Method for enriching and separating listeria monocytogenes
A technology of Listeria monocytogenes, which is applied in the field of food-borne pathogenic bacteria separation, can solve the problems of separation failure, large concentration of miscellaneous bacteria, changes, etc., to increase the chance of contact, stabilize the reaction solution, and improve separation. The effect of efficiency
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Embodiment 1
[0030] 1. The dendrimer-antibody complex is prepared according to the following steps:
[0031] (1) Dissolve 1.0 mg dendrimer in 2 mL phosphate buffer (PBS, 0.02mol / L, pH 6.5), add 0.6 mg N-hydroxysuccinimide NHSS, 0.4 mg ethyl 3-(3) -Dimethylamino) carbodiimide hydrochloride EDC, placed on a mixer at room temperature and stirred, activated for 15 min;
[0032] (2) Take 10.5 mg Lm The specific antibody is added to the above reaction solution and placed on the mixer at room temperature and stirred for 30 min;
[0033] (3) The above solution was spin-dried solvent under reduced pressure, dissolved in deionized water, and dialyzed in PBS and deionized water for 1 d; after the dialysis, the obtained solution was freeze-dried.
[0034] 2. The long-chain biotin-dendrimer-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 0.53 mg of dendrimer-antibody compl...
Embodiment 2
[0040] Example 2 Enrichment effect experiment
[0041] (1) Take 1 mL concentration as 10 4 cfu / mL Lm Centrifuge at 12000 rpm for 5 min in a 1.5 mL sterile centrifuge tube, discard the supernatant, and resuspend with an equal volume of sterile PBS solution.
[0042] (2) Enrichment and capture: Set up the technical solution groups of the present invention ( Lm Dendrimer group modified by antibody and long-chain biotin), Lm Nano magnetic beads modified by specific antibodies, Lm The micron magnetic beads modified by specific antibodies enrich the target bacteria.
[0043] (3) After magnetic separation, pour the supernatant into a sterile centrifuge tube, and the separated Lm The immunomagnetic beads were washed twice with PBST, mixed well, and resuspended the immunomagnetic bead complex with 1 mL sterile PBS solution.
[0044] (4) Calculation of capture rate: After gradient dilution of the resuspension of the target bacteria enriched in each group, count each gradient with a plate, ...
Embodiment 3
[0057] Example 3 Enrichment and capture experiment
[0058] The conventional magnetic stand separation time is 30 min, and the rest is the same as in Example 2.
[0059] The capture rate of each group is as follows:
[0060] Lm Capture rate of micron magnetic beads modified by specific antibody Lm Capture rate of specific antibody-modified nano magnetic beads Lm Capture rate of dendrimers co-modified by antibody and long-chain biotin 60.1% 40.6% 92.8%
[0061] The experimental results show that the separation of 3 minutes in Comparative Example 2 and when the separation time reaches 30 minutes, the capture efficiency of the three groups has been improved, especially Lm The capture efficiency of the nanomagnetic bead group modified by the specific antibody has the most obvious improvement, which shows that the capture efficiency of the nanomagnetic bead group can be greatly improved by prolonging the time, but it is still lower than the short time separation (3 min). Lm Captu...
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