Infectious hematopoietic necrosis virus (INHV)RAA (recombinase-aid amplification) thermostatic fluorescence detection method and reagent

A hematopoietic organ necrosis and detection kit technology, applied in the field of molecular biology, can solve the problems of high false positives, limited application, and few, etc., and achieve the effects of simple operation and simple identification.

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.
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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  • Infectious hematopoietic necrosis virus (INHV)RAA (recombinase-aid amplification) thermostatic fluorescence detection method and reagent
  • Infectious hematopoietic necrosis virus (INHV)RAA (recombinase-aid amplification) thermostatic fluorescence detection method and reagent
  • Infectious hematopoietic necrosis virus (INHV)RAA (recombinase-aid amplification) thermostatic fluorescence detection method and reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The present invention searches the gene sequence of the infectious hematopoietic organ necrosis virus strain 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.

[0032] Table 1 primers and probe sequences:

[0033]

[0034] 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 i...

example 3

[0048] Example 3: Kit infectious hematopoietic necrosis virus according to the present invention

[0049] 1. Extraction of positive sample nucleic acid

[0050] 1.1. Nucleic acid extraction: use traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

[0051] 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.

[0052] table 3:

[0053] 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

[0054] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0055] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and perform RAA amplification according to the following pr...

Embodiment 4

[0058] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0059] The kit of the present invention was used to carry out clinical blind experiment, and 60 prawns were detected; the experimental results showed that the fourth primer pair of the present invention can distinguish infectious hematopoietic organ necrosis virus, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 copies, 33 were positive results and 27 were negative results by reverse transcription PCR. The results detected by the RAA method were 33 positive and 27 were also negative results. The results were completely consistent with the actual samples. Consistent, indicating that the RAA detection reagent of the present invention has a higher accuracy rate.

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Abstract

The invention discloses an infectious hematopoietic necrosis virus (INHV) RAA (recombinase-aid amplification)thermostaticfluorescence detection method and a detection kit. The detection kit includes aforward primer SEQ ID NO. 1,a reverse primer SEQ ID NO. 2,a specific fluorescent probe SEQ ID NO. 3,reaction liquid, reverse transcriptase, recombinant polymerase, and reference substance. The detection kit is high in specificity; detection sensitivity is high and can reach 0.1fg / [mu]L; high accuracyand reliability are realized; operation is easy, convenient, and quick, and the detection kit is suitable for field testing and has a wide range of application scenarios.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for marine aquaculture industry, in particular to a RAA constant-temperature fluorescence detection method and a kit for infectious hematopoietic organ necrosis virus. Background technique [0002] Infectious haematopoietic necrosis virus (IHNV) belongs to Rhabdoviridae (Rhabdoviridae), Nora Rhabdovirus (Novirhabdovirus), and IHNV is the representative species of this genus. IHNV virus particles are bullet-shaped, enveloped, and unstable to heat, acid, and ether. The IHNV virion contains a linear, antisense, single-stranded RNA, and its genome structure contains six genes, N-P(M1)-M2-G-NV-L, from the 3' end to the 5' end, which encode viral nucleoproteins respectively , phosphoproteins, matrix proteins, glycoproteins, nonstructural proteins and polymerase proteins, the current genome sequencing shows that the full length of the IHNV genome is about 11k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2522/101C12Q2563/107C12Q2521/107
Inventor 刘荭钱冬程奇刘莹黄震巨张建勋肖文余国君史秀杰郑晓聪贾鹏王津津于力何俊强温智清
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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