Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling

A food-borne pathogenic bacteria, fluorescent labeling technology, applied in the field of nanomaterials and analytical chemistry, can solve the problems of high fluorescence background value, unstable nature, influence of detection sensitivity, etc., to improve sensitivity, accuracy and stability Effect

Active Publication Date: 2014-07-23
JIANGNAN UNIV
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  • Application Information

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Problems solved by technology

However, most fluorescent dyes are unstable and easy to be photobleached, which affects the detection sensitivity. Although quantum dots and other fluorescent nanomaterials have improved the fluorescence quenching phenomenon, their excitation light is still in the ultraviolet-visible region, so the biological samples tested It

Method used

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  • Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling
  • Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling
  • Method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0024] Example 1: Establishment of standard detection curves for Staphylococcus aureus, Vibrio parahaemolyticus and Salmonella

[0025] Take 200μL of the upconversion fluorescent nanoparticles functionalized with aptamers of three pathogenic bacteria, and incubate them with the magnetic nanoparticles modified by the corresponding complementary strands for half an hour at 37°C to form three pairs of upconversion-magnetic bead nanoparticles through base complementation. Complex. The unbound upconversion nanoparticles were washed away by magnetic field separation, and the upconversion fluorescence intensities at 477nm, 550nm and 660nm were recorded using a 980nm light source. Then add three kinds of pathogenic bacteria at different concentrations, the concentration range is from 1cfu / mL to 1×10 8 cfumL. Incubate for another half an hour at 37°C, wash off the upconverting nanoparticles after magnetic field separation, measure the fluorescence intensity of the remaining nanocomposite...

Example Embodiment

[0029] Example 2: Simultaneous detection of three pathogenic bacteria in actual milk samples

[0030] Buy 4 kinds of aseptic milk from the local supermarket and add them to the concentration range of 1×10 2 cfu / mL to 1×10 5 Three pathogenic bacteria of cfu / mL. Take 5 mL of milk separately at 7000 rpm at 10°C for 10 minutes, then remove the upper layer of milk fat, then dilute it by 20 times, filter with ultrafine glass fiber filter paper after standing still, and collect the filtrate for use. Using this discovery method to detect the concentration of three pathogenic bacteria in milk is similar to Example 1. Record the upconversion fluorescence intensity at 477nm, 550nm and 660nm obtained by excitation at 980nm, and use the linear equation of pathogenic bacteria and fluorescence intensity to obtain The concentration of the three pathogenic bacteria, the results are shown in the table below:

[0031] Table 2: Detection results of three pathogenic bacteria in actual milk samples

[0...

Example Embodiment

[0034] Example 3: Simultaneous detection of three pathogenic bacteria in actual shrimp meat samples

[0035] Buy 4 kinds of frozen shrimp meat from a local supermarket. After thawing, weigh 25g of shrimp meat and soak it in 225mL alkaline peptone containing 3% NaCl, and add them to the concentration range of 1×10. 2 cfu / mL to 1×10 5 The three kinds of pathogenic bacteria with cfu / mL are mixed evenly. Centrifuge the sample at rest for 30 minutes, filter the supernatant with ultrafine glass fiber filter paper, and collect the filtrate for use. Using this discovery method to detect the concentration of three pathogenic bacteria in shrimp meat is similar to Example 1. Recording the upconversion fluorescence intensity at 477nm, 550nm and 660nm obtained by excitation at 980nm, using the linear equation between pathogenic bacteria and fluorescence intensity, Obtain the concentration of three pathogenic bacteria, the results are shown in the following table:

[0036] Table 3: Detection re...

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Abstract

The invention provides a method used for simultaneous detection of three food-borne pathogenic bacteria based on multicolor upconversion fluorescence labeling. According to the method, three upconversion materials with differentiable fluorescence spectrums are used for forming multicolor upconversion fluorescent nanoprobes via respective connection with aptamers of staphylococcus aureus, vibrio parahaemolyticus, and salmonella, and complementary oligonucleotide single chains of the aptamers are connected with magnetic nanoparticles so as to form nano-composites. When bacteria to be tested are in a detection system, double chain unwinding is realized because of specific binding of the pathogenic bacteria with corresponding aptamers; it is possible to realize simultaneous quantitative determination of staphylococcus aureus, vibrio parahaemolyticus, and salmonella by monitoring upconversion fluorescence signal strength at 477nm, 550nm, and 660nm, detection linear range ranges from 50 to 1000000cfu/ml, and detection limits are 25cfu/ml, 10cfu/ml, and 15cfu/ml respectively. The method is used for detection of pathogenic bacteria, is high in sensitivity, is rapid and convenient, and can be used for detection of the three pathogenic bacteria in food such as milk and shrimp meat; and results are accurate and reliable.

Description

technical field [0001] A method for simultaneous detection of three food-borne pathogens based on multicolor up-conversion fluorescent labels, involving the technical fields of nanomaterials and analytical chemistry, for the detection of Staphylococcus aureus, Vibrio parahaemolyticus and Salmonella in food . Background technique [0002] Food-borne pathogenic bacteria are one of the important food hazard substances. They have strong survival and reproduction ability and are easy to spread, which greatly endanger human life, health and safety. Such as Escherichia coli, Salmonella, Staphylococcus aureus, etc. can infect the human body through food and cause diseases. More importantly, there are often a variety of foodborne pathogenic bacteria in food at the same time. Although the respective levels are not high, they can also cause foodborne diseases. Therefore, it is necessary to develop a detection method with high sensitivity and good specificity that can simultaneously d...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/533
Inventor 王周平吴世嘉段诺史召方聪聪
Owner JIANGNAN UNIV
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