LAMP-HNB rapid detection method for aeromonas salmonicida
A LAMP-HNB, Aeromonas salmonicidal technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of complex operation and long time
Pending Publication Date: 2021-05-11
QINGDAO AGRI UNIV
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Problems solved by technology
[0005] Aiming at the deficiencies in the prior art, the present invention provides a rapid detection method of Aeromonas salmonicida LAMP-HNB, which solves the problem of rapid detection of 16S rRNA PCR method and ELISA with the development of molecular biology and the development of various technologies. Methods such as real-time quantitative PCR and RAPD methods can quickly detect pathogens, but these methods are time-consuming and complicated to operate
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[0047] Take Aeromonas salmonicida DNA and carry out ten-fold gradient dilution to 3×10 1 -10 -6 ng / μL 7 concentrations, each take 1 μL as a template, mix the above-mentioned primer mixture and reaction mixture evenly, perform amplification according to the optimized reaction conditions, observe the color change after the reaction, as shown in figure 1 shown;
[0048] Take 5 μL of the product and add 1 μL 6×LoadingBuffer, perform 2% agarose gel electrophoresis, observe and take pictures with a gel instrument, such as figure 2 As shown, it can be seen that the first 6 samples have the characteristic bands of LAMP, and the sensitivity can reach 3×10 -5 ng / μL;
[0049] At the same time, conventional PCR was performed using the above-mentioned diluted DNA as a template. The system was as follows: 2×TaqPlus PCRMasterMix 12.5 μL, F3 / B3 each 1 μL, template 2 μL sterilized double-distilled water 8.5 μL; PCR reaction conditions were 94°C pre-denaturation for 5 minutes; 94°C denatura...
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The invention discloses an LAMP-HNB rapid detection method for aeromonas salmonicida, and relates to the technical field of LAMP-HNB rapid detection methods for the aeromonas salmonicida. The method comprises the following steps: S1, design and synthesis of primers: designing six specific primers through primer design software Primer Explore V4 according to a known aeromonas salmonicida virulence array protein gene (vap A) sequence in GenBank. According to the LAMP-HNB rapid detection method for the aeromonas salmonicida, the lowest detection concentration of the aeromonas salmonicida is 2*10<1> CFU / mL through a set LAMP method, the detection sensitivity is 102 times higher than that of common PCR, and the detection sensitivity is effectively improved; and the LAMP-HNB rapid detection method for the aeromonas salmonicida has the advantages that by means of the six designed specific primers, the system can specifically detect the aeromonas salmonicida, other strains are not amplified, and moreover, agarose gel electrophoresis results show that only the aeromonas salmonicida is amplified, so that the detection specificity of the aeromonas salmonicida is relatively high.
Description
technical field [0001] The invention relates to the technical field of a rapid detection method for Aeromonas salmonicida LAMP-HNB, in particular to a rapid detection method for Aeromonas salmonicida LAMP-HNB. Background technique [0002] Aeromonas salmonicida (Aeromonas salmonicida) was first isolated and described from trout by Emmerich and Weible in 1890; Aeromonas genus, the bacterium does not move, and is facultatively anaerobic; Aeromonas salmonicida is currently divided into five subspecies: colorless subspecies, salmonicidal subspecies, Japanese salmonid subspecies, pectin subspecies, and historical subspecies species; this fungus can cause furunculosis in salmon and trout, and red abscesses can form on the surface of diseased fish, which can develop into ulcers in severe cases, which seriously restricts the healthy development of the aquaculture industry; in 2005, Bjrnsdóttir et al. Bacteria had certain pathogenicity to turbot, and the pathological changes showed ...
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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844
Inventor 周顺于明明修云吉黄晴李影
Owner QINGDAO AGRI UNIV