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Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor

A metal-organic framework, food-borne pathogenic bacteria technology, applied in the field of food-borne pathogenic bacteria analysis and detection, can solve the problems of cumbersome and time-consuming operation, low sensitivity, and low specificity of pathogenic bacteria detection technology.

Inactive Publication Date: 2015-11-04
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing pathogenic bacteria detection technology, such as low specificity, low sensitivity, cumbersome and time-consuming operation, and high false positive rate, and apply metal organic framework materials to the field of food safety analysis. The fluorescence quenching characteristics of the material and the adsorption of nucleic acid aptamers, combined with the high affinity and high specificity recognition ability of nucleic acid aptamers, provide a fast, accurate, reliable, sensitive and specific detection of foodborne pathogens method

Method used

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  • Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor
  • Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor
  • Method for detecting food-borne pathogenic bacteria on basis of metal organic framework material and aptamer fluorescence sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Application of the method of the present invention in the detection of Salmonella

[0021] First synthesize organic framework materials containing Cd, the process is as follows:

[0022] Step 1: Add CdCl 2? 2.5H 2 O (0.046g, 0.2mmol) and H 2 BTC (0.105g, 0.5mmo) was dissolved in 8.0mL of DMF, 3.0mL of cyclohexanol and 2.0mL of water, mixed and stirred at room temperature for 4 hours, for later use.

[0023] Step 2: Transfer the solution obtained in step 1 to a beaker, and slowly add 0.2 mL of triethylamine dropwise while stirring. After 10 minutes, the dropwise addition is complete. Seal the large beaker with a plastic film and place the beaker in an ultrasonic reactor at 60°C Ultrasound for 15 minutes. The product was taken out by centrifugal filtration, washed with 60 mL DMF, and dried under vacuum at 70°C for 24 hours to obtain [Cd 4 (BTC) 3 (DMF) 2 (H2O) 2 ] Porous Cd-containing organic framework materials.

[0024] The Cd-containing organic framework material ...

Embodiment 2

[0029] Example 2: Application of the method of the present invention in detecting Salmonella and other pathogenic bacteria

[0030] In order to evaluate the specificity of the method of the present invention, common food-borne pathogens in food such as Salmonella, Staphylococcus aureus, Escherichia coli, Shigella and Listeria were selected, and the detection steps described in Example 1 were followed. Measure these pathogenic bacteria, record their respective fluorescent signals and compare them, the results are as follows Figure 4 Shown. Since the target bacteria of the aptamer is Salmonella, Salmonella (5.5 × 10 3 cfu / mL) has the strongest fluorescence signal, while other pathogenic bacteria at a concentration of 10 times that of Salmonella, the fluorescence intensity is one-tenth or even lower than that of Salmonella. This result shows that the method based on metal organic framework material-nucleic acid aptamer fluorescence sensor to detect pathogenic bacteria is highl...

Embodiment 3

[0031] Example 3: The method of the present invention is applied to the detection of Salmonella in beef samples

[0032] Ground 10g of frozen fresh beef, homogenize it with 100mL Tris-HCl (pH 7.4) buffer solution for 15 minutes, filter to remove large particles and suspended matter, take the supernatant for use, and determine the supernatant according to the detection procedure described in Example 1. Salmonella in the serum. Take 5 beef samples for parallel testing.

[0033] Different concentrations of Salmonella (1.0 × 10) were injected into the supernatant of 5 beef samples. 3 ~ 5.0 × 10 3 cfu / mL), each concentration was confirmed by the plate counting method, and then the Salmonella in the sample was detected again according to the detection steps described in Example 1. The detection results are shown in Table 1. Salmonella was not detected in 5 beef samples, the relative standard deviation (RSD) of the spiked experiment was 3.6% ~ 7.5%, and the recovery rate was in the range...

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Abstract

The invention provides a method for detecting food-borne pathogenic bacteria on the basis of a metal organic framework material and aptamer fluorescence sensor. The fluorescence quenching characteristic of a metal organic framework material and the adsorbability of the framework material to aptamer are utilized, when the aptamer marked by a fluorescence probe is adsorbed to the metal organic framework material, fluorescence of the probe is quenched, a target bacterium is added into a system, the aptamer marked by the fluorescence probe leaves the metal organic framework material and is combined with the target bacterium to enhance fluorescence signals of the probe, and by means of the combination with high appetency and a high specific recognition ability of the aptamer, the method is constructed. Salmonellas are used as model analyte, the fluorescence intensity of the probe and the logarithm of target bacterium concentration form a good linear relation, the linearity range is 18-3.2*10<4> cfu / mL, the detection limit is 5 cfu / mL (S / N=3), the relative standard deviation (RSD) of standard adding experiments ranges from 3.6% to 7.5%, and the recovery rate is within the range of 90.0%-106.0%. The method for detecting the food-borne pathogenic bacteria has the advantages of being accurate, sensitive, high in specificity and the like.

Description

Technical field [0001] The invention relates to the field of food-borne pathogenic bacteria analysis and detection, in particular to a method for detecting food-borne pathogenic bacteria by using the fluorescence quenching characteristics of metal organic framework materials and nucleic acid aptamer recognition technology. Background technique [0002] Food-borne pathogens are a large group of bacteria that cause diseases in humans as carriers. They have the characteristics of diverse sources and rapid reproduction, and are the primary food safety hazards. [0003] Currently, the commonly used detection techniques for pathogenic bacteria in food include conventional culture method, immunoassay, polymerase chain reaction (PCR), gene probe method, etc. (1) The conventional culture method is based on microbial enrichment, isolation culture and biochemical identification (Crit Rev Microbiol, 2011, 37 (1): 40). It cannot detect difficult or non-cultivable pathogenic bacteria, and it...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 蒋晓华丁文捷代建国
Owner SHENZHEN POLYTECHNIC
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