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Biosensor and method of detecting vibrio parahemolyticus thereby

A biosensor, hemolytic vibrio technology, applied in the direction of instruments, measuring devices, scientific instruments, etc., can solve the problems that cannot meet the requirements of fast and simple food safety detection, antibodies are easily affected by external conditions, and the operation is cumbersome, etc., to achieve The effects of shortened detection time, easy labeling, and low preparation cost

Inactive Publication Date: 2018-06-05
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 and cumbersome, which can no longer meet the requirements of fast and simple food safety detection; although molecular biology technology can shorten the detection time, it is necessary to extract the total bacterial DNA in the early stage, and the detection object is the target DNA rather than the bacteria itself , the detection results need to be converted into data; immunological methods have the advantages of strong specificity, high sensitivity, and easy observation, but antibodies as recognition molecules are easily affected by external conditions and have low stability

Method used

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  • Biosensor and method of detecting vibrio parahemolyticus thereby
  • Biosensor and method of detecting vibrio parahemolyticus thereby
  • Biosensor and method of detecting vibrio parahemolyticus thereby

Examples

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Embodiment 1

[0022] Embodiment 1: the preparation of biosensor

[0023] (1) Preparation of chromophore:

[0024] Rhodospirillum rubrum ATCC 11170 (purchased from the American Type Culture Collection) was cultured in tryptone soybean broth, and cultured statically at 26°C for 72 hours. 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) resuspended cells, centrifuged at 8000r / min for 10min at 4°C, and removed the supernatant. The precipitate was resuspended with the above-mentioned extraction buffer (approximately 1 g was added to 10 mL of extraction buffer), and then PMSF (final concentration was 1 mmol / L) was added, and ultrasonically crushed on ice for 30 min. Centrifuge the crushed bacteria at 4°C, 25,000r / min for 90min, take the supernatant and continue to centrifuge at 4°C, 50,000r / min for 90min, the precipitate is the chromophore, which contains the F0F1-ATP...

Embodiment 2

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

[0036] Take 300 μL of the aptamer-molecular motor complex, add 100 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 , ADP 0.35mM, NADH 2mM, pH 9.0) were incubated at 37°C for 10min, then 450μL of PBS was added, mixed evenly, and the fluorescence intensity was measured (excitation wavelength 585nm, emission wavelength 610nm). Such as image 3As 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 minimum at this time; when there is Vibrio parahaemolyticus in the system, the aptamer specificity and The combination of Vibrio increases the load of F0F1-ATPase, cause...

Embodiment 3

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

[0038] Purchase fresh shrimp from a local supermarket, open the shell with aseptic operation, take 25g of shrimp meat sample, mince, mix and homogenize with 225mL 3% sodium chloride alkaline peptone water for 10min, then filter with a 0.45μm filter membrane, and take the supernatant 200 μL was used as the actual sample in a centrifuge tube, and a total of 4 samples were taken, and a known concentration of Vibrio parahaemolyticus was added (the concentration was obtained by plate counting), followed by 300 μL of the aptamer-molecular motor complex, and incubated at 37°C 1h, then centrifuge at 4°C, 15000r / min for 30min, discard the supernatant; add 30μL of ATP synthesis buffer (0.1mM trimethylglycine, 10% glycerol, 5mM NaH 2 PO 4 , 5mMMgCl 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 (excit...

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Abstract

The invention provides a method of detecting vibrio parahemolyticus based on an aptamer recognizing quantum dot labelled molecular motor biosensor. The aptamer of vibrio parahemolyticus is specifically recognized as a recognizing probe by labeling a pH sensitive quantum dot fluorescent nanomaterial to the surface of a color carrier. An F0F1 ATPas molecular motor aptamer sensing system is constructed by connecting the recognizing probe to a F0F1-ATPase molecular motor by means of an epsilon subunit antibody-biotin-avidin-5' end biotinylated vibrio parahemolyticus aptamer system. The lowest detection limit to the vibrio parahemolyticus by the method can reach 7cfu / mL, and the method can be applied to detecting vibrio parahemolyticus in shrimp meat samples, and is accurate and reliable in 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 an aptamer-quantum dot-molecular motor biosensor. Background technique [0002] Vibrio parahaemolyticus is a halophilic bacterium that widely exists in seafood such as shrimps, crabs, and shellfish, as well as seawater and seabed sediments. After people eat food infected by Vibrio parahaemolyticus, it will cause acute gastroenteritis, diarrhea, sepsis, etc., which will cause great harm to people's health. At present, food poisoning incidents caused by Vibrio parahaemolyticus have surpassed Salmonella poisoning incidents, especially in coastal areas, food poisoning caused by Vibrio parahaemolyticus accounts for more than 60% of bacterial food poisoning, and Vibrio parahaemolyticus The outbreak of foodborne disease caused by it is one of the important public health problems facing at present. In view of this, my country ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486
Inventor 段诺吴世嘉邹颖沈默斐王周平
Owner JIANGNAN UNIV
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