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RAA constant temperature fluorescence detection method and reagents for prawn covert mortality nodavirus (CMNV)

A technology of Nodamura virus and detection kit, applied in the field of molecular biology, can solve the problems of high false positive, limited application and low accuracy, and achieve the effect of simple operation and simple identification.

Inactive Publication Date: 2018-09-28
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 routine pathogen detection in aquaculture animals.
LAMP isothermal amplification technology also has high false positives and low accuracy, and 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 reagents for prawn covert mortality nodavirus (CMNV)
  • RAA constant temperature fluorescence detection method and reagents for prawn covert mortality nodavirus (CMNV)
  • RAA constant temperature fluorescence detection method and reagents for prawn covert mortality nodavirus (CMNV)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The present invention searches the Genebank database for the gene sequence of the prawn stealing Nodamura virus strain, uses DNAMAN 6.0 software to compare multiple sequences, and finds out the conserved segments. 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.

[0031] Table 1 primers and probe sequences:

[0032]

[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 is ...

example 3

[0047] Example 3: Kit of the present invention kills wild field village virus from shrimp

[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 perform RAA amplification accor...

Embodiment 4

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

[0058] Adopt kit of the present invention to carry out the experiment of clinical blind sample, detect 50 prawns; Experimental result shows, the 4th primer pair of the present invention can distinguish the wild field village virus of stealing prawns, and the positive coincidence rate with reverse transcription PCR is very high. Among the 50 samples, reverse transcription PCR, 31 were positive results, 19 were negative results, 31 were positive by the RAA method, 18 were also negative results, and one positive result was different. The sample nucleic acid 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 a prawn covert mortality nodavirus (CMNV) RAA constant temperature fluorescence detection method and a prawn covert mortality nodavirus (CMNV) detection kit, wherein 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, reverse transcriptase, recombinant polymerase and a controlsubstance. According to the present invention, the kit has characteristics of strong specificity, high detection sensitivity, high accuracy, reliability, simple and rapid operation and wide application scene, and is suitable for on-site detection, wherein the detection sensitivity can reach 100 fg / [mu]L.

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 test kit for stealing dead wild field village virus of prawns. Background technique [0002] Covert mortality disease (CMD) of prawns is a viral disease caused by "covert mortality nodavirus (CMNV)". CMNV belongs to Nodaviridae α Nodavirus genus (Nadaviridae, Alphanodavirus), is a kind of single-stranded RNA virus, virion is spherical (icosahedral), without capsule, diameter 32 nanometers. CMD is a new disease that has occurred in my country's cultured prawns in recent years. In 2003 or earlier, the disease appeared in high-density cultured Litopenaeus vannamei in Hainan, Guangxi and other places. From 2008 to 2009, a large-scale outbreak occurred in cultured prawns in various provinces and cities in southern my country, and it began to sprea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/701C12Q2563/107C12Q2521/507C12Q2522/101
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泓郑晓叶郑天伦沈伟良吕文浩
Owner HANGZHOU ZHONGCE BIO SCI&TECH CO LTD
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