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Method for detecting infectious spleen and kidney necrosis viruses by using hyper-branched rolling circle amplification

A technology of spleen-kidney necrosis virus and rolling circle amplification, which is applied in the detection field of infectious spleen-kidney necrosis virus, can solve the unreported problems in the detection of aquatic animal pathogens, and achieve short detection time, strong specificity and high sensitivity Effect

Inactive Publication Date: 2010-12-08
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, HRCA technology has been applied to the detection of animal and plant genes and their pathogens, but there is no report on the detection of aquatic animal pathogens. This invention applies HRCA technology to the detection of ISKNV for the first time

Method used

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  • Method for detecting infectious spleen and kidney necrosis viruses by using hyper-branched rolling circle amplification
  • Method for detecting infectious spleen and kidney necrosis viruses by using hyper-branched rolling circle amplification
  • Method for detecting infectious spleen and kidney necrosis viruses by using hyper-branched rolling circle amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Preparation of ISKNV genome DNA sample template and ISKNV-DPOL gene sequencing verification

[0038] 1.1 Materials and methods

[0039] 1.1.1 Materials

[0040] Sick and dying large yellow croaker and perch were collected from Xiangshan Harbor Aquatic Breeding Center in Zhejiang Province. Escherichia coli strain TG1 was preserved in our laboratory. The Extraction kit was purchased from QIAGEN, the plasma Mini kit was purchased from OMEGA, and the BIPospin Tissue Genomic DNA Extraction Kit was purchased from BIPoFlux.

[0041] 1.1.2 Method

[0042] 1.1.2.1 Preparation of DNA sample template of ISKNV genome:

[0043] Take 0.5 g of the spleen tissue of the fish to be tested, use BIPospin Tissue Genomic DNA Extraction Kit (BIPoFlux Company) to extract and purify tissue DNA, and prepare a DNA sample template for HRCA reaction.

[0044] 1.1.2.2 Sequencing verification of ISKNV-DPOL gene

[0045] According to the genome sequence of infectious spleen and kidney necrosis...

Embodiment 2

[0070] Sensitivity determination of ISKNV hyperbranched rolling circle amplification detection technology of the present invention

[0071] 1. The original concentration of the ISKNV genome DNA template that will be obtained (10 9 copies / μl) were serially diluted 10 times, respectively as sample templates.

[0072] 2. Use the optimized condition method obtained in Example 1 to detect the templates of each concentration so as to draw the sensitivity of the present invention (such as Figure 6 ).

[0073] 2.1 Connection of lock probe

[0074] The ligation reaction system is as follows: padlock probe 0.1 μM, 10×T4 DNA Ligase Buffer 1 μl, sample template 2 μl, add double-distilled water to a total reaction volume of 10 μl, denature at 94-100°C for 3-5 minutes, and ice-bath for 3-5 minutes , plus 1U / μl T4 DNA Ligase, reacted at 37°C for 30min.

[0075] 2.2 HRCA response

[0076] The final concentrations of the reaction system were: dNTPs 0.4mM, CF1 0.4μM, CF2 0.4μM, Tris-HCl (...

Embodiment 3

[0079] The present invention is based on the specificity determination of HRCA detection ISKNV method

[0080] Extraction of Infectious spleen and kidney virus, white spot syndrome virus, Lymphocystis disease virus and spring viremia of carp virus , that is, the genomic DNAs of four different related viruses were used as templates for the specificity verification experiment of the present invention, wherein double distilled water was used as a negative control. The DNA extraction method and the HRCA reaction system and conditions are described above. Amplified products were detected by agarose gel electrophoresis. The results show that see Figure 7 , indicating that the ISKNV-HRCA detection method provided by the present invention can ensure the specific detection of ISKNV and does not cross-react with other related viruses.

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Abstract

The invention discloses a method for detecting infectious spleen and kidney necrosis viruses by using hyper-branched rolling circle amplification, which is characterized by comprising the following steps of: (1) designing one locking type probe and a pair of universal primers for the connection sequence of the locking type probe; (2) making a connecting reaction system of the locking type probe for connecting reaction; and (3) making an HRCA (Hyper-branched Rolling Cycle Amplification) reaction system for HRCA reaction, and finally detecting the products of the HRCA reaction. Compared with the method for detecting the infectious spleen and kidney necrosis viruses by using PCR (Polymerase Chain Reaction) in the prior art, the method has the advantages of higher sensitivity, specificity and convenience, can be applied to actual production sites and is beneficial to detecting and controlling the cross infection of the infectious spleen and kidney necrosis viruses in the cultivation of aquatic animals.

Description

technical field [0001] The invention relates to a detection method for infectious spleen and kidney necrosis virus, in particular to a hyperbranched rolling circle amplification detection method for infectious spleen and kidney necrosis virus. Background technique [0002] Infectious spleen and kidney necrosis virus (ISKNV) is a member of the cytomegalovirus genus of the family Iridoviridae and is a cytoplasmic DNA virus. It is one of the important viral pathogens of farmed fish and can cause systemic, Systemic infection can cause serious damage to the spleen, kidney and other hematopoietic organs and tissues of susceptible fish, leading to anemia, multiple organ failure and death of the diseased fish. Its host range includes grouper, sea bass, red sea bream, large Important aquaculture fish such as turbot, mandarin fish, flounder, rock bream, and large yellow croaker, and important ornamental fish such as blue-eyed lantern and cichlid. Molecular biology studies have shown ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 陈炯史雨红孔诚将陆新江李登峰李明云
Owner NINGBO UNIV
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