A rapid diagnostic test paper for flounder tarda Edwardsiosis and its preparation method
A slow Edwardian and rapid diagnosis technology, applied in the fields of microbiology and diagnostic reagents, immunology, can solve problems such as interference, and achieve the effect of convenient storage, high sensitivity and accurate diagnosis
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Embodiment 1
[0047] Example 1: Screening of Edwardsiella tarda specific antigens
[0048] 1. Preparation of Antisera
[0049] The pathogenic strains used in the experiment were frozen in a -80°C ultra-low temperature refrigerator and thawed on ice before use. Pick up the bacteria liquid with an inoculation loop in the ultra-clean workbench, streak the Edwardsiella tarda bacteria liquid on the common agar (LB) plate, and culture it at 30°C, and streak the Vibrio and Streptococcus on the 2216E plate and the brain respectively. Heart infusion (BHI) solid medium, cultured at 28°C. After a single colony grows on the plate, pick a single colony and inoculate them into BHI liquid medium respectively, culture Edwardsiella tarda at 30°C, Vibrio and streptococcus at 28°C overnight with shaking. The culture solution was centrifuged at 8 000 rpm for 10 min, the bacterial pellet was collected, and washed three times with sterile phosphate buffered saline (PBS, pH 7.4), 10 min each time. The concentr...
Embodiment 2
[0082] Example 2: Preparation of rapid diagnostic test paper for flounder tarda Edwardsiosis
[0083] 1. Preparation and purification of ascites fluid of mouse anti-flounder IgM monoclonal antibody
[0084] (1) Preparation of ascites: The hybridoma cell line JF-IgM-H was taken out of the -80°C ultra-low temperature refrigerator, and then immediately placed in a 37°C water bath and shaken rapidly to thaw the cells within 1 min. Thawed cells were centrifuged at 200 g for 4 min, and the supernatant was discarded under sterile conditions. Add 500 μL of GIT medium to the cryovial to resuspend the cells, transfer the cells into cell culture wells, and store in 5% CO 2 Concentrated incubator culture. Observe the growth state of the hybridoma cells under an inverted microscope. The hybridoma cells in the logarithmic growth phase are round, translucent, uniform in size, neatly arranged, and semi-densely distributed. Generally, subculture is carried out every 3-4 days, and cells in t...
Embodiment 3
[0095] Example 3: Detection method of rapid serological diagnostic test paper for flounder tarda Edwardsiosis
[0096] 1. Preparation of Assay Samples
[0097] The diseased flounder was collected, and 1 mL of blood was collected from the tail vein. After the blood sample was allowed to stand at room temperature for 1 h, the supernatant was drawn, which was the fish serum to be tested. The serum is diluted 10 times to obtain the test sample solution.
[0098] 2. Application of rapid serological test strips for Edwardsiella tarda in flounder
[0099] Lay the test paper test card of the present invention flat, add 100 μL of the treated test sample solution dropwise into the sample hole, wait for about 10 minutes, observe the test result with the naked eye, and judge the disease condition according to the color development of the test line T1 and T2.
[0100] 3. Result judgment
[0101] Ⅰ: The detection line T, T2 and the quality control line all appear red, which is a positive...
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Abstract
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