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Method for detecting salmonella with fluorescence method on basis of enzymatic remediation isothermal cycle amplification

A cyclic amplification and Salmonella technology, applied in the field of detection, can solve the problems of long detection cycle, low specificity and sensitivity, etc., and achieve the effects of fast detection speed, improved sensitivity and improved reaction speed

Active Publication Date: 2016-07-13
UNIV OF JINAN
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Problems solved by technology

[0004] In order to solve the problems of relatively low specificity and sensitivity and long detection period in the method for detecting Salmonella in the above prior art, the present invention provides a fluorescence method based on enzymatic repair cycle amplification with high specificity and sensitivity and fast detection speed How to detect Salmonella

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  • Method for detecting salmonella with fluorescence method on basis of enzymatic remediation isothermal cycle amplification

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Embodiment 1

[0037] The preparation method of described fluorescent biosensor comprises the following steps:

[0038] (1) Add sterilized water, 10× buffer (buffer), 1 μL of 20 μM aptamer chain and Trigger chain to the pre-prepared sterilized EP tube; shake for 30 seconds, incubate at 95°C for 5 minutes, and slowly cool to room temperature;

[0039] (2) Add 1 μL of target bacteria to the EP tube (the concentration is 10 6 cfu / mL, 10 5 cfu / mL, 10 4 cfu / mL, 10 3 cfu / mL, 10 2cfu / mL), UDG (1 μL), 10 μM HAP (1 μL), 10 μSignalprobe (1 μL), endonuclease IV (1 μL), BstDNA polymerase (1 μL) and dNTP (1 μL), shake for 30 seconds, and place in a 37°C incubator Incubate in medium for 2h;

[0040] (3) Dilute the above-mentioned reacted solution (10 μL) to 70 μL, and detect the fluorescence at 518 nm with a fluorometer;

[0041] The excitation wavelength of the fluorescence instrument is set to 486nm, the emission wavelength is 518nm, the detection range is 450nm-530nm, and the change of the fluor...

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Abstract

The invention relates to the technical field of biological detection, in particular to a fluorescence biological detection method based on enzymatic remediation cycle three-time amplification. The method comprises steps as follows: an arched probe is constructed; enzymatic remediation cycle signal amplification and fluorescence detection are performed; high-specificity detection is performed on a target object, namely, salmonella, by the aid of a nucleic acid aptamer through specific recognition of the nucleic acid aptamer; the signal amplification effect is realized through enzymatic remediation cycle amplification.

Description

technical field [0001] The invention relates to a detection method, in particular to a method for detecting Salmonella with a circular fluorescence method. Background technique [0002] Salmonella is a common foodborne pathogen, a Gram-negative, intracellular parasitic enteric bacterium. The bacteria widely exist in nature, not only can cause acute, chronic or latent infection of livestock, poultry and other animals, but also cause food poisoning by contaminating food, posing a great threat to human beings. According to statistics, among the types of bacterial food poisoning in various countries in the world, food poisoning caused by Salmonella often ranks first. Salmonella sickens about 40,000 Americans and kills about 600 each year. Salmonella is the most common pathogenic bacteria in food poisoning in my country, accounting for the first place in food poisoning. The clinical symptoms of salmonellosis mainly include headache, abdominal pain, fever, etc., and the mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10G01N33/569C12R1/42
CPCC12Q1/6804G01N33/56916C12Q2521/101C12Q2521/301C12Q2521/531C12Q2537/155C12Q2563/107
Inventor 黄加栋冷雪琪王玉刘素王虹智郭玉娜
Owner UNIV OF JINAN
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