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 met

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

Examples

Experimental program
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Example Embodiment

[0028] Example 1:

[0029] The present invention searches the Genebank database for the mandarin fish infectious spleen and kidney necrosis virus gene sequence, uses DNAMAN 6.0 software to compare the multiple sequences, and finds out the conservative segment. Four sets of primers and probes were designed in the conserved regions, and BLAST alignment was performed in the NCBI database. The sequences of the primers and probes are shown in Table 1. Amplification curves of positive samples such as figure 1 shown.

[0030] Table 1 Primer and probe sequences:

[0031]

[0032] Depend on figure 1 The results show that the amplification curve of the fourth group of primers and probes is the most typical, with obvious exponential phase and plateau phase, high fluorescence intensity (ordinate value), and small CT value (the intersection of the curve and the threshold line). The corresponding abscissa) results are shown in Table 2. Other primer-probe curves have lower rising hei...

Example Embodiment

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

[0036] The nucleic acid detection kit of the present invention further includes primer mixture, specific fluorescent probe, A Buffer, BBuffer, RAA dry powder reagent, standard substance of mandarin fish infectious spleen and kidney necrosis virus and ddH 2 O.

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

[0038]In the kit of the present invention, the components of the RAA dry powder reagent are as follows: 1mmol / L dNTP, 90ng / μL SSB protein, 120ng / μL recA recombinase protein (SC-recA / BS-recA) or 30ng / μL 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 ...

Example Embodiment

[0044] Example 3: kit of the present invention mandarin fish infectious spleen and kidney necrosis virus

[0045] 1. Extraction of sample nucleic acid

[0046] 1.1. Nucleic acid extraction: DNA extraction kits were used for DNA extraction from marine animal tissues.

[0047] 2. 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 prepared reaction system in the ABI7500 amplifying instrument, and perform RAA amplification according to the follow...

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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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