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Methods for detecting pathogen in coldwater fish

A pathogenic bacteria detection and pathogenic bacteria technology, applied in the field of pathogenic bacteria detection, can solve problems such as loss

Active Publication Date: 2015-11-04
SCHWEITZER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cold water fish diseases can cause a mortality rate of up to over 90%, but the more common mortality rate is 10-30%. Such mortality is still a huge negative economic factor for the aquaculture industry, causing hundreds of millions of dollars in losses a year

Method used

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  • Methods for detecting pathogen in coldwater fish
  • Methods for detecting pathogen in coldwater fish
  • Methods for detecting pathogen in coldwater fish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The detection of embodiment 1 pathogen Candidatus Branchiomonas cysticola

[0081] The 16S ribosomal RNA gene (GenBank accession No. JQ723599) of the pathogen Candidatus Branchiomonas cysticola was inserted into the pUC57 cloning vector (Thermo Company, Massachusetts, USA) to obtain the pUC57-BC plasmid.

[0082] 1. Perform traditional PCR with primers BCF1 and BCR1

[0083] The 50 μl PCR mix used for conventional PCR contains: Diluted pUC57-BC plasmid (10 10 ,10 9 ,10 8 ,10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 ,10 1 ,10 0 copy number), 0.01-2 μM pre-primer BCF1 (SEQ ID NO:1), 0.01-2 μM post-primer BCR1 (SEQ ID NO:2), 0.2 μM dNTP and 1.25U Taq DNA polymerase. The amplification reaction was carried out in a thermal cycler (such as, but not limited to, PC818, Astec Co. Ltd., Japan), and included an initial cycle of denaturation at 94° C. for 3 minutes, and 35 cycles of 94° C. for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds. Amplified products were then...

Embodiment 2

[0104] The detection of embodiment 2 pathogen fish respiratory tract and enterovirus (PRV)

[0105] The L1 segment gene (GenBank accession number GU994013) of the pathogenic bacterium fish respiratory and enterovirus (PRV) was inserted into the pUC57 cloning vector (Thermo) to obtain the pUC57-PRV plasmid.

[0106] In addition, by using a commercial kit (such as, but not limited to, Kit; Thermo Fisher Scientific, Massachusetts, USA) transcribed the DNA sequence of the L1 segment gene of PRV to prepare the RNA template for in vitro transcription of PRV.

[0107] 1. Perform traditional PCR with primers PRVF1 and PRVR1

[0108] The 50 μl PCR mix used for conventional PCR contains: Diluted pUC57-PRV plasmid (10 6 ,10 5 ,10 4 ,10 3 ,10 2 ,10 1 copy number), 0.01-2 μM pre-primer PRVF1 (SEQ ID NO:4), 0.01-2 μM post-primer PRVR1 (SEQ ID NO:5), 0.2 μM dNTP and 1.25U Taq DNA polymerase. The amplification reaction was carried out in a thermal cycler (such as, but not limited to,...

Embodiment 3

[0125] The detection of embodiment 3 pathogen infectious pancreatic necrosis virus (IPNV)

[0126] The A segment gene (GenBank accession number AY379740) of the pathogen infectious pancreatic necrosis virus (IPNV) was inserted into T&A TM Selection vector (Yeastern biotech, Taiwan) to obtain pTA-IPNV plasmid.

[0127] By using a commercial kit (such as, but not limited to, Kit; Thermo Fisher Scientific, Massachusetts, USA) transcribed the DNA sequence of the A segment gene of IPNV to prepare the in vitro transcription RNA template of IPNV.

[0128] In addition, salmon virus samples from farms were collected and tested to evaluate the performance of iiPCR versus real-time PCR on field samples. Infectious pancreatic necrosis virus (IPNV) was diagnosed in salmon from a salmon farm. By using commercial kits such as, but not limited to, FavorPrep TM Viral Nucleic Acid Extraction Kit; Favorgen Biotech Corp., Taiwan) was used to extract RNA extracts from tissues. The purity of ...

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Abstract

The present invention relates to a method for detecting a pathogen in coldwater fish using insulated isothermal polymerase chain reaction (iiPCR). In addition, the present invention also relates to pairs of oligonucleotides for detecting pathogens in coldwater fish.

Description

technical field [0001] The invention relates to a method for detecting pathogenic bacteria in cold-water fish, in particular to a method for using oligonucleotide pairs to detect pathogenic bacteria in cold-water fish. Background technique [0002] The economic value of cold-water fish has reached billions of dollars, of which Atlantic salmon is the largest single cultured species, and its population exceeded 1.5 million tons in 2013. Cold water fish diseases can cause a mortality rate of up to over 90%, but the more common mortality rate is 10-30%. Such mortality is still a huge negative economic factor for the aquaculture industry, causing hundreds of millions of dollars in losses a year . These diseases can occur both early and late in the production cycle, with the greatest impact on the economy occurring later. [0003] The key to solving the above problems is to diagnose the pathogenic bacteria of cold-water fish and take necessary measures to avoid high mortality. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/70C12N15/11C12R1/93
CPCC12Q1/701C12Q1/689C12Q2527/101C12Q2600/158C12Q1/686
Inventor 郭村勇谢旺儒
Owner SCHWEITZER
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