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Loop-mediated isothermal amplification combined lateral flow test strip method for detecting carp spring viremia virus

A carp spring viremia and ring-mediated isothermal technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem that antibodies cannot be used to detect Asian strains and sample detection workload Huge, high requirements for antibodies, etc., to achieve the effect of short detection time, friendly experimenters and the environment, and low requirements for equipment

Inactive Publication Date: 2017-10-20
北京科弘生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of method has high sensitivity, but it also has high requirements for antibodies. It has been reported that antibodies prepared against European strains of SVCV cannot be used to detect Asian strains
In addition, the operation of this type of method is relatively cumbersome, and the workload of detecting a large number of samples is huge

Method used

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  • Loop-mediated isothermal amplification combined lateral flow test strip method for detecting carp spring viremia virus
  • Loop-mediated isothermal amplification combined lateral flow test strip method for detecting carp spring viremia virus
  • Loop-mediated isothermal amplification combined lateral flow test strip method for detecting carp spring viremia virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1, LAMP-LFD technology detection SVCV method establishment

[0075] 1. LAMP primer probe design

[0076] The homology analysis of the G gene of SVCV (AB375390) published in NCBI was carried out, and the sequence of the G gene of similar species of SVCV was compared, and several primers for detecting SVCV were obtained. Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally a set of primer probes for detecting SVCV (upstream outer primer SVCV-G-F3, downstream outer primer SVCV-G-B3, upstream inner primer SVCV -G-FIP, downstream inner primer SVCV-G-BIP and probe SVCV-G-HP).

[0077] The sequence is as follows:

[0078] SVCV-G-F3 (Sequence 1 of the Sequence Listing): 5'-GACCCCAATATATAACCCACAG-3';

[0079] SVCV-G-B3 (sequence 2 of the sequence listing): 5'-ATCGATCCAGTGTCCTCC-3';

[0080] SVCV-G-FIP (SEQ ID NO: 3 of the SEQUENCE LISTING):

[0081] 5'-CAGTTCCTGATGCAATCCTTGATTCATCCAATCAGT...

Embodiment 2

[0101] Embodiment 2, LAMP-LFD detects the evaluation of SVCV sensitivity

[0102] 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) was extracted to obtain a DNA solution with a concentration of 8.6 ng / μL;

[0103] 2. 10-fold gradient dilution of the DNA solution obtained in step 1 to obtain each dilution;

[0104] 3. Take each DNA dilution in step 2 as a template, and use LAMP and LAMP-LFD to detect and compare the sensitivity of SVCV respectively. Double distilled water was used as a negative control.

[0105] In the 25 μL reaction system, the following reaction systems with different initial DNA contents were formed:

[0106] In reaction system 1, the initial templ...

Embodiment 3

[0120] Embodiment 3, LAMP-LFD detects the evaluation of SVCV specificity

[0121] 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).

[0122] 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).

[0123] 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 loop-mediated isothermal amplification combined lateral flow test strip method for detecting a carp spring viremia virus. A loop-mediated isothermal amplification primer group provided by the invention comprises an upstream outer primer, a downstream outer primer, an upstream inner primer and a downstream inner primer; the nucleotide sequence of the upstream outer primer is represented by sequence 1; the nucleotide sequence of the downstream outer primer is represented by sequence 2; the nucleotide sequence of the upstream inner primer is represented by sequence 3; and the nucleotide sequence of the downstream inner primer is represented by sequence 4. Compared with traditional PCR method for detecting the SVCV, the method disclosed in the invention has the advantages of high practicality, high convenience, realization of practical field onsite instant detection, and facilitation of prevention and control the outbreak of the SVCV, and is of great significance to protect the economy development of Chinese aquatic product culture.

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 method for detecting carp spring viremia virus. Background technique [0002] Spring viraemia of carp (SVC) is an acute epidemic with hemorrhage as the main clinical symptom and highly contagious, and its pathogen is Spring viraemia of carpvirus (SVCV) . The disease has acute onset and high mortality rate, which can be as high as 90%. It seriously endangers fishery production. The World Organization for Animal Health (OIE) lists it as an important epidemic disease that must be declared. In 2008, the Ministry of Agriculture’s Announcement No. 1125 even listed the disease as a “Class I Infectious Disease of Imported Animals of the People’s Republic of China”, and it is also the only fish disease listed as a Class I infectious disease, so it has become a fish port quarantine The first category...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625C12Q2563/107
Inventor 肖勤刘露王建梅李慧欣
Owner 北京科弘生物技术有限公司
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