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Nano/ALISA method and kit used for rapid detection of Salmonella

A detection method, Salmonella technology, applied in the direction of measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems of low concentration, cumbersome labeling, dependence, etc., and achieve the effects of sensitive detection, simple operation, and low cost

Inactive Publication Date: 2012-07-11
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity and specificity of ELISA technology often depend on the affinity between the antibody and the antigen, and the antibody has disadvantages such as difficult preparation, cumbersome labeling, and easy denaturation, which are the key points we need to improve; in addition, the target detected by ELISA is difficult Like nucleic acid, it is directly amplified by PCR, so the signal amplification efficiency is greatly limited, and the concentration of Salmonella in actual samples is often low

Method used

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  • Nano/ALISA method and kit used for rapid detection of Salmonella
  • Nano/ALISA method and kit used for rapid detection of Salmonella
  • Nano/ALISA method and kit used for rapid detection of Salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Aptamer modification of magnetic particles

[0045] Specific aptamers such as Salmonella typhimurium modified with 5'-terminal amination were synthesized and modified by Sangong Bioengineering (Shanghai) Co., Ltd. Use Maldi-Tof mass spectrometry for quality control. The aptamers were dissolved in sterilized water, packed in aliquots, and stored at -20°C. Before use, denature at 95°C for 5 minutes and pretreatment at 4°C for 10 minutes.

[0046] The amino magnetic particle binding kit was purchased from Bangs Laboratories, the particle diameter is about 1 ~ 1.5 μm, and the solid content is 50 mg ml -1 . According to the product instructions, take 100μl of amino magnetic particles into a 1.5ml EP tube; add 0.8ml of Pyridine Wash Buffer (PWB), mix, magnetically separate, and repeat washing 3 times; add 0.4ml of 5% glutaraldehyde to the above In the magnetic particles, mix well; mix at room temperature for 3 hours; magnetically separate, discard the supernatant, and...

Embodiment 2

[0047] Example 2 Preparation of Nano-Gold Coupling

[0048] 1. Determination of the optimal stable amount of the protein to be labeled

[0049] Prepare a series of equal-volume protein solutions (2μl) of different concentrations, add 100μl (pH adjusted to 8.0-8.5) of nano gold, and mix quickly, then add 20μl of 10% NaCl solution, shake well, and let it stand for 5 min. Each tube. According to the size of the gold nanoparticles, the absorbance value at 520nm is measured, the absorbance value is the ordinate and the protein dosage is the abscissa to draw a curve, and the protein dosage at the point where the curve first reaches the equilibrium point is taken as the optimum stable amount.

[0050] image 3 As shown in the figure, the optimal stable amounts of the secondary antibody (goat anti-mouse IgG, purchased from Fitzgerald) and horseradish peroxidase (HRP, purchased from SIGMA) in the 15nm nano-gold solution are 25μg ml respectively -1 And 35μg ml -1 .

[0051] 2. Optimization of ...

Embodiment 3

[0054] Example 3 Optimization of primary antibody concentration

[0055] 1. Take 2μl of aptamer-modified magnetic particles, magnetically separate and discard the supernatant;

[0056] 2. Add 20μl of blocking solution 1% BSA-PBS, 37℃, 650rpm, 0.5h, shake and mix;

[0057] 3. Add 200μl of bacteria solution diluted with sterilized PBS, incubate at 650rpm, 37℃ for 1h, discard the reaction solution, and wash with 200μl washing solution 3 times;

[0058] 4. Add primary antibodies of different concentrations diluted with blocking solution (0.1, 0.5, 2, 10, 50μg ml -1 ) 10μl, 650rpm, 37℃ incubate for 1h

[0059] 5. Discard the reaction solution and wash 3 times with the washing solution;

[0060] 6. Add 10 μl of 10-fold diluted gold nano conjugate and incubate at 37°C for 1 hour;

[0061] 7. Discard the reaction solution and wash 5 times with the washing solution;

[0062] 8. Add 100μl TMB, shake at room temperature for 10 minutes, read the absorbance at 595nm with a microplate reader.

[0063] Su...

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Abstract

The invention relates to a novel method used for rapid detection of Salmonella based on nano / aptamer-linked immobilized sorbent assay (Nano / ALISA) and a kit used for rapid detection of Salmonella, and the method is established by using an aptamer to substitute an antibody for capture of Salmonella and using gold nanoparticle couplet as a signal reporting group. The method comprises the following steps: (1) modifying the surface of magnetic particles with a specific aptamer of Salmonella; (2) adding a specimen for a reaction; (3) adding a specific primary antibody of Salmonella for reaction; (4) adding the couplet of secondary antibody-gold nanoparticle-reporting group for reaction; and (5) adding a specific substrate for reaction or directly recording light signals at the time. The kit comprises (1) the magnetic particles modified by the specific aptamer, (2) the specific primary antibody of Salmonella, (3) the couplet of secondary antibody-gold nanoparticle-reporting group, (4) confining liquid, (5) rinsing liquid, (6) the substrate, (7) positive control bacteria liquid and negative control bacteria liquid, etc.

Description

Technical field [0001] The invention belongs to the technical field of biological detection, and relates to the use of aptamers instead of antibodies for Salmonella capture and nano-gold conjugates as signal reporting groups, and particularly relates to a nano / aptamer combined immunoadsorption based on the application of this technology A novel method for rapid detection of Salmonella using nano / aptamer-linked immobilized sorbent assay, Nano / ALISA and a kit for rapid detection of Salmonella. Background technique [0002] Salmonella is a common zoonotic pathogen. It is widely present in the intestines of various animals and in water and soil contaminated by animal feces. It can cause enteritis, enteric fever and even sepsis. Its infection and incidence occupies the main position of bacterial food poisoning. The typhoid fever caused by it has been listed as one of the Class B infectious diseases in my country's "Infectious Disease Prevention and Control Law". It is still a serious ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/532G01N21/31G01N21/33
Inventor 吕建新吴文鹤张杰
Owner WENZHOU MEDICAL UNIV
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