Method for detecting grass carp reovirus by loop-mediated isothermal amplification combined with lateral flow dipstick

A reovirus, ring-mediated isothermal technology, applied in the detection/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of less research on rapid visual detection, and achieve the protection of aquaculture economic development , the result is obvious, the test result is clear and obvious

Inactive Publication Date: 2018-07-27
北京科弘信息技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the research on its detection methods, such as electron microscope observation, serum neutralization experiment, PCR and other technologies have also been reported, but there are few researches on its rapid visual detection

Method used

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  • Method for detecting grass carp reovirus by loop-mediated isothermal amplification combined with lateral flow dipstick
  • Method for detecting grass carp reovirus by loop-mediated isothermal amplification combined with lateral flow dipstick
  • Method for detecting grass carp reovirus by loop-mediated isothermal amplification combined with lateral flow dipstick

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, LAMP-LFD technique detection GCRV method establishment

[0073] 1. Design of LAMP primers

[0074] After performing homology analysis on the S8 gene fragment (HQ231204) of GCRV published in NCBI, and comparing the sequence of the S8 fragment with the GCRV similar species, the following 2 pairs of primers and a probe were designed and screened. The sequence is as follows :

[0075] GCRV-S8-F3: 5'-TGCGCGTATGTGTGGTAC-3' (SEQ ID NO: 1);

[0076] GCRV-S8-B3: 5'-GACAGACGAGGCAGAGCT-3' (SEQ ID NO: 2);

[0077] GCRV-S8-FIP: 5'-BIO-CGTTTGGCAGATTGCGTTAGCA-CTACCCTTCGACGCCTCTA-3' (SEQ ID NO: 3);

[0078] GCRV-S8-BIP:

[0079] 5'-GACTAACGCTTCCTCTTCCGCC-CAAAACTGGTCGTAGCCGAG-3' (SEQ ID NO: 4);

[0080] GCRV-S8-HP: 5'-FITC-GTCTGCACTGCAACTGTTTC-3' (SEQ ID NO: 5).

[0081] The 5' end of GCRV-S8-FIP is labeled with biotin, and the 5' end of GCRV-S8-HP is labeled with FITC with fluorescein isothiocyanate.

[0082] The S8 gene fragment LAMP amplification primer sequence ...

Embodiment 2

[0097] Embodiment 2, LAMP-LFD detects the evaluation of GCRV sensitivity

[0098] 1. Extract the total RNA of GCRV and reverse transcribe it into cDNA to obtain a cDNA solution with a concentration of 97 ng / μL.

[0099] 2. Dilute the cDNA solution obtained in step 1 by 10 times to obtain each dilution.

[0100] 3. Take each cDNA dilution in step 2 as a template, and use LAMP and LAMP-LFD to detect GCRV and compare the sensitivity. Double distilled water was used as a negative control.

[0101] In the 25 μL reaction system, the following reaction systems with different initial DNA contents were formed:

[0102] In reaction system 1, the initial template cDNA content is: 0.97ng (diluted 10 times);

[0103] In reaction system 2, the initial template cDNA content is: 97pg (diluted 10 2 times);

[0104] In reaction system 3, the initial template cDNA content is: 9.7pg (diluted 10 3 times);

[0105] In reaction system 4, the initial template cDNA content is: 0.97pg (diluted 1...

Embodiment 3

[0117] Embodiment 3, LAMP-LFD detects the evaluation of GCRV specificity

[0118] 1. Construction of recombinant plasmid containing SVCV-specific gene: Insert SVCV-specific fragment (double-stranded DNA molecule shown in sequence 6 of the sequence table, NCBI accession number: AY527273) between the SmaI restriction sites of the pUC57 vector to obtain SVCV-specific gene-containing The recombinant plasmid of the gene (sequenced and verified).

[0119] 2. Construction of recombinant plasmid containing KHV-specific gene: Insert KHV-specific fragment (double-stranded DNA molecule shown in sequence 7 of the sequence listing, NCBI accession number: AB375390) between the SmaI restriction sites of pUC57 vector to obtain KHV-specific The recombinant plasmid of the gene (sequenced and verified).

[0120] 3. Construction of recombinant plasmid containing GCRV-specific gene: Insert the GCRV-specific fragment (double-stranded DNA molecule shown in sequence 8 of the sequence listing, NCBI a...

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Abstract

The invention discloses a method for detecting grass carp reovirus (GCRV) by loop-mediated isothermal amplification combined with a lateral flow dipstick. The invention provides a complete set of primers for loop-mediated isothermal amplification, which comprises an upstream outer primer, a downstream outer primer, an upstream inner primer and a downstream inner primer; a nucleotide sequence of the upstream outer primer is a sequence 1; a nucleotide sequence of the downstream outer primer is a sequence 2; a nucleotide sequence of the upstream inner primer is a sequence 3; a nucleotide sequenceof the downstream inner primer is a sequence 4. Compared with a traditional method for detecting the GCRV through a PCR technology, the method disclosed by the invention has higher specificity, sensitivity, practicability and convenience, can immediately detect on the actual site, is favorable for prevention and control of the outbreak of the GCRV and is of great significance in protecting the development of aquaculture economy in China.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a loop-mediated isothermal amplification combined with a lateral flow test strip for detecting grass carp reovirus. Background technique [0002] Grass carp is one of the "four major fish" in my country, and its aquaculture volume accounts for about 20% of the total freshwater aquaculture output in China, and is an important part of my country's agricultural economy. However, grass carp is very susceptible to disease during the breeding process, and its annual survival rate is only about 30%, which brings huge economic losses to the grass carp farming industry in my country. Among the common viral diseases of grass carp, grass carp viral hemorrhagic disease is considered to be the most harmful infectious disease to grass carp farming. Grass carp reovirus (GCRV) is the main pathogen of grass carp hemorrhagic disease, which is highly lethal and contagious. Over the years,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625
Inventor 肖勤刘露李慧欣
Owner 北京科弘信息技术有限公司
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