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RAA (Recombinase-aid Amplification) constant-temperature fluorescence detection method and reagent of cyprinid herpesvirus II (CyHV-2)

A carp herpes virus and detection kit technology, applied in the field of molecular biology, can solve the problems of few, high cost, and time-consuming real-time fluorescent PCR, and achieve the effects of simple identification and simple operation.

Pending 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.

Method used

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  • RAA (Recombinase-aid Amplification) constant-temperature fluorescence detection method and reagent of cyprinid herpesvirus II (CyHV-2)
  • RAA (Recombinase-aid Amplification) constant-temperature fluorescence detection method and reagent of cyprinid herpesvirus II (CyHV-2)
  • RAA (Recombinase-aid Amplification) constant-temperature fluorescence detection method and reagent of cyprinid herpesvirus II (CyHV-2)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The invention searches the Genebank database for the carp herpesvirus type II MCP gene sequence, 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.

[0030] Table 1 Primer 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 the curve is lower, the CT value is larger, and ...

Embodiment 2

[0035] Embodiment 2: Said kit carp herpesvirus type Ⅱ

[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 herpesvirus type II standard 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 of the present invention carp herpesvirus type Ⅱ

[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°C, 40...

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Abstract

The invention discloses a RAA (Recombinase-aid Amplification) constant-temperature fluorescence detection method and detection reagent kit of cyprinid herpesvirus II (CyHV-2). The detection reagent kit comprises a forward primer SEQID NO.1, a reverse primer SEQID NO.2, a specific fluorescence probe SEQID NO.3, reaction liquid, recombinant polymerase and a reference substance. The reagent kit disclosed by the invention is high in specificity, high in detection sensitivity which can reach 100 fg / [mu]L, high in accuracy, reliable, simple, convenient and quick to operate and suitable for on-site detection, and has broad application scene.

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 reagent kit of carp herpes virus type II. Background technique [0002] Cyprinid herpesvirus Ⅱ (Cyprinid herpesvirus Ⅱ, CyHV-2) is the second herpesvirus isolated from carps. The genus also includes carp herpesvirus type I (CyHV-1), carp herpesvirus type III (CyHV-3) and eel herpesvirus type I (AngHV-1). CyHV-2 is highly pathogenic to goldfish (Carassius arratus) and crucian carp (Carassius aumtus), with a mortality rate of almost 100%. It mainly occurs in spring and autumn, and the incidence rate is greatly affected by water temperature. It is easy to develop the disease at 15-25°C, and the incidence rate decreases when the water temperature is higher than 25°C. The disease occurs in Japan, the United States, Australia, the United Kingdom an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/705C12Q1/6844C12Q2563/107C12Q2521/507
Inventor 贾鹏钱冬程奇何俊强刘荭黄震巨张建勋肖文余国君史秀杰郑晓聪王津津于力刘莹温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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