Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead

A technology of immune magnetic beads and antibodies, which is applied in the field of microbial detection, can solve the problems of low sensitivity, complex detection methods, and inability to achieve detection in complex environments, and achieve the effects of improved sensitivity, convenient detection process, fast, accurate and efficient detection

Inactive Publication Date: 2016-08-17
SHANTOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the sensitivity of the current vibriosis detection method is not high, the detection method is complicated, and only one type of Vibrio can be detected at a time, which cannot be detected in a complex environment

Method used

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  • Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead
  • Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead
  • Immunomagnetic bead containing OmpU antibody, preparation of immunomagnetic bead and method for capturing pathogenic vibrios and detecting pathogenic vibrios through multiplex PCR by utilizing immunomagentic bead

Examples

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Effect test

Embodiment 1

[0058] Take the detection of Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, and Vibrio mimicus as examples.

[0059] 1. Preparation of immunomagnetic beads containing OmpU antibody, such as figure 1 As shown, it mainly includes the following steps:

[0060] 1) Preparation of immune antigen: using the genomes of Vibrio parahaemolyticus ATCC17802, Vibrio vulnificus ATCC27562, Vibrio cholerae Vb0 and Vibrio mimicus ATCC33653 as templates to amplify the OmpU gene by PCR, and the PCR product was connected to pET-32a to construct a heterologous expression vector, Transformed into E.coli BL21; Inoculate OmpU recombinant bacteria into LB medium (containing Amp 100 μg / mL), culture with shaking at 37°C until logarithmic growth phase, and add IPTG with a final concentration of 1 mmol / L to induce for 6 hours. The bacteria were collected by centrifugation, and the recombinant protein was purified by affinity chromatography (Nickel Sepharose FF) to obtain a relatively pure Om...

Embodiment 2

[0077] Take the detection of Vibrio parahaemolyticus, Vibrio cholerae, Vibrio alginolyticus, Vibrio vulnificus and Vibrio mimicus as an example.

[0078] 1. Preparation of immunomagnetic beads containing OmpU antibody, such as figure 1 As shown, it mainly includes the following steps:

[0079] 1) Antigen preparation: such as Figure 4 , Figure 5 , Image 6 , Figure 7 with Figure 8 As shown, the intraspecies and interspecies conservation and cross-immunity characteristics of Vibrio OmpU protein were analyzed by bioinformatics analysis, and the results indicated that OmpU protein is a common antigen widely existing in Vibrio genus, and it is also a good Immunomagnetic beads capture the target. Using the online signal peptide analysis software Signal 3.0, the signal peptide was predicted for the amino acid sequence of the ompU gene, and PCR expression primers were designed to remove the nucleotide sequence encoding the signal peptide. The primers are shown in Table 3:

...

Embodiment 3

[0101] Take the detection of any one of the five pathogenic Vibrios of Vibrio parahaemolyticus, Vibrio cholerae, Vibrio alginolyticus, Vibrio vulnificus and Vibrio mimicus as an example.

[0102] 1. Preparation of immunomagnetic beads containing OmpU antibody, such as figure 1 As shown, it mainly includes the following steps:

[0103] 1) Antigen preparation: PCR amplification is carried out with the genome of one of Vibrio parahaemolyticus ATCC17802, Vibrio alginolyticus ATCC33787, Vibrio vulnificus ATCC27562, Vibrio cholerae Vb0 and Vibrio mimicus ATCC33653 as a template to be detected, The PCR product was connected to pET-32a to construct a heterologous expression vector, transformed into E.coli BL21(DE3), and each clone was sequenced at least twice. The OmpU recombinant bacteria were inoculated into LB medium (containing Amp 100 μg / mL), cultured with shaking at 37°C to the logarithmic growth phase, and induced by adding IPTG with a final concentration of 1 mmol / L for 6 h. ...

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Abstract

The invention relates to an immunomagnetic bead containing an OmpU antibody, preparation of the immunomagnetic bead and a method for capturing pathogenic vibrios and detecting the pathogenic vibrios through multiplex PCR by utilizing the immunomagnetic bead. PCR amplification of an OmpU gene is conducted by taking genomes of the vibrios as templates, pure OmpU recombinant protein is obtained to serve as an antigen to prepare the antibody, and the immunomagnetic bead containing the OmpU antibody is prepared through the coupling-washing process; the immunomagnetic bead can capture multiple vibrios, and then the vibrios are identified by multiplex PCR specific primers which are designed on the basis of a danJ gene. The immuomagnetic bead can simultaneously detect the vibrio parahaemolyticus, the vibrio cholera, the vibrio alginolyticus, the vibrio vulnificus and the vibrio mimicus at most and can be widely applied to environment and import and export food inspection. The immuomagnetic bead combines the specificity of immune separation and the high efficiency of PCR amplification, has the capacities of separation, concentration and enrichment, can efficiently separate and enrich the low-concentration pathogenic bacteria in a sample, reduces or eliminates inhibition and influences of the sample to PCR, improves detection limitation and has the very good application prospect.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and in particular relates to an immunomagnetic bead containing OmpU antibody and its preparation and capture-multiple PCR detection method for pathogenic Vibrio. Background technique [0002] Vibriosis can cause great harm to humans and the aquaculture industry and spread widely. With the continuous expansion of aquaculture scale in our country, the increase of aquaculture density, and the deterioration of the marine aquaculture environment, once various pathogenic bacteria, mainly Vibrio, break out in marine aquaculture animals, it will be difficult to control, and the aquaculture industry will suffer a lot. Bring huge economic losses, so it is necessary to monitor Vibrio diseases in the breeding environment during the breeding process. Once the precursors of bacterial diseases are found, they should be diagnosed in time, and corresponding prevention and control measures should be taken to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/569G01N33/531C12Q1/68
CPCC07K16/1239C12Q1/686C12Q1/689G01N33/531G01N33/56911Y02A50/30
Inventor 胡忠伦镜盛刘丹张设熙董亚萍袁传飞
Owner SHANTOU UNIV
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