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Method for detecting Vibrio parahemolyticus based on aptamer identification-molecular motor biosensing

A hemolytic vibrio and molecular motor technology, which is applied in measuring devices, analysis materials, material excitation analysis, etc., can solve the problems that antibodies are easily affected by external conditions, high selectivity requirements, cumbersome operation, etc., and shorten the detection time , broad application prospects and low preparation cost

Inactive Publication Date: 2018-06-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods are time-consuming, laborious, and cumbersome to operate, which can no longer meet the requirements of fast and simple food safety detection; although molecular biology technology can shorten the detection time, non-specific binding between primers and templates is prone to occur, and the detection results are prone to variation due to steps. It is cumbersome to produce false positives; immunological methods have the advantages of high sensitivity, strong specificity, and short detection time, but immunological methods require too high selectivity of reagents, and antibodies as recognition molecules are easily affected by external conditions and have low stability

Method used

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  • Method for detecting Vibrio parahemolyticus based on aptamer identification-molecular motor biosensing
  • Method for detecting Vibrio parahemolyticus based on aptamer identification-molecular motor biosensing
  • Method for detecting Vibrio parahemolyticus based on aptamer identification-molecular motor biosensing

Examples

Experimental program
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Embodiment 1

[0022] Example 1: Construction of a method for detection of Vibrio parahaemolyticus based on aptamer-molecular motor biosensing

[0023] (1) Preparation of chromophore:

[0024] Rhodospirillum rubrum was cultivated in tryptone soybean liquid medium at 26°C and 150r / min for 72h. Then at 4°C, centrifuge at 5000r / min for 30min to collect the bacterial cells. With extraction buffer (20mmol / L Tris-HCl (pH 8.0), 100mmol / L NaCl, 2mmol / L MgCl 2 , 1mmol / L DTT) to resuspend the bacteria, then centrifuge at 8000r / min for 10min at 4°C, and remove the supernatant. The precipitate was resuspended with the above-mentioned extraction buffer, then added with phenylmethylsulfonyl fluoride (final concentration: 1 mmol / L), and sonicated on ice for 30 min. Centrifuge the broken bacteria at 4°C, 25000r / min for 90min, take the supernatant and continue to centrifuge at 4°C, 50000r / min for 90min, the precipitate is the chromophore, which contains F 0 f 1 -ATPase molecular motor.

[0025] (2) Pre...

Embodiment 2

[0035] Embodiment 2: establishment of Vibrio parahaemolyticus standard detection curve

[0036] Take 300 μL of aptamer-molecular motor complex, add 200 μL of Vibrio parahaemolyticus liquid of different concentrations, incubate at 37°C for 1 hour, then centrifuge at 4°C, 15,000 r / min for 30 minutes, discard the supernatant; add 30 μL of ATP to synthesize Buffer (0.1 mM trimethylglycine, 10% glycerol, 5 mM NaH 2 PO 4 , 5 mM MgCl 2 , 0.35mM ADP, 2mM NADH, pH 9.0) were incubated at 37°C for 30min, then 470μL of PBS was added, mixed evenly, and the fluorescence intensity was measured (excitation wavelength 485nm, emission wavelength 512nm). Such as image 3 As shown, when there is no target strain in the system, the load of the molecular motor F0F1-ATPase does not change, so the fluorescence intensity is the largest at this time; when there is Vibrio parahaemolyticus in the system, the aptamer specificity and vibrio binding, making F 0 f 1 - The load of ATPase increases, caus...

Embodiment 3

[0037] Example 3: Detection of Vibrio parahaemolyticus in salmon samples

[0038] Purchase fresh salmon from a local supermarket, take 25g of salmon meat sample and mince, mix and homogenize with 225mL 3% sodium chloride alkaline peptone water for 10min, then filter with a 0.45μm filter membrane, take 200μL of the supernatant to a centrifuge tube as the actual sample, A total of 6 samples were taken, and a known concentration of Vibrio parahaemolyticus was added (the concentration was obtained by plate counting), followed by 300 μL of aptamer-molecular motor complex, and the final concentration of Vibrio parahaemolyticus in the system was 1.5 ×10 2 ~1.5×10 3 cfu / mL, incubate at 37°C for 1h, then centrifuge at 15000r / min for 30min at 4°C, discard the supernatant; add 30μL of ATP synthesis buffer (0.1mM trimethylglycine, 10% glycerol, 5mM NaH 2 PO 4 , 5 mM MgCl 2 , 0.35mM ADP, 2mM NADH, pH 9.0) were incubated at 37°C for 30min, then 470μL of PBS was added, mixed evenly, and ...

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Abstract

The invention provides a method for detecting Vibrio parahemolyticus based on aptamer identification-molecular motor biosensing. According to the method, the interior of a chromatophore is labeled with a pH-sensitive fluorescent dye F1300 so that a signal probe is obtained, an aptamer specifically recognizing Vibrio parahemolyticus is used as a recognition probe, and an epsilon subunit antibody-biotin-avidin-end 5' biotinylated Vibrio parahemolyticus aptamer system connects the recognition probe and an F0F1-ATPase molecule motor so that the F0F1-ATPase molecule motor aptamer sensing system isconstructed. The method has the lowest detection limit of 15cfu / mL to Vibrio parahemolyticus, can be used for detection of Vibrio parahemolyticus in a salmon sample and can produce an accurate and reliable result.

Description

technical field [0001] The invention relates to the field of food safety detection, in particular to a method for detecting Vibrio parahaemolyticus based on aptamer-molecular motor biosensing. Background technique [0002] Vibrio parahaemolyticus is a Gram-negative bacterium with halophilicity. It is mainly distributed in seawater, fish, shellfish, shrimp, jellyfish and other seafood, followed by salt pickled products. In addition, livestock and poultry meat, salty Eggs, freshwater fish, etc. can also spread the bacteria due to cross-contamination. People eating food infected by Vibrio parahaemolyticus can cause acute gastroenteritis, diarrhea, sepsis, and even life-threatening in severe cases. Vibrio parahaemolyticus has not only caused great harm to people's health, but also brought serious losses to the mariculture industry. It has become a food-borne pathogen that is of great concern in my country and even the world. In this regard, my country refers to the limit stand...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N21/64
Inventor 段诺吴世嘉沈默斐邹颖王周平
Owner JIANGNAN UNIV
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