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
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[0018] Example 1
[0019] 1. Preparation of polyclonal antibodies against Vibrio parahaemolyticus:
[0020] Take the fresh bacterial liquid of Vibrio parahaemolyticus ATCC33846, collect the bacterial cells by centrifugation, and after inactivation treatment under ultraviolet light, immunize New Zealand white rabbits (purchased from Sipuer Bica), and detect the serum by indirect ELISA, until the titer is> 1: 4000, stop immunization, and take blood after 10 days. The rabbit antiserum was purified by ammonium sulfate multi-step precipitation method, and the molecular weight and purity of the antiserum were detected by SDS-PAGE.
[0021] 2. Preparation of immunomagnetic beads:
[0022] The specific operation of carboxyl magnetic beads coupled with polyclonal antibody of Vibrio parahaemolyticus is as follows:
[0023] Take 1ml magnetic beads in a 2ml centrifuge tube, and wash the magnetic beads twice with 25mM 2-(N-morpholino)ethanesulfonic acid MES (pH5). Then add 500 μl EDC (carbodiimid...
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[0031] Example 2
[0032] Optimal selection of immunomagnetic beads for enrichment of Vibrio parahaemolyticus:
[0033] Refer to Example 1 for the preparation steps of carboxyl immunomagnetic beads. 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 (the three magnetic beads are purchased from Invitrogen, USA). use Refer to Example 1 for the system's enrichment and capture of Vibrio parahaemolyticus and its color development and culture steps.
[0034] The detection results of the immunomagnetic beads' ability to capture Vibrio parahaemolyticus are shown in Table 1. When the initial bacterial volume is 6.64 log CFU / ml, the amount of Vibrio parahaemolyticus captured by each magnetic bead is 4-5 log CFU / ml level. The carboxyl magnetic beads have the best capture ability, which is 5.76log CFU / ml, so the carboxyl immunomagnetic beads are used to enrich Vibrio parahaemolyticus.
[0035] Table 1: Dete...
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[0048] Example 3
[0049] Specificity test:
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