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RAA constant-temperature fluorescence detection method and kit for infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish

A technology of spleen and kidney necrosis virus and a detection kit, which is applied in the field of molecular biology, can solve the problems of high false positives, application limitations, and long detection time, and achieve the effects of simple operation, simple identification, and high sensitivity

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

Among them, the PCR detection method can be detected relatively quickly and accurately, but because conventional PCR detection requires expensive PCR instruments, gel electrophoresis and imaging systems, and the detection time is long, it also greatly limits the popularization and application of PCR detection methods in practice
qPCR detection method has strong specificity and high sensitivity, but qPCR is time-consuming and costly, and currently it is not widely used in routine pathogen detection 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 infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish
  • RAA constant-temperature fluorescence detection method and kit for infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish
  • RAA constant-temperature fluorescence detection method and kit for infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish

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Experimental program
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Effect test

Embodiment 1

[0029] The present invention searches the gene sequence of the mandarin fish infectious spleen and kidney necrosis 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 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 the ...

Embodiment 2

[0035] Embodiment 2: The kit mandarin fish infectious spleen and kidney necrosis virus

[0036] The nucleic acid detection kit of the present invention also includes primer mixture, specific fluorescent probe, A Buffer, BBuffer, RAA dry powder reagent, mandarin fish infectious spleen and kidney necrosis 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 rever...

Embodiment 3

[0044] Embodiment 3: kit mandarin fish infectious spleen and kidney necrosis virus according to the present invention

[0045] 1. Extraction of 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 t...

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Abstract

The invention discloses an RAA constant-temperature fluorescence detection method and kit for infectious spleen and kidney necrosis virus (ISKNV) in mandarin fishes. 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, recombinant polymerase and a reference substance. The kit disclosed by the invention is strong in specificity, high in detection sensitivity which can reach 300 fg / [mu]L, high in accuracy, reliable, simple, convenient and rapid to operate, suitable for field detection and wide in application prospect.

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 mandarin fish infectious spleen-kidney necrosis virus. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) belongs to the cytomegalovirus genus of the family Iridoviridae. It is an enveloped icosahedral virus parasitic in the cytoplasm, with a diameter of 150 nm and a viral genome of double-stranded DNA. The body of the diseased fish turns black, and sometimes has cramping-like tremors; the head is congested, bleeding around the mouth and eyes; the spleen, kidney and liver have symptoms such as swelling, erosion and congestion, which are purple-black; ascites is common, and the intestines are sometimes full Yellow viscous matter, gallbladder enlargement; gill anemia, whitish, accompanied by parasite or bac...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2521/507C12Q2563/107
Inventor 郑晓聪钱冬程奇温智清刘荭黄震巨张建勋肖文余国君史秀杰贾鹏王津津于力何俊强刘莹
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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