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Immunomagnetic bead separation and detection method of escherichia albertii

A technology of immunomagnetic beads and detection method, which is applied in the biological field, can solve the problems such as the lower detection rate of Escherichia albert, and achieve the effects of reducing the interference of bacteria, simple operation, and improving sensitivity and detection rate

Pending Publication Date: 2019-04-12
自贡市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Escherichia albert and Escherichia coli belong to the genus Escherichia, they have many similarities in biochemical and strain culture characteristics. In the traditional plate culture isolation method, the interference and inhibition of E. coli make E. The detection rate of Escherichia burt was greatly reduced

Method used

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  • Immunomagnetic bead separation and detection method of escherichia albertii
  • Immunomagnetic bead separation and detection method of escherichia albertii
  • Immunomagnetic bead separation and detection method of escherichia albertii

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Preparation of Escherichia albert O1~O7 serotype-specific immune serum

[0056] According to the difference of serotype, MLST type, PFGE type and strain source, SP140089, SP150020, SP140724, D140513, T150248, T150072 and ZG141049 were selected from strains isolated from multiple sources in Zigong City, Sichuan Province from 2014 to 2015 Seven strains were used as representative strains of O1~O7 serotypes (Hong Wang, Han Zheng, Qun Li, et al. Defining the Genetic Features of O-Antigen Biosynthesis GeneCluster and Performance of an O-Antigen Serotyping Scheme for Escherichia albertii. Frontiers in Microbiology, 26 September 2017, https: / / doi.org / 10.3389 / fmicb.2017.01857).

[0057] Each strain of SP140089, SP150020, SP140724, D140513, T150248, T150072 and ZG141049 were respectively immunized with three New Zealand white rabbits (female, weighing 1.5kg-2kg), and each rabbit was immunized with 2.5×10 10 CFU / ml bacteria, immunized four times with the same dose. Th...

Embodiment 2

[0064] The preparation of embodiment 2 Escherichia albert immunomagnetic beads

[0065] The immune sera of Escherichia albert O1-O7 serotypes were purified by n-octanoic acid-saturated ammonium sulfate precipitation, and the concentration and purity of the purified antibodies were determined with a protein analyzer. The concentration range of the purified antibodies of O1-O7 serotypes was finally determined to be 5.19-13.76 mg / ml, and the purity was >95%.

[0066] Select Invitrogen's M-280 Tosylactivated magnetic beads, using purified O1-O7 serotype polyclonal antibodies to label immunomagnetic beads, the specific steps are as follows:

[0067] 1. Wash magnetic beads:

[0068] (1) Mix magnetic beads;

[0069] (2) Put 5ml of magnetic beads in a 50ml centrifuge tube on a magnetic rack, let it stand for 1min, and suck out the supernatant;

[0070] (3) Add 5ml Buffer B (H 3 BO 3 3.09g, add water to 400ml, add 2.2ml 10M NaOH to pH 9.5, dilute to 500ml, autoclave at 121°C fo...

Embodiment 3

[0082] Example 3 Verification of Sensitivity, Specificity and Enrichment Effect of Immunomagnetic Beads

[0083] 1. Activity verification of immunomagnetic beads

[0084] Take 1ml of bacterial solution of O1~O7 serotype strains 1×10 1 cfu / ml was subjected to an adsorption reaction with the immunomagnetic beads prepared in Example 2 to verify whether the magnetic beads were successfully coupled to the antibody. The specific steps were as follows:

[0085] (1) Add 20 μl of immunomagnetic beads to 1ml of bacterial solution, and mix for 10 minutes at room temperature;

[0086] (2) Place it on a magnetic stand, turn it upside down several times, let it rest for 3 minutes, and discard the supernatant;

[0087](3) Add 1ml of PBST (phosphate buffer saline containing 0.05% (V / V) Tween-20), mix well, place on a magnetic stand, turn it upside down several times, let stand for 3min, and discard the supernatant;

[0088] (4) Repeat step (3);

[0089] (5) Add 100 μl of PBST, mix well, s...

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Abstract

The invention relates to an immunomagnetic bead separation and detection method of escherichia albertii. A polyclonal antibody of O1-O7 serotype of specific binding escherichia albertii is coupled with magnetic beads, so that immunomagnetic beads of the O1-O7 serotype of the escherichia albertii can be specially adsorbed. The escherichia albertii immunomagnetic beads can specially and flexibly adsorb and separate the escherichia albertii from intestinal flora. The invention further provides a detection method of the escherichia albertii. based on immunomagnetic bead separation. The immunomagnetic beads are used for specially separating and enriching the escherichia albertii. in samples, and is combined with PCR detection of eae genes. Compared with a conventional separation and detection method, the method disclosed by the invention can notably increase the positive detection rate of the escherichia albertii, the cost of materials, time and human resources is greatly saved, and quick and accurate detection of the escherichia albertii is realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an immunomagnetic bead separation and detection method for Escherichia albert. Background technique [0002] Escherichia albertii is a new enteropathogenic bacterium discovered and named in recent years, which is related to sporadic intestinal infection and sudden food-borne diarrhea in humans. In 1991, Escherichia albert was isolated for the first time by Albert et al. In 2003, Huys et al. identified it as a new species in the genus Escherichia through phenotype and genotype analysis, and named it E.albertii (Escherichia albertii). In addition, foreign scholars have successively isolated Escherichia albert from animal hosts such as poultry, livestock, wild animals, and birds. After years of research, the inventors of the present application confirmed the existence of Escherichia albert in diarrhea patients, healthy people, wild egrets, poultry, and animal-derived food in China for...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12Q1/686C12Q1/10C12R1/19
CPCC12N1/02C12N1/20C12Q1/686C12Q2537/143C12Q2563/143C12Q2563/149
Inventor 李群王红熊衍文郑翰邹年莉闫国栋张玲赵艺曾妮陈弘汐
Owner 自贡市疾病预防控制中心