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Method for enriching and capturing vibrio patahaemolyticus with high throughput

A hemolytic vibrio, high-throughput technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., to achieve the effect of short time consumption, high sensitivity and simple preparation

Inactive Publication Date: 2012-07-18
SHANGHAI OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

with the help of Systematic research on high-throughput enrichment and capture of Vibrio parahaemolyticus has not been reported

Method used

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  • Method for enriching and capturing vibrio patahaemolyticus with high throughput
  • Method for enriching and capturing vibrio patahaemolyticus with high throughput
  • Method for enriching and capturing vibrio patahaemolyticus with high throughput

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1. Preparation of polyclonal antibodies against Vibrio parahaemolyticus:

[0020] Take fresh Vibrio parahaemolyticus ATCC33846 bacterial liquid, collect the bacterial cells by centrifugation, inactivate them under ultraviolet light, immunize New Zealand white rabbits (purchased from Sipulbicam), and detect the serum by indirect ELISA until the titer is >1: 4000, the immunization was terminated, and blood was collected 10 days later. Rabbit antiserum was purified by ammonium sulfate multi-step precipitation method, and the molecular weight and purity of antiserum were detected by SDS-PAGE.

[0021] 2. Preparation of immunomagnetic beads:

[0022] The specific operation of carboxyl magnetic beads coupling Vibrio parahaemolyticus polyclonal antibody is as follows:

[0023] Take 1 ml of magnetic beads in a 2 ml centrifuge tube, and wash the magnetic beads twice with 25 mM 2-(N-morpholino)ethanesulfonic acid MES (pH 5). Then add 500 μl EDC (carbodiimide, 50 mg / ml) and 500...

Embodiment 2

[0032] Optimal selection of immunomagnetic beads for enrichment of Vibrio parahaemolyticus:

[0033] The preparation steps of carboxyl immunomagnetic beads refer to Example 1. The preparation steps of tosyl magnetic beads D-280-T, streptavidin magnetic beads D-280-S and D-100-S refer to the instructions for use (all three kinds of magnetic beads were purchased from Invitrogen, USA). use Refer to Example 1 for the systematic enrichment and capture of Vibrio parahaemolyticus and the chromogenic culture steps.

[0034]The detection results of the ability of immunomagnetic beads to capture Vibrio parahaemolyticus are shown in Table 1. When the initial bacterial liquid volume is 6.64log CFU / ml, the amount of Vibrio parahaemolyticus captured by each magnetic bead is in the range of 4-5log CFU / ml. ml level. Carboxyl magnetic beads had the best capture ability, which was 5.76log CFU / ml, so carboxyl immunomagnetic beads were chosen to enrich Vibrio parahaemolyticus.

[0035] Table...

Embodiment 3

[0049] Specific test:

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Abstract

The invention relates to a method for enriching and capturing vibrio patahaemolyticus with high throughput. The method is characterized by comprising the following steps: coupling a polyclonal antibody of the vibrio patahaemolyticus to a magnetic bead, whose surface is coated with carboxyl, by using an EDC / NHS activation method so as to obtain an immunomagnetic bead of the vibrio patahaemolyticus; suspending the immunomagnetic bead coupled with polyclonal antibody; adding the immunomagnetic bead to a reaction tube; adding a reaction solution and a sample solution to the reaction tube; adding a washing solution to an elution tube; and carrying out immune enriching on the vibrio patahaemolyticus by using a system.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a method for high-throughput enrichment and capture of Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus is an important food-borne pathogen. Once people eat aquatic products contaminated by this bacteria, it is easy to cause typical gastrointestinal symptoms such as diarrhea, intestinal cramps, nausea, vomiting, and fever. Therefore, the detection of Vibrio parahaemolyticus is an important part of the safety inspection of aquatic products. For example, in the inspection of imported and exported aquatic products, in the work of drinking water, food inspection, food poisoning source, infectious disease source investigation, etc. Bacteria are tested. The traditional detection of Vibrio parahaemolyticus needs to go through the process of bacterial enrichment, selective culture, physiological and biochemical identification, and serological reaction. T...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/63
Inventor 孙晓红苏晨曦卢瑛赵勇潘迎捷
Owner SHANGHAI OCEAN UNIV
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