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Method for rapidly detecting and screening Enterobacter sakazakii

A rapid technology of Enterobacter sakazakii, applied in the field of microbial detection, can solve the problems of high false positive and false negative rates of PCR reaction, and achieve the effect of low requirements for supporting equipment, good reliability and convenient operation

Inactive Publication Date: 2010-10-20
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 rapidly detecting and screening Enterobacter sakazakii
  • Method for rapidly detecting and screening Enterobacter sakazakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of immunized superparamagnetic nano-magnetic beads and connection of antibodies

[0037] 1. Magnetic core γ-Fe 2 o 3 Synthesis and wrapping of

[0038] Separately prepare FeSO with double distilled water 4 ·7H 2 O and FeCl 3 ·6H 2 O solution and NaOH solution, mix the two iron salt solutions. Fe in the mixed solution of iron salt 2+ The concentration of ions is 0.15mol / L, Fe 3+ The concentration of NaOH solution is 0.25mol / L, and the concentration of NaOH solution is 2.5mol / L. Slowly add the above prepared NaOH solution dropwise into the mixed iron salt solution under vigorous stirring, age the resulting precipitate at 60°C for 2 hours, wash the precipitate with twice distilled water for 3 to 5 times, and filter it in Dry at 60°C to 70°C for 24 hours, grind in an agate mortar, then dry and store at room temperature for later use.

[0039] Due to the magnetic core γ-Fe 2 o 3 The dispersion of magnetic beads is not good, the particle siz...

Embodiment 2

[0044] Embodiment 2 The connection of fluorescent quantum dots and antibodies

[0045] Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC) as a crosslinking agent, N-hydroxysulfosuccinimide (NHSS) as a protective agent, the surface Quantum dots modified with carboxyl groups (QDs-COOH) cross-link with the terminal amino groups of antibodies (Ab) to form amide bonds, and then immune quantum dots are obtained. The molar ratio of antibody to quantum dot is 1:10 to 1:30.

[0046] The specific method is: 40ul carboxy-modified quantum dots (red light emitting) (5.2×10 -6 mol) was added to 80ul (0.01mol / L, pH=7.4) sterile phosphate buffer solution, 3mg of EDAC was added to the solution (final concentration was 15mg / mL), and vortexed at room temperature for 10min to fully activate the functionalization Carboxyl groups on the surface of quantum dots, then add 1mg of NHSS to the above solution, and vortex at room temperature for 30min, after fully reacting, add 80u...

Embodiment 3

[0047] Embodiment 3: the detection of sample

[0048] The immunized superparamagnetic nanoparticles prepared in Example 1 were dispersed in phosphate buffer (0.01mol / L, pH=7.2) at a concentration of 50mg / ml, and 50ul was dripped into 3ml of different concentrations (10 3 ~10 5 cfu / ml) of Enterobacter sakazakii bacterial liquid sample solution, shaker at room temperature to fully react for 0.5h, and under the effect of an external magnetic field (3000Gs), collect the immune superparamagnetic nanoparticles, and use phosphate buffer ( 0.01mol / L, pH=7.2) wash 2 to 3 times, and dilute to 0.2ml system.

[0049] Then, 50 ul of the immune fluorescent quantum dots prepared in Example 2 were added dropwise to the washed system, and shaken for 0.5 h under a shaker at room temperature, so that the antigen on the surface of Enterobacter sakazakii and the anti-sakazakii linked to the quantum dots Enterobacter sakazakii polyclonal antibody is fully immunocombined. Under the action of an ex...

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Abstract

The invention discloses a method for rapidly detecting and screening Enterobacter sakazakii, which comprises the following steps: adding immunized super-paramagnetic nanoparticles in a sample to be detected to enable the immunized super-paramagnetic nanoparticles to be specifically combined with Enterobacter sakazakii; collecting the immunized super-paramagnetic nanoparticles, and after washing the immunized super-paramagnetic nanoparticles, adding immunized luminescent quantum dots to enable the immunized luminescent quantum dots to be specifically combined with Enterobacter sakazakii carried by the immunized super-paramagnetic nanoparticles; and using an applied magnetic field to adsorb and collect Enterobacter sakazakii and immunized luminescent quantum dot combinations carried by the immunized super-paramagnetic nanoparticles, and after washing the combinations, conducting qualitative or quantitative fluorescence detection. By adopting the method, the target bacteria can be rapidly separated from all kinds of samples and can be efficiently enriched, the operation is convenient and simple, the reliability is high and the requirement on the supporting equipment is low.

Description

technical field [0001] The invention relates to the field of microorganism detection, and discloses a method for rapidly detecting and screening Enterobacter sakazakii by using immunized superparamagnetic nanoparticles and immunized fluorescent quantum dots. Background technique [0002] The rapid detection of food-borne pathogens has always been the focus of research. The "Food Safety Law of the People's Republic of China" was passed at the seventh meeting of the Standing Committee of the Eleventh National People's Congress on February 28, 2009. 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] There are mainly three kinds of detection methods widely used now: plate culture separation and counting method, polymerase chain reaction (polymerase chain reaction, PCR)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/52G01N33/533
Inventor 支援孟谨顾鸣韩奕奕沈鹤柏
Owner SHANGHAI NORMAL UNIVERSITY
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