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Kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of kit

A detection kit and hepatopancreas technology are applied in the field of rapid detection kits for the pathogens of acute hepatopancreatic necrosis of shrimp, which can solve the problems of false positives, expensive instruments and equipment, and lack of equipment, and achieve reliable results, good specificity and high sensitivity. Effect

Inactive Publication Date: 2016-02-10
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This PCR method can lead to false-positive detection results because the detection target site is against other genes of the 70kb plasmid pVA1, but not the PirA and PirB virulence proteins; because some V. parahaemolyticus strains carry the pVA1 plasmid but naturally lack PirA and PirB Virulence protein
In addition, the PCR detection method requires expensive instruments and equipment, high detection costs, and high technical requirements for detection personnel, making it unsuitable for the popularization and use of prawns.

Method used

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  • Kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of kit
  • Kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of kit
  • Kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 : The kit for detecting the pathogen of prawn AHPND is established:

[0042] The kit for detecting the AHPND pathogen of prawns is established based on LAMP technology, including LAMP primer set, LAMP reaction solution, BstDNA polymerase, positive control and negative control.

[0043] (1), LAMP primer design: with the virulence protein pirB gene (GenBank accession number KP324996) of the shrimp AHPND pathogen as the target site, use the online design software PrimerExplorerversion4 (http: / / primerexplorer.jp / e) to carry out the LAMP primer design. The primer sequences are listed in Table 1.

[0044] Table 1 Primer sequence list

[0045]

[0046] (2) LAMP reaction liquid: containing 10mMdNTP, 10×ThermoPol reaction buffer, and 150mM MgSO4 aqueous solution, the volume ratio of the three is 8:5:2.

[0047] (3), positive control is the plasmid DNA that contains prawn AHPND pathogenic toxin gene pirB fragment, and its preparation method is: take the artifici...

Embodiment 2

[0049] Example 2 : Use a turbidimeter to establish a method for the detection of pathogens in shrimp AHPND:

[0050] Utilize the method for the kit detection prawn AHPND pathogen of embodiment 1, comprise the steps:

[0051] (1), Purification and isolation of Vibrio strains: from the prawn tissue to be tested, use a sterile inoculation loop to take a sample and streak it on a TCBS plate for overnight culture, and purify and isolate the Vibrio strains;

[0052] (2) Preparation of Vibrio DNA: Add 50 μL sterile TE solution (10mM Tris-HCl, 1mM EDTA, pH 8.0) into the centrifuge tube, pick a single colony of Vibrio into the above TE solution, place the centrifuge tube on the vortex Fully shake on the mixer for 10 seconds, cook in boiling water for 10 minutes, cool to room temperature, shake again for 10 seconds, then centrifuge at 12000r / min for 2 minutes, the supernatant is the Vibrio DNA and store it at -20°C for later use;

[0053] (3) Constant temperature gene amplification r...

Embodiment 3

[0055] Example 3 : Use ESE-Quanttubescanner to establish a detection method for shrimp AHPND pathogens:

[0056] Use the above kit to detect the AHPND pathogen of prawns to be tested according to the following method, except that the chromogenic agent (SYTO-9) that is not in Example 1 is added in the kit, the others are the same as Example 1.

[0057] (1), Purification and isolation of Vibrio strains: from the prawn tissue to be tested, use a sterile inoculation loop to take a sample and streak it on a TCBS plate for overnight culture, and purify and isolate the Vibrio strains;

[0058] (2) Preparation of Vibrio DNA: Add 50 μL sterile TE solution (10mM Tris-HCl, 1mM EDTA, pH 8.0) into the centrifuge tube, pick a single colony of Vibrio into the above TE solution, place the centrifuge tube on the vortex Fully shake on the mixer for 10 seconds, cook in boiling water for 10 minutes, cool to room temperature, shake again for 10 seconds, then centrifuge at 12000r / min for 2 minute...

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Abstract

The invention relates to a kit for rapidly detecting pathogeny of acute hepatopancreatic necrosis disease of prawns and application of the kit, belonging to technical field of biotechnology. The kit comprises an LAMP primer group, DNA polymerase, LAMP reaction liquid, a positive control and a negative control. The kit further comprises a color developing agent. The LAMP primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. The application of the kit in the pathogeny detection of the acute hepatopancreatic necrosis disease of the prawns comprises the steps of purifying and separating vibrio strains, preparing vibrio DNA, carrying out constant temperature gene amplification reaction, and carrying out result judgment by virtue of a turbidity meter or an ESE-Quant Tube Scanner. The kit has the advantages of good specificity, high sensitivity, simple operation, reliable result and the like, and is applicable to the rapid detection of prawn farming sites.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a molecular detection method for seawater cultured animal pathogens, in particular to a rapid detection reagent for acute hepatopancreatic necrosis (AHPND) of prawns developed by using a loop-mediated isothermal amplification technique (LAMP) boxes and their applications. Background technique [0002] Shrimp aquaculture is the pillar industry of marine aquaculture in my country. Shrimp production and trade have made important contributions to the development of my country's rural economy, increased farmers' income, and created employment opportunities. At the same time, they have also made contributions to providing more high-quality protein foods for global consumers. Actively contribute. However, the healthy and sustainable development of the shrimp farming industry has been facing challenges from many factors such as frequent disease occurrence, germplasm degradation and deteriorati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2545/113
Inventor 赵哲胡超群任春华江晓
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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