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RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for SIV (shrimp iridovirus)

A technology of iridescent virus and detection kit, which is applied in the field of molecular biology, can solve problems such as low sensitivity, complicated operation, and unsuitable technical requirements for detection personnel, and achieve the effect of simple identification and simple operation

Active Publication Date: 2018-05-04
HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Electron microscope observation is time-consuming, expensive, and requires special experimental equipment and a narrow field of view; histopathological observation is not suitable for early detection, usually when the virus seriously infects the whole body of the body and produces obvious and irreversible tissue lesions. Can be confirmed; immunohistochemistry and in situ hybridization are relatively cumbersome, the sensitivity is not high, and it is not tender for quantitative research on viruses
PCR-electrophoresis is also commonly used now, but the operation is complicated
In addition, the PCR detection method requires expensive instruments and equipment, high detection costs, and high technical requirements for detection personnel, making it unsuitable for the popularization and use of prawns.

Method used

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  • RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for SIV (shrimp iridovirus)
  • RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for SIV (shrimp iridovirus)
  • RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and reagent for SIV (shrimp iridovirus)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The shrimp iris virus of the present invention searches the gene sequence of the shrimp iris virus strain in the Genebank database, and uses DNAMAN 6.0 software to compare multiple sequences to find out the conservative segments. Four sets of primers and probes were designed in the conserved region, and BLAST alignment was performed in the NCBI database. The sequences of the primers and probes are shown in Table 1. The positive sample amplification curve is as figure 1 Shown.

[0030] Table 1 Primer and probe sequence:

[0031]

[0032] by figure 1 The results show that the amplification curve of the fourth set of primers and probes is the most typical, with obvious exponential phase and plateau phase, high fluorescence intensity (ordinate value), and small CT value (the intersection of the curve and the threshold line) The corresponding abscissa) result analysis is shown in Table 2. Other primer probe curves have a low rise height, a large CT value, and the plateau period...

example 3

[0053] Example 3: The kit shrimp iridescent virus of the present invention

[0054] 1. Extraction of nucleic acid from positive samples

[0055] 1.1. Nucleic acid extraction: Use marine animal tissue DNA extraction kit for DNA extraction.

[0056] 2. The configuration of the RAA reaction system: each test sample corresponds to a RAA reaction dry powder tube. The reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.

[0057] table 3:

[0058] RAA reaction system components

Volume (μL)

A Buffer

12.5μL

B Buffer

2.5μL

Primer mix

4μL

Specific fluorescent probe

0.6μL

DNA template

2μL

ddH 2 O

28.4μL

total capacity

50μL

[0059] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0060] 3. Place the RAA reaction tube equipped with the reaction system in the ABI7500 thermal cycler, and perform RAA amplification according to the following procedures: 39°C, 40s; 39°C, 20min, a total of 40 cycles. The fluorescence of the FAM channel...

Embodiment 4

[0063] Example 4: Evaluation of the RAA detection kit of the present invention in clinical practical applications

[0064] The test kit of the present invention is used to conduct a clinical blind sample experiment to detect 500 prawns; the experimental results show that the fourth primer pair of the present invention can distinguish shrimp iridescent virus, and the positive coincidence rate with nested PCR is very high. Among 500 copies of nested PCR, 318 copies were positive results, 182 copies were negative results, 318 copies were positive results by RAA method, and 181 copies were negative results. One of them may not be tested due to contamination Out, there are 182 negative results, and all of them can be one-to-one correspondence.

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Abstract

The invention dislcoses an RAA (recombinase-aid amplification) constant-temperature fluorescence detection method and a detection kit for SIV (shrimp iridovirus). The detection kit comprises a forwardprimer SEQ ID NO. 1, a reverse primer SEQ ID NO. 2, a specific fluorescence probe SEQ ID NO. 3, reaction liquid, recombinant polymerase and a reference substance. The kit has the advantages that thespecificity is strong; the detection sensitivity is high, and can reach 2fg / mu L; high accuracy and reliability are achieved; the operation is simple and rapid, and the kit is suitable for field detection and has wide application scenarios.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology, relates to a detection method in the marine aquaculture industry, and specifically relates to an RAA constant temperature fluorescent detection method and a kit for shrimp iridescent virus. Background technique [0002] Iridescent virus family viruses form icosahedral virus particles with a diameter of 120-300 nm and a linear double-stranded DNA genome with a genome size of about 102-212 kbp, which replicates in the cytoplasm. The iridescent virus is rich in the major capsid protein (MCP), and the protein region is highly conserved, and is usually used to identify different species. The Iridovirus family has recently been divided into 5 genera: Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus and Megalocytivirus. The iridescent virus has a very wide host and can infect fish, amphibians and invertebrates. Among them, invertebrateiridescent viruses (IIVs) mainly infect insects and te...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2563/107C12Q2521/507C12Q2522/101
Inventor 程奇钱冬黄震巨张建勋肖文余国君陶智勇徐锦余霍胜楠沈弘郑晓叶郑天伦沈伟良吕文浩
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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