Method for rapid detection of enterobacter sakazakii

An Enterobacter sakazakii, fast technology, applied in measuring devices, instruments, analysis by nuclear magnetic resonance, etc., can solve the problems of high false positive and false negative rates in PCR reactions, and achieve good reliability, simple processing, and low requirements. Effect

Inactive Publication Date: 2012-01-18
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the PCR reaction presents a high false positive and false negative rate

Method used

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  • Method for rapid detection of enterobacter sakazakii
  • Method for rapid detection of enterobacter sakazakii
  • Method for rapid detection of enterobacter sakazakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of Immunized (Biologically Functionalized) Superparamagnetic Nano Magnetic Beads

[0031] 1. Ferric oxide (Fe 3 o 4 ) synthesis and encapsulation and amino functionalization

[0032] ①, 20mM (3.25g) of ferric chloride, 120mM (12.3mL) of acetylacetone, and 20mL of distilled water were mixed and stirred for 15 minutes, then 6mL of triethylamine was added and filtered to obtain a red powder, which was dissolved in 30mL of absolute ethanol and distilled water ( V:V 7:3), heated to 80°C, cooled to room temperature and recrystallized to obtain iron acetylacetonate.

[0033] Add 1.06g of ferric acetylacetonate into the system of 15mL of phenyl ether and 15mL of oleylamine, stir it with a magnetic force, and continuously raise the temperature to 115°C (the temperature range can be 110-120°C), keep it for 30 minutes, vacuumize, and continue to heat up to 180°C (Temperature range can be at 175~185°C), keep for 30 minutes, continue to heat up to 300°C (tem...

Embodiment 2

[0038] The immunized superparamagnetic nano-magnetic beads prepared in Example 1 were dispersed in phosphate buffer (0.01 mol / L, pH=7.2) at a concentration of 0.8 mg / mL (experimentally optimized data).

[0039] Add 225uL of the above-mentioned magnetic beads to 75uL of E. sakazakii containing different concentrations (1cfu / ml, 10cfu / ml, 25cfu / ml, 50cfu / ml, 10 2 cfu / ml, 10 3 cfu / ml, 10 4 cfu / ml) dairy product samples, add 2.7 mL of stabilizer 2% milk, and incubate at 37° C. for 45 min to 60 min (data optimized in the experiment). A non-bacterial dairy product sample was used as a control.

[0040] Add the processed sample into the NMR tube with a diameter of 3cm, put the NMR tube into the NMR analyzer, and adjust the parameters, D1(us): 100; D2(us): 200; D0(ms): 100; TD : 20000; SW (kHz): 75.0; DFW (kHz): 30.0; RG: 2; Ns: 8; C1: 2000; (T 2 ), calculate ΔT 2 value. The result is as figure 1 shown.

Embodiment 3

[0042] The immunized superparamagnetic nano-magnetic beads prepared in Example 1 were dispersed in phosphate buffer (0.01 mol / L, pH=7.2) at a concentration of 0.8 mg / mL (experimentally optimized data).

[0043] Take 225uL of the above-mentioned magnetic beads and add them to 75uL of dairy product samples that have been incubated for 2h (Enterobacter sakazakii concentration 10 2 cfu / ml), add 2.7 mL of stabilizer 2% milk, and incubate at 37° C. for 45 min to 60 min (data optimized in the experiment). A non-bacterial dairy product sample was used as a control.

[0044] Add the processed sample into the NMR tube with a diameter of 3cm, put the NMR tube into the NMR analyzer, and adjust the parameters, D1(us): 100; D2(us): 200; D0(ms): 100; TD : 20000; SW (kHz): 75.0; DFW (kHz): 30.0; RG: 2; Ns: 8; C1: 2000; (T 2 ), calculate ΔT 2 value.

[0045] And use the same conditions to detect the same concentration (10 2 cfu / ml) of Escherichia coli O157, Shigella, Salmonella, Vibrio p...

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Abstract

The invention relates to the field of microbial detection and discloses a method for rapid detection of enterobacter sakazakii. The method comprises the steps of: adding immunized superparamagnetism nanometer magnetic beads into a sample, adding a stabilizer, and incubating for 45-60min at 30-40 DEG C, wherein the magnetic beads are specifically bonded and enriched on microbial food-borne pathogenic bacteria, namely the enterobacter sakazakii, in the sample; and measuring spinning-spinning relaxation time and calculating a value deltaT2 by taking a bacteria-free sample as a blank control. The method disclosed by the invention has high specificity and sensitivity to the detection of the enterobacter sakazakii, is rapid, and is particularly suitable to detection of low-concentration enterobacter sakazakii, especially the enterobacter sakazakii in dairy products; in addition, the method is convenient and simple to operate and has good reliability and low requirement on assorted equipment.

Description

technical field [0001] The invention relates to the field of microorganism detection, in particular to the field of food-borne microorganism detection, and discloses a method for detecting biologically functionalized superparamagnetic nanoparticles immune recognition target microorganisms by means of nuclear magnetic resonance to realize highly sensitive detection of Enterobacter sakazakii. Background technique [0002] Many foodborne diseases are mostly caused by foodborne pathogens. Rapid detection of foodborne pathogens has always been the focus of research. Rapid detection of foodborne pathogenic bacteria is to use microbiological, chemical, biochemical, biophysical and immunological methods to isolate, detect, identify and count pathogenic bacteria in food and its processing, storage and other environments. [0003] Enterobacter sakazakii is a newly discovered pathogen in dairy products, which has been identified as an important conditional pathogen causing infant mort...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531G01N24/08
Inventor 赵渝陈艳黄慧心郭鲁申
Owner SHANGHAI NORMAL UNIVERSITY
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