A kind of test paper for the detection of pathogenic Vibrio alginolyticus
A technology of Vibrio alginolyticus and detection test paper, which is applied in the fields of microbiology and diagnostic reagents, and immunology, can solve the problems of accurate determination of test results, increase the difficulty of screening for common antigens, etc., and achieve the effect of accurate distinction.
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[0029] One, the preparation method of test paper of the present invention is as follows:
[0030] (1) Preparation of gold standard pad
[0031] ① Preparation of colloidal gold and gold-labeled rabbit antibody
[0032] Colloidal gold with a particle size of about 20nm was prepared by microwave oven-trisodium citrate reduction method, and the pH value of the colloidal gold was adjusted to 8.4 with 0.1mol / L potassium carbonate solution; rabbit anti-Vibrio alginolyticus was added to the colloidal gold in an appropriate proportion , then add BSA, after centrifugation and purification, suspend with gold-labeled preservation solution, which is the gold-labeled rabbit anti-liquid;
[0033] ② Preparation of gold standard pad
[0034] The prepared gold standard rabbit anti-liquid was sprayed on the glass fiber and freeze-dried.
[0035] (2) Design and preparation of detection layer of nitrocellulose membrane
[0036] The detection layer is to arrange multiple antigenic components of...
Embodiment 1
[0053] Embodiment 1: the preparation of Vibrio alginolyticus rapid detection test paper gold standard pad
[0054] 1. Preparation and purification of rabbit anti-Vibrio alginolyticus antibody
[0055] (1) Preparation of rabbit anti-Vibrio alginolyticus antibody
[0056] ①Expand culture Vibrio alginolyticus at 37°C for 24 hours, wash 3 times with 0.01mol / L sterile PBS, resuspend the bacteria, and adjust to 1×10 8 CFU / mL. A certain amount of formalin was added to the bacterial solution to inactivate it for 24 hours. The next day, take the bacterial liquid plate and streak it, and cultivate it for 48 hours to check the degree of inactivation.
[0057] ②The New Zealand white rabbits were immunized four times with one week interval between each time. For the first immunization, the bacterial solution was mixed with Freund's complete adjuvant at a ratio of 1:2 (v:v), and injected at 6 points on the back, 0.2 mL per point. For the second immunization, the bacterial solution was ...
Embodiment 2
[0077] Embodiment 2: Preparation of the detection layer of Vibrio alginolyticus rapid detection test paper
[0078] 1. Preparation of Vibrio alginolyticus Outer Membrane Protein
[0079] ① Cultivate Vibrio alginolyticus at 37°C, centrifuge at 8,000×g for 15 min at 4°C, wash off the culture medium, resuspend and wash with 0.01mol / L PBS for 3 times, and finally add PBS to concentrate to 1 / 25 of the original volume.
[0080] ② Add a small amount of 10% sucrose solution to the collected bacterial liquid, mix well, and treat at -20°C for 15 minutes. Add an equal volume of 0.4% Triton X-100 (PBS), mix well at room temperature, and treat for 10 min. Centrifuge at 12,000×g for 15 min at 4°C, and take the supernatant. Add 2.5 times volume of ice ethanol, mix well, and place overnight at -20°C. The next day, centrifuge at 12,000×g for 15 min at 4°C to collect the precipitate.
[0081] ③ After the precipitate was washed with Tris-NaCl (pH = 7.4), an equal volume of 0.6 mol / L KI solut...
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