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Carp edema virus (CEV) RAA constant-temperature fluorescence detection method and kit

A detection kit and carp technology, applied in the field of molecular biology, can solve the problems of lack of high-sensitivity detection reagents for carp edema disease, can not meet the needs of disease quarantine and prevention work, and achieve the effect of simple operation and simple identification

Pending Publication Date: 2020-03-20
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, there is a lack of highly sensitive detection reagents for carp edema disease in the world, which cannot meet the quarantine and prevention work for diseases caused by the virus.

Method used

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  • Carp edema virus (CEV) RAA constant-temperature fluorescence detection method and kit
  • Carp edema virus (CEV) RAA constant-temperature fluorescence detection method and kit
  • Carp edema virus (CEV) RAA constant-temperature fluorescence detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The invention searches carp edema disease TK gene sequence 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.

[0029] Table 1 Primer and probe sequences:

[0030]

[0031] 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, the CT value is larger, and the plateau is n...

Embodiment 2

[0035] Embodiment 2: said kit carp edema disease

[0036] The nucleic acid detection kit of the present invention also includes primer mixture, specific fluorescent probe, A Buffer, BBuffer, RAA dry powder reagent, carp edema disease standard substance and ddH 2 O.

[0037] In the kit of the present invention, the A Buffer is 20% PEG; the B Buffer is 280mM MgAc.

[0038] The kit of the present invention, wherein, the composition of the RAA dry powder reagent is as follows: 1mmol / L dNTP, 90ng / μL SSB protein, 120ng / μL recA recombinase protein (SC-recA / BS-recA) or 30ng / L μL Rad51, 30ng / μL Bsu DNA polymerase, 100mmol / L Tricine, 20% PEG, 5mmol / L dithiothreitol, 100ng / μL creatine kinase, Exo exonuclease.

[0039] In the primer mixture of the present invention, the base sequence of the forward primer is shown in SEQ ID NO.1, the base sequence of the reverse primer is shown in SEQ ID NO.2, the forward primer and reverse primer The molar ratio of the primers is 1:1 for SEQ ID NO.1:S...

Embodiment 3

[0044] Embodiment 3: kit carp edema disease according to the present invention

[0045] 1. Extraction of positive sample nucleic acid

[0046] 1.1. Nucleic acid extraction: DNA extraction was performed using a marine animal tissue DNA extraction kit.

[0047] 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.

[0048] table 3:

[0049] 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

[0050] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0051] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and perform RAA amplification according to the following procedure: 37°...

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Abstract

The invention discloses a carp edema virus (CEV) RAA constant-temperature fluorescence detection method and a detection kit. The detection kit comprises a forward primer SEQ ID NO. 1, a reverse primerSEQ ID NO. 2, a specific fluorescent probe SEQ ID NO. 3, reaction liquid, recombinant polymerase and a reference substance. The kit of the invention has strong specificity; the detection sensitivityis high and can reach 2.02 fg / mu L; the accuracy is high and reliable; and the detection kit is simple, convenient and quick to operate, is suitable for field detection, and has wide application scenes.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method of marine aquaculture industry, in particular to a RAA constant temperature fluorescence detection method and a kit for carp edema disease. Background technique [0002] Carp edema virus (CEV) is a virus that infects carp and koi carp and causes disease and death. Both adult and juvenile fish will be sick. The affected fish had symptoms such as lethargy, gill damage, sunken eyes, and skin damage, and died within 2 weeks after the onset. In recent years, with the rapid development of the koi breeding industry in China, the occurrence of koi diseases has become increasingly serious. Because carp edema disease and Koi herpers virus (KHV) infection cause similar symptoms, both can cause high mortality of farmed carp and koi, and it is difficult to distinguish them clinically. Carp edema disease, as the pathogen of emerging infectious diseases in my countr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2563/107C12Q2522/101
Inventor 郑晓聪钱冬程奇史秀杰刘荭张建勋肖文余国君贾鹏王津津于力何俊强刘莹温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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