Recombinase-aid amplification (RAA) constant-temperature fluorescence detection method and reagent for Koi herpesvirus (KHV)

A koi herpes virus and detection kit technology, applied in the field of molecular biology, can solve the problems of high false positives, limited application, and few, and achieve the effects of high sensitivity, simple identification, and simple operation

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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  • Recombinase-aid amplification (RAA) constant-temperature fluorescence detection method and reagent for Koi herpesvirus (KHV)
  • Recombinase-aid amplification (RAA) constant-temperature fluorescence detection method and reagent for Koi herpesvirus (KHV)
  • Recombinase-aid amplification (RAA) constant-temperature fluorescence detection method and reagent for Koi herpesvirus (KHV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The present invention searches the TK gene sequence of the koi herpes virus in the 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.

[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,...

Embodiment 2

[0035] Embodiment 2: The kit koi herpes virus

[0036] The nucleic acid detection kit of the present invention also includes primer mixture, specific fluorescent probe, ABuffer, BBuffer, RAA dry powder reagent, koi herpes virus 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:SEQ ID NO.2.

[0...

Embodiment 3

[0044] Embodiment 3: kit koi herpes virus of 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°C, 40s; 37°C...

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Abstract

The invention discloses recombinase-aid amplification (RAA) constant-temperature fluorescence detection method and reagent for Koi herpesvirus (KHV). The RAA constant-temperature fluorescence detection reagent for the KHV 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, recombinant polymerase and a reference substance. The RAA constant-temperature fluorescence detection reagent for the KHV disclosed by the invention is high in specificity, high in detection sensitivity (up to 100fg / microliter), high in accuracy and reliable; and moreover, the reagent is easy and convenient to operate as well as suitable for field testing. Thus, wide range of application scenarios is ensured.

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 of koi herpes virus. Background technique [0002] Koi herpers virus (KHV), also known as carp herpes virus type III (CyHV-3), is the main pathogen of koi herpes virus disease. In 1998, it was isolated from sick carp and koi in the United States and Israel for the first time and was confirmed. It has been spread and popular in more than a dozen countries and regions such as Sweden, Britain, Germany, Indonesia and Taiwan. KHV mainly infects carps and koi carps, and can infect fry, juveniles, and adult fish. They die 24-48 hours after the onset of symptoms, and the mortality rate is as high as 80% to 100%. Because KHV is highly contagious, has a high mortality rate, and has no effective treatment method, it greatly affects the enthusiasm of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6844C12Q2563/107C12Q2521/507C12Q2522/101
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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