RAA constant-temperature fluorescence detection method and kit for grass carp reovirus type 2 (GCRV-2)

A technology of grass carp hemorrhagic disease and type 2 virus, which is applied in the field of molecular biology, can solve the problems of high false positives, limited application, and few, and achieve the effect of simple operation and simple identification

Inactive Publication Date: 2019-12-20
HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, real-time fluorescent PCR is time-consuming and expensive, and it is not widely used in the routine detection of pathogens in aquaculture animals.
LAMP isothermal amplification technology also has high false positives and low accuracy, so its application in the detection of aquatic pathogens is still relatively limited

Method used

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  • RAA constant-temperature fluorescence detection method and kit for grass carp reovirus type 2 (GCRV-2)
  • RAA constant-temperature fluorescence detection method and kit for grass carp reovirus type 2 (GCRV-2)
  • RAA constant-temperature fluorescence detection method and kit for grass carp reovirus type 2 (GCRV-2)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The present invention searches the gene sequence of grass carp hemorrhagic disease type 2 virus strain in Genebank database, uses DNAMAN 6.0 software to compare multiple sequences, and finds out the conserved segment. 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.

[0033] Table 1 primers and probe sequences:

[0034]

[0035] 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 the curve is lower,...

example 3

[0048] Example 3: Kit grass carp hemorrhagic disease type 2 virus according to the present invention

[0049] 1. Extraction of positive sample nucleic acid

[0050] 1.1. Nucleic acid extraction: use traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

[0051] 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.

[0052] table 3:

[0053] 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 DEPC treated water 28.4μL total capacity 50μL

[0054] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0055] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and perform RAA amplification according to the followin...

Embodiment 4

[0058] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0059] The kit of the present invention was used to carry out a clinical blind sample experiment, and 48 prawns were detected; the experimental results showed that the fourth primer pair of the present invention can distinguish grass carp haemorrhagic disease type 2 virus, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 samples, reverse transcription PCR, 26 samples were positive results, 22 samples were negative results, 27 samples were positive results detected by RAA method, 21 samples were also negative results, and one positive result was different. This sample was amplified by reverse transcription PCR and sequenced, and the sequencing result showed that the sample was positive, indicating that the RAA detection reagent of the present invention has a higher accuracy rate.

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Abstract

The invention discloses an RAA constant-temperature fluorescence detection method and detection kit for a grass carp reovirus type 2 (GCRV-2). The detection kit comprises a forward primer SEQ ID NO.1,a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, a reaction solution, reverse transcriptase, recombinant polymerase and a reference substance. The kit disclosed by the invention is high in specificity; the detection sensitivity is high and can reach 1 fg / [mu]L; the accuracy is high and reliable; and the operation is simple and quick, suitability of field detection is achieved and the application prospect is wide.

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 grass carp hemorrhagic disease type 2 virus. Background technique [0002] Grass carp reovirus type 2 (GCRV) belongs to the genus Aquatic Reovirus, a new member of Reoviridae, and the first fish virus isolated from mainland China. The virus mainly causes haemorrhagic disease in freshwater cultured grass carp species in China, Vietnam, Myanmar and other Asian countries at the fingerling stage, and the mortality rate can be as high as 60%. The virus is widespread, harmful, high in mortality, and has a long onset season, which seriously threatens fishery production. Grass carp reovirus has a double-layer capsid, the average diameter of the virus particles is 60nm-70nm, icosahedral symmetry, no envelope, and the genome consists of 11 seg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 郑晓聪钱冬程奇于力刘荭黄震巨张建勋肖文余国君史秀杰贾鹏王津津何俊强刘莹温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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