Acetylcholin esterase marked engineering phage for rapidly detecting microorganisms

A technology of acetylcholinesterase and bacteriophage, which can be applied in the direction of bacteriophage, microorganism, virus/phage, etc., can solve the problems of complicated operation steps and the like

Inactive Publication Date: 2017-08-01
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For microbial gene analysis technology, it is widely used in microbial identification. The target primers used are usually designed based on the characteristic sequence of the microbial 16S rRNA gene, and its detection limit can reach 1 cfu mL-1. It requires special sample processing and purification, the operation steps are cumbersome, and there are high technical requirements

Method used

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  • Acetylcholin esterase marked engineering phage for rapidly detecting microorganisms
  • Acetylcholin esterase marked engineering phage for rapidly detecting microorganisms
  • Acetylcholin esterase marked engineering phage for rapidly detecting microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Detection of sulfate-reducing bacteria:

[0018] The microorganisms used in the experiment were cultured in suspension in lysed broth (1% peptone, 1% sodium chloride, 0.5% yeast extract, 100 mL water), and a single colony was cultured overnight at 30°C and 200 rpm on a shaking table at 4500 rpm. / Centrifuge for ten minutes, and dilute to different concentrations with PBS buffer solution.

[0019] 100 µL concentration (10 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to the disposable medium, and reacted at 37 °C for 8 hours. Add acetylcholinesterase-responsive iodothioacetylcholine and nano-gold colloids, incubate for 30 minutes, use the excitation light source of the microbial diagnostic instrument to excite the sensor platform to generate light signals, and measure the microbial signals at this concentration.

[0020] 100 µL concentration (10 2 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to th...

Embodiment 2

[0029] E. coli detection

[0030] The microorganisms used in the experiment were cultured in suspension in lysed broth (1% peptone, 1% sodium chloride, 0.5% yeast extract, 100 mL water), and a single colony was cultured overnight at 30°C and 200 rpm on a shaking table at 4500 rpm. / Centrifuge for ten minutes, and dilute to different concentrations with PBS buffer solution.

[0031] 100 µL concentration (10 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to the disposable medium, and reacted at 37 °C for 8 hours. Add acetylcholinesterase-responsive iodothioacetylcholine and nano-gold colloids, incubate for 30 minutes, use the excitation light source of the microbial diagnostic instrument to excite the sensor platform to generate light signals, and measure the microbial signals at this concentration.

[0032] 100 µL concentration (10 2 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to the disposable medium, a...

Embodiment 3

[0041] Staphylococcus aureus detection

[0042] The microorganisms used in the experiment were cultured in suspension in lysed broth (1% peptone, 1% sodium chloride, 0.5% yeast extract, 100 mL water), and a single colony was cultured overnight at 30°C and 200 rpm on a shaking table at 4500 rpm. / Centrifuge for ten minutes, and dilute to different concentrations with PBS buffer solution.

[0043] 100 µL concentration (10 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to the disposable medium, and reacted at 37 °C for 8 hours. Add acetylcholinesterase-responsive iodothioacetylcholine and nano-gold colloids, incubate for 30 minutes, use the excitation light source of the microbial diagnostic instrument to excite the sensor platform to generate light signals, and measure the microbial signals at this concentration.

[0044] 100 µL concentration (10 2 cfu ml -1 ) bacteria solution and acetylcholinesterase-labeled phage were added to the dispos...

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Abstract

The invention plans to research and develop an acetylcholin esterase marked engineering phage kit for directly analyzing microorganisms in an environment biological sample, and an acetylcholin esterase marked engineering phage can be used for specifically recognizing pathogenic microorganisms. When the engineering phage recognizes and combines with the microorganism, a genetic engineering modified DNA (deoxyribonucleic acid) of the engineering phage is injected into the microorganism, and a great number of new phages with acetylcholin esterase protein are recombined, expressed and amplified in the microorganism. The main content of the project is as follows: laying emphasis on the designing of an engineering phage and microorganism response material recognition mechanism and reaction kinetics, investigating an action rule of recognition responsive functional modules in microorganism rapid detection and microorganism real-time expression and analysis. The invention has the innovativeness that reference is provided for solving online, portable and full-automatic electromechanical engineering and biology composite problems related to the microorganism detection methods.

Description

technical field [0001] The invention designs an acetylcholinesterase-labeled engineered phage for rapid detection of microorganisms. Background technique [0002] In the marine environment, pathogenic microbial pollution, water eutrophication, microbial corrosion, and microbial fouling are all apparent forms of microorganisms that threaten human production and life, and are also objective conditions for rapid microbial detection technology. Existing data show that the loss of pathogenic microbial contamination is closely related to the speed of microbial identification, the longer the identification time, the greater the loss. This is because, on the one hand, the rapid growth and spread of microorganisms aggravates environmental pollution and human diseases; on the other hand, the inability to identify the types of microorganisms makes it impossible to implement targeted protection programs, which in turn leads to the abuse of certain drugs or antibacterial agents. In the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12Q1/70C12Q1/46C12Q1/06C12Q1/10C12Q1/14C12R1/19C12R1/445C12R1/42C12R1/63C12R1/01C12R1/145C12R1/085
CPCC12N7/00C12N9/18C12N2795/10121C12N2795/10221C12N2795/10321C12Q1/06C12Q1/10C12Q1/14C12Q1/46C12Y301/01007G01N2333/918
Inventor 万逸周腾许强葛鉴
Owner HAINAN UNIVERSITY
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