shown in SEQ ID NO: 2; outer primer F3, its nucleotide sequence is shown in SEQ ID NO: 3; outer primer B3, its The nucleotide sequence is shown as SEQ ID NO: 4, and the concentrations of the above four primers are 10uM respectively;

[0075] (2) Configure LAMP reaction solution: containing ...

Embodiment 3

[0089] The preparation of embodiment 3 rapid diagnostic kits

[0090]The rapid diagnostic kit of this embodiment can be used for the rapid diagnosis of whether red-spotted grouper neuronecrosis virus is contained in the sample by the loop-mediated isothermal amplification technique. Reaction solution, Bst DNA polymerase, positive control substance, chromogenic solution, Trizol, chloroform, isopropanol, 70% ethanol, RNase-free water, RT reaction solution, reverse transcriptase and foam plate, in which:

[0091] (1) Primer set for detection of red grouper neuronecrosis virus: DNA synthesizer, 1 tube, inner primer F3, outer primer B3, inner primer FIP and inner primer BIP, inner primer FIP, its nucleotide sequence As shown in SEQ ID NO: 1; inner primer BIP, its nucleotide sequence is shown in SEQ ID NO: 2; outer primer F3, its nucleotide sequence is shown in SEQ ID NO: 3; outer primer B3, its The nucleotide sequence is shown as SEQ ID NO: 4, and the concentrations of the above f...

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Abstract

The invention discloses primer pairs, a detection method and a fast diagnostic kit for detecting red-spotted grouper nervous necrosis viruses. The primer pairs comprise the following four primers: an inner primer FIP, an inner primer BIP, an external primer F3 and an external primer B3. Both the primer pairs and the kit of the invention can detect the red-spotted grouper nervous necrosis viruses with high efficiency and high specificity, as well as are based on loop-mediated isothermal amplification technology, apply six segments, four primers and one constant temperature, complete an amplification reaction within 1 hour, and are low in detection cost, short in detection time, high in yield and specificity, obvious in negative and positive result color developing difference, high in verification rate, obvious and reliable in verification. The kit is provided with a set of optimized reverse transcription-loop-mediated isothermal amplification reaction system can be used for detecting red-spotted grouper nervous necrosis viruses at different stages in a fish culture process, avoids viral spread and prevalence, improves scientific management efficiency and has high practical value.

Description

technical field [0001] The invention relates to the biological identification of aquatic organism diseases, in particular to the detection of fish viral neuronecrosis, in particular to a primer set, a detection method and a rapid diagnostic kit for detection of red grouper neuronecrosis virus. Background technique [0002] Fish viral neuronecrosis, also known as viral encephalopathy and retinopathy, is an epidemic infectious disease of fish worldwide. The pathogen is fish neuronecrosis virus, which belongs to Nodamuraviridae type II Nodamuravirus genus. Fish neuronecrosis virus is divided into four genotypes: red fin puffer neuronecrosis virus (tiger puffer NNV, TPNNV), yellow banded trevally neuronecrosis virus (striped jack NNV, SJNNV), striped star flounder neuronecrosis virus (barfin flooder NNV, BFNNV) and red-spotted grouper neuronecrosis virus (red-spotted grouper NNV, RGNNV). [0003] The pathogen of fish viral neuronecrosis found in China is mainly red grouper neu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/78C12R1/93
Inventor 郭志勋许海东区又君冯娟苏友禄
Owner SOUTH CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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