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RAA constant temperature fluorescence detection method and reagent for infectious myonecrosis virus (IMNV)

A technique for muscle necrosis, detection kit

Inactive Publication Date: 2018-06-01
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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  • RAA constant temperature fluorescence detection method and reagent for infectious myonecrosis virus (IMNV)
  • RAA constant temperature fluorescence detection method and reagent for infectious myonecrosis virus (IMNV)
  • RAA constant temperature fluorescence detection method and reagent for infectious myonecrosis virus (IMNV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]In the present invention, the gene sequence of the shrimp infectious myonecrosis virus strain is searched in the Genebank database, and DNAMAN 6.0 software is used to compare multiple sequences to find 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...

example 3

[0047] Example 3: Kit prawn infectious myonecrosis virus according to the present invention

[0048] 1. Extraction of positive sample nucleic acid

[0049] 1.1. Nucleic acid extraction: use traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

[0050] 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.

[0051] table 3:

[0052] 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

[0053] A Buffer is 20% PEG; B Buffer is 280mM MgAc

[0054] 3. Place the RAA reaction tube with the reaction system in the ABI7500 amplification instrument, and carry out RAA amplification ac...

Embodiment 4

[0057] Embodiment 4: Evaluation of the RAA detection kit of the present invention in clinical practice

[0058] The kit of the present invention is used to carry out a clinical blind sample experiment, and 50 prawns are detected; the experimental results show that the fourth primer pair of the present invention can distinguish Hepatocystis prawns, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 samples, reverse transcription PCR, 38 samples were positive results, 12 samples were negative results, 39 samples were positive results by RAA method, 11 samples were also negative results, and one positive result was different. This sample was amplified by reverse transcription PCR and sequenced, and the sequencing result showed that the sample was positive, indicating that the RAA detection reagent of the present invention has a higher accuracy rate.

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Abstract

The invention discloses an RAA constant temperature fluorescence detection method and a detection kit for infectious myonecrosis virus (IMNV). The detection kit includes 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, reverse transcriptase, a recombinant polymerase, and a reference substance. The kit has high specificity and high detection sensitivity reaching 0.10 fg / [mu]L, is high in accuracy and reliability, has simple and convenient operations, is suitable for on-site detection, and has extensive application scenes.

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 infectious myonecrosis virus of prawns. Background technique [0002] Shrimp infectious myonecrosis virus (IMNV) is a double-stranded RNA virus with a diameter of 40nm, an icosahedron, and no envelope; a length of 7560bp, including two open reading frames (ORF1, ORF2), in which ORF1 encodes An RNA-binding protein and a capsid protein, ORF2 encodes an RNA-dependent RNA polymerase of 736 amino acids. It first broke out in Brazil in 2002. IMNV mainly infects Litopenaeus vannamei, and the virus can infect adult shrimp, larvae and seedlings, and mainly infects 60-80-day-old juvenile shrimp. The virus destroys the musculature of the whole body, but the course of the disease is slow and the mortality rate is not high. In severe cases,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/701C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 程奇钱冬黄震巨张建勋肖文余国君陶智勇徐锦余霍胜楠沈弘郑晓叶郑天伦沈伟良吕文浩
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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