High-efficiency rapid vibrio parahemolyticus enrichment and separation method
A hemolytic vibrio, fast technology, applied in the biological field, can solve the problems of separation failure, high concentration of bacteria, poor monodispersity of micron magnetic beads, etc., to increase the chance of contact, improve the separation efficiency, and shorten the separation time.
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Embodiment 1
[0030] 1. The dendritic hyperbranched polymer-antibody complex is prepared according to the following steps:
[0031] (1) Dissolve 1 mg of dendritic hyperbranched polymer in 2 mL, 0.02 M, pH 6.5 PBS, add 0.6 mg NHSS, 0.4 mg EDC, stir on a mixer at room temperature, and activate for 15 min;
[0032] (2) Take 2.68 mg of Vibrio parahaemolyticus-specific antibody and add it to the above reaction solution, place it on a mixer at room temperature and stir for 30 min;
[0033] (3) The above solution was spin-dried under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 day; after the dialysis, the obtained solution was freeze-dried.
[0034] 2. The long-chain biotin-dendritic hyperbranched polymer-antibody complex is prepared according to the following steps:
[0035] (1) Dissolve 15 mg long-chain biotin, 3.6 mg NHSS, and 2.4 mg EDC in 2 mL 0.02 M pH 6.5 PBS buffer;
[0036] (2) Add 2.24 mg of dendritic hyperbranched polymer-antibody comp...
Embodiment 2
[0040] Example 2 Enrichment effect experiment
[0041] (1) Take 1 mL of concentration as 10 4 cfu / mL Vibrio parahaemolyticus in a 1.5 mL sterile centrifuge tube, centrifuge at 12000 rpm for 5 min, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0042] (2) Enrichment and capture: Set up the technical solution group of the present invention (dendritic hyperbranched polymer group co-modified with Vibrio parahaemolyticus antibody and long-chain biotin), and nano magnetic beads modified with Vibrio parahaemolyticus-specific antibody The target bacteria were enriched by micron magnetic beads modified with Vibrio parahaemolyticus specific antibody.
[0043] (3) After magnetic separation, the supernatant was poured into a sterile centrifuge tube, and the isolated immunomagnetic beads that captured Vibrio parahaemolyticus were washed twice with PBST, mixed evenly, and washed with 1 mL sterile PBS solution Resuspend the immunomagnetic bead compl...
Embodiment 3
[0057] Example 3 Enrichment capture experiment
[0058] Conventional magnetic stand separation time is 30min, and all the other are with embodiment 2.
[0059] The catch rate of each group is as follows:
[0060] Capture rate of Vibrio parahaemolyticus-specific antibody-modified micron magnetic bead set Capture efficiency of nano-magnetic bead group modified with specific antibody of Vibrio parahaemolyticus Capture efficiency of dendritic hyperbranched polymer groups co-modified with Vibrio parahaemolyticus antibody and long-chain biotin 58.7% 39.1% 92.3%
[0061] The experimental results show that compared with the separation of 3min in Example 2, when the separation time reaches 30min, the capture efficiency of the three groups has been improved, especially the capture efficiency of the nano-magnetic bead group modified by Vibrio parahaemolyticus specific antibody is the most Obviously, this shows that the capture efficiency of the nanomagnetic b...
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