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Method for detecting N-acylated homoserine lactone

A technology for acylated homoserine lactone and sample detection, which is applied in the direction of material analysis by observing the influence on chemical indicators, and analysis by making materials undergo chemical reactions, etc., to achieve high sensitivity, broad application prospects, and good specificity Effect

Inactive Publication Date: 2011-07-20
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many kinds of aquatic bacterial pathogens, and it is not predictable and accurate to warn the outbreak of bacterial diseases through the signs of sick fish, changes of pathogenic bacteria, water quality indicators, etc. Bacteria: N-acylated homoserine lactone (AHLs) activity concentration to regulate the "toxicity switch" of pathogenic bacteria, different pathogenic bacteria may have differences in toxicity effects, but due to the commonality of chemical structure and receptor specificity of AHLs, through the AHLs The monitoring of active concentration can accurately warn the outbreak of bacterial diseases

Method used

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  • Method for detecting N-acylated homoserine lactone
  • Method for detecting N-acylated homoserine lactone
  • Method for detecting N-acylated homoserine lactone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the preparation of ahyr receptor protein

[0053] 1. Extraction of total DNA from Aeromonas hydrophila ATCC 7966 and cloning of the gene of ATCC 7966 receptor protein AhyR

[0054] The total DNA of Aeromonas hydrophila ATCC 7966 was extracted according to the instructions of the bacterial DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China). The primers AhyR-EF (5′-ccggaattca tgaccgttaa aaaactttac-3′) (SEQ ID: 3) and AhyR-ER (5′-attgcggccg cttataaaaa ttcaggaaat g- 3′) (SEQ ID: 4), respectively introduce endonuclease EcoR I and Not I restriction sites (underlined part) at the end of the primer, carry out PCR amplification with total DNA of Aeromonas hydrophila ATCC 7966 as template . The PCR reaction parameters were as follows: denaturation at 94°C for 5 minutes and cooling to 4°C; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, and incubation at 72°C for 10 min after 30 cycles. The obt...

Embodiment 2

[0059] Embodiment 2, the application of acceptor protein

[0060] 1. Binding function with target DNA

[0061] 1. The gene recognition DNA (inter) sequence of Ah ATCC 7966 receptor protein AhyR was obtained

[0062] The primers AhyR-BE (5'-CTCGTTTTTCTACCTCCCATC-3') (SEQ ID: 5) and AhyR-BR (5'-CAGTTGGTCTTGTTTCATATGC-3') (SEQ ID : 6), using Aeromonas hydrophila ATCC 7966 total DNA as a template for PCR amplification. The PCR reaction parameters were as follows: denaturation at 94°C for 5 minutes and cooling to 4°C; denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and incubation at 72°C for 10 min after 30 cycles. Purified and preserved. The nucleotide sequence of the obtained DNA fragment is shown in SEQ ID NO:7.

[0063] 2. Gel retardation experiment of the interaction between the signal molecule receptor protein (AhyR) of AhATCC 7966 and its recognition DNA (inter) sequence

[0064] The receptor protein (AhyR) and its recognition...

Embodiment 3

[0075] Example 3, Ah ATCC 7966 signal molecule receptor protein (AhyR) nano-magnetic bead surface modification

[0076] Nanomagnetic beads were purchased from Shanghai Aorun Micro-Nano New Material Technology Co., Ltd. (Shanghai, China), AllMag TM PM3-008.

[0077] 1. Preparation of modified nano-magnetic beads:

[0078] 1. Magnetic beads activated by carbodiimide N-hydroxysuccinimide method (EDC / NHS method)

[0079] (1) Take 2mg PM3-008 magnetic beads into a 1.5ml centrifuge tube, wash twice with 500μl MEST buffer, and remove the supernatant after magnetic separation. The MEST buffer was prepared as follows: 100 ml of MES buffer (2-(N-morpholine)ethanesulfonic acid) with a concentration of 10 mM and pH 6.0 was mixed with 0.05 ml of Tween 20 to obtain a MEST buffer.

[0080] (2) Preparation of EDC solution: Dissolve the EDC solid (1ml: 5mg) in MES buffer with a concentration of 10mM and a pH of 6.0 stored at 4°C to obtain an EDC solution (ready for immediate use);

[0081]...

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Abstract

The invention discloses a method for detecting N-acylated homoserine lactone. The method for detecting whether N-acylated homoserine lactone exists in an auxiliary detection sample comprises the following steps of: (1) preparing experimental liquid, namely mixing a sample to be tested, magnetic nanoparticles coupled with an N-acylated homoserine lactone receptor and a buffer solution and incubating the mixture to obtain the experimental liquid, wherein the pH value of the buffer solution is 7.4; and (2) detecting whether the experimental liquid becomes blue after meeting agrobacteriumtumefaciens KYC55, and if so, determining that the sample to be detected contains the N-acylated homoserine lactone as a candidate. The novel application of the method disclosed by the invention is application of the N-acylated homoserine lactone receptor in detecting the N-acylated homoserine lactone in the sample. The method for detecting the N-acylated homoserine lactone by using N-acylated homoserine lactone receptor molecules has high sensitivity, good specificity and wide application prospect in the fields of detecting N-acylated homoserine lactone single molecules and pathogenic bacteria toxicity.

Description

technical field [0001] The invention relates to a method for detecting N-acylated homoserine lactone. Background technique [0002] Aeromonas hydrophila has a wide range of pathogenicity in nature and is an important pathogenic bacteria of zoonosis. Occurs naturally in aquatic environments and poses a serious threat to the aquaculture industry. The pathogenicity of the bacteria is related to its production of various virulence factors, mainly exotoxin, endotoxin, hemolysin, cytotoxin, protease and so on. [0003] The Quorum Sensing (QS) system means that bacteria can detect changes in the number of themselves or other bacteria in the surrounding environment according to the concentration of specific signal molecules. When the signal molecules reach a certain concentration threshold, they can start the expression of related genes in the bacteria to adapt to the environment. phenomena of change. Many Gram-negative bacteria produce acyl-homoserine lactones (acyl-homoserine l...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 周志刚曹雅男姚斌毛玮崔浩何夙旭刘玉春张美超
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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