Vibrio parahemolyticus detection kit and detection method thereof
A detection kit, a technology for hemolytic Vibrio, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as pre-treatment process affecting detection efficiency, and achieve labor saving, short time consumption, and operation process. easy effect
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Embodiment 1
[0056] The specific content of the present invention is described further below:
[0057] 1. Using the polyclonal antibody of Vibrio parahaemolyticus, using the EDC / NHS activation method to couple the polyclonal antibody of Vibrio parahaemolyticus to magnetic beads coated with carboxyl groups on the surface to obtain the preparation of Vibrio parahaemolyticus Bacterial immunomagnetic beads.
[0058] The specific operation of carboxyl magnetic beads coupling Vibrio parahaemolyticus polyclonal antibody is as follows:
[0059] 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 μl NHS (N-hydroxysuccinimide, 50 mg / ml), mix well, and react at room temperature for 30 minutes. Add 200 μl of 6.39mg / ml polyclonal antibody to the activated magnetic beads, then add MES to make up 1ml, mix well, and incubate at room temperature for 3h. Add...
Embodiment 2
[0085] Embodiment 2 Sensitivity detection:
[0086] The Vibrio parahaemolyticus culture solution of 8.79log CFU / ml is carried out 10 times of gradient dilutions, gets each dilution (10 -1 -10 -6 ) each 1ml of the bacterial solution was inoculated on the surface of the boiled shrimp body. After drying, add 225ml of APW (alkaline peptone water), and beat at a low speed for 90s. Take 25ml of the homogeneous solution and add it to a 50ml centrifuge tube, centrifuge at 4000rpm for 5min, remove the supernatant, add 50ml of Tris-HCl (pH6.4), add 100μl of immunomagnetic beads, add 35ml of PBS (+0.05% Tween -20), install the reaction kit, and carry out immune enrichment. After the reaction, the result was detected by the LAMP method, the color change was observed and the result was detected by electrophoresis. The electrophoresis detection result shows that the sensitivity that LAMP can detect the artificial contamination shrimp sample is 3.79log CFU / 25g (see image 3 ). Using th...
Embodiment 3
[0089] Embodiment 3 specific detection:
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