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Raa constant temperature fluorescence detection method and reagents of shrimp iridescent virus (siv)

A technology of iridescent virus and detection kit, which is applied in the field of molecular biology, can solve the problems of low sensitivity, complicated operation, and not suitable for early detection, etc., and achieve the effect of simple identification and simple operation

Active Publication Date: 2021-04-06
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 constant temperature fluorescence detection method and reagents of shrimp iridescent virus (siv)
  • Raa constant temperature fluorescence detection method and reagents of shrimp iridescent virus (siv)
  • Raa constant temperature fluorescence detection method and reagents of shrimp iridescent virus (siv)

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Experimental program
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Effect test

Embodiment 1

[0029] The shrimp iridescent virus of the present invention searches the gene sequence of the shrimp iridescent virus strain in the Genebank database, uses DNAMAN 6.0 software to compare multiple sequences, and find out the conserved segments. Four sets of primers and probes were designed in the conserved regions, and BLAST comparison 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 follows figure 1 shown.

[0030] Table 1 primers and probe sequences:

[0031]

[0032] Depend on figure 1 The results show that the amplification curves of the fourth group of primers and probes are the most typical, with obvious exponential phase and plateau phase, higher fluorescence intensity (ordinate value), and smaller CT value (the intersection of the curve and the threshold line Corresponding abscissa) result analysis is shown in Table 2. For other primers and probes, the rising height of ...

example 3

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

[0054] 1. Extraction of positive sample nucleic acid

[0055] 1.1. Nucleic acid extraction: DNA extraction was performed using a marine animal tissue DNA extraction kit.

[0056] 2. The configuration of the RAA reaction system: each test sample corresponds to a RAA reaction dry powder tube, and 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 wxya 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 with the reaction system in the ABI7500 amplification instrument, and carry out RAA amplification according to the following procedure: 39°C, 40s; 39°C...

Embodiment 4

[0063] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0064] The kit of the present invention is used to carry out a clinical blind sample experiment, and 500 copies of prawns are detected; 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. Of the 500, nested PCR, 318 were positive and 182 were negative, 318 were positive by the RAA method, 181 were also negative, one was not tested probably due to contamination It was found that 182 were also negative results, and all of them could be matched one by one.

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Abstract

The invention discloses a shrimp iridescent virus (SIV) RAA constant temperature fluorescence detection method and a detection kit. The detection kit includes a forward primer of SEQ ID NO.1, a reverse primer of SEQ ID NO.2, a specific fluorescent probe of SEQ ID NO.3, a reaction solution, a recombinant polymerase and a control substance. The kit of the invention has strong specificity; high detection sensitivity, which can reach 2fg / μL; high accuracy and reliability; simple and fast operation, suitable for on-site detection, and has wide application scenarios.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a detection method for marine aquaculture industry, in particular to a RAA constant-temperature fluorescence detection method and a kit for shrimp iridescent virus. Background technique [0002] Iridoviridae viruses form icosahedral virus particles with a diameter of 120-300nm, have a linear double-stranded DNA genome with a genome size of about 102-212kbp, and replicate in the cytoplasm. Iridoviruses contain an abundant major capsid protein (MCP), and the protein region is highly conserved, which is often used to identify different species. The family Iridoviridae has recently been divided into five genera: Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus and Megalocytivirus. Iridoviruses have a wide range of hosts and can infect fish, amphibians and invertebrates. Among them, invertebrate iridescent viruses (IIVs) mainly infect insects and terrestrial amphibian...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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泓郑晓叶郑天伦沈伟良吕文浩
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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