Closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp.

A technique for Aeromonas hydrophila, ring-mediated isothermal

Active Publication Date: 2017-11-10
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report on the application of pH in

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  • Closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp.
  • Closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp.
  • Closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp.

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Detection of samples by novel loop-mediated isothermal amplification technology

[0034] 1) Eel genome extraction: Genome extraction was performed using the method provided by GB / T 28630.2-2012. The specific operation is as follows: collect about 100 mg of eel muscle, gills, liver and other lesion tissues, add 300 μL CTAB solution to grind and homogenize, then transfer to a 1.5 mL centrifuge tube, then add 450 μL CTAB solution to mix well, and stand at 25 °C for 2 hours . Add 600 μL of phenol-chloroform-isoamyl alcohol to the tissue sample, mix thoroughly, and centrifuge at 12000 r / min for 5 min. Aspirate the supernatant into a new 1.5ml centrifuge tube, add 700 μL phenol-chloroform-isoamyl alcohol to the tissue sample, mix well, and centrifuge at 12000r / min for 5min. Aspirate the supernatant into a new 1.5ml centrifuge tube, add 900 μL of pre-cooled absolute ethanol (-20°C), mix well and let it stand, and place it at -20°C for more than 8 hours. Centrifug...

Embodiment 2

[0041] Example 2: Sensitivity of closed-tube visualized loop-mediated isothermal amplification technique for Aeromonas hydrophila from eel origin

[0042] The positive plasmids of Aeromonas hydrophila gyrB gene and hlyA gene were respectively used as templates, and the 10-fold ratio dilution was carried out, and the copy numbers of the target gene gyrB in the templates were 2.0×10 6 , 2.0×10 4 , 2.0×10 3 , 2.0×10 2 , 2.0×10 1 , 2.0×10 0 (copy / μL); the target gene hlyA copy number is 1.1×10 6 , 1.1×10 4 1.1×10 3 , 1.1×10 2 , 1.1×10 1 , 1.1×10 0 (copy / μL) and the negative control, while using the optimized LAMP reaction system and program for LAMP reaction, the reaction system is 25 μL. The results of the detected samples were judged by visually observing changes and electrophoretic detection in Example 1. The results showed that templates with a copy number of the target gene gyrB greater than or equal to 20 copy / μL or a copy number of the target gene hlyA greater th...

Embodiment 3

[0043] Example 3: Specificity of the Loop-Mediated Isothermal Amplification Technique for the Visualization of Closed Tubes of Aeromonas hydrophila from Eels

[0044]Take 10 positive samples and 10 negative samples by PCR, and the corresponding eel tissue DNA as a template; or 5 strains of Aeromonas hydrophila (4 of which contain the hlyA gene), 1 strain of Aeromonas temperatus A.sobria, 1 strain of A.Veronii, 1 strain of Aeromonas caviae (A.caviae), 2 strains of Staphylococcus aureus, 1 strain of parahaemolyticus The genomes of Vibrio parahemolyticus, 1 strain of Pseudomonas aeruginosa and 1 strain of Klebsiella pneumoniae were used as templates, using the optimized LAMP reaction system and reaction procedures, prepared in Example 1 The negative and positive controls were used as reference. The results of the detected samples were judged by visually observing changes and electrophoretic detection in Example 1. The results showed that only 20 Aeromonas hydrophila positive sp...

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Abstract

The invention relates to a closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp. and relates to detection of pathogenic microorganisms of aquatic animals. The method comprises steps as follows: extracting a genome of Anguilla spp.; extracting a genome of pathogenic bacteria; preparing a positive control; preparing a negative control; detecting a sample and determining a result. LAMP primers are designed by taking conserved sequences of housekeeping gene gyrB of Aermonas hydrophila and pathogenic factor hlyA, an LAMP reaction system is established, and whether a target gene is amplified effectively can be determined in a closed-tube visualized manner by adding different pH indicators to an LAMP reaction. With adoption of the closed-tube visualization for rapid detection of pathogenic Aermonas hydrophila sourced from Anguilla spp., a reaction product is not needed to be identified through electrophoresis and/or open-tube addition of fluorescent dyes and the like, so that the pollution risk of the LAMP product is reduced.

Description

technical field [0001] The invention relates to the detection of pathogenic microorganisms in aquatic animals, in particular to a method for detecting the pathogenic Aeromonas hydrophila Aeromonas of eels based on a loop-mediated constant temperature amplification technology with rapid closed-tube visualization. Background technique [0002] Eel (Anguilla spp.) is a valuable economic fish in the world. Eel farming has a history of nearly 30 years in my country, and its aquaculture output accounts for about 70% of the world's total. It is one of the important industries for exporting and earning foreign exchange in my country. However, in the process of intensive high-density farming, the outbreak of eel bacterial diseases has seriously restricted the healthy and sustainable development of China's eel aquaculture industry (Xiong Jing et al. 2017. Comparison of exogenous products and their activities, Journal of Huazhong Agricultural University, 2017, 36(1):76-85). Aeromonas ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2545/113
Inventor 熊静黄文树徐继松赖晓健刘爱原
Owner JIMEI UNIV
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