Primer and probe for detecting fish anaphylactogen in food
An allergen and fish technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as high price and limited fish species to be detected
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[0031] Example 1
[0032] Check according to the following procedure:
[0033] (1) Extraction of DNA from fish ball samples to be tested
[0034] A. Weigh 0.5g of fish balls, cut into pieces, and transfer to a 1.5mL centrifuge tube. Add 500 μL of extraction buffer lysis solution (0.05M Tris-HCl (tris-hydroxymethylaminomethane-hydrochloric acid), 0.1M EDTA (ethylenediaminetetraacetic acid), 0.2M sodium chloride, pH 7.6 sterile double distilled water) , fully oscillating;
[0035] B. Add proteinase K 60 μL, 10% SDS 2 μL, invert and mix. 57°C water bath for 10h until the raw material is digested into a supernatant, centrifuged at 4000rpm for 2min, and the supernatant is taken;
[0036] C. Add 500 μL equal volume of Tris-phenol, invert and mix for 10 min, centrifuge at 12000 rpm for 10 min, and take the supernatant;
[0037] D. Repeat the previous step;
[0038] E. Add Tris-phenol, chloroform and isoamyl alcohol (volume ratio is 25:24:1) in the supernatant, invert and mix fo...
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[0052] Example 2
[0053] Check according to the following procedure:
[0054] (1) DNA extraction of fish pine samples to be tested
[0055] A. Weigh 0.5g of fish floss, cut into pieces, and transfer to a 1.5mL centrifuge tube. Add 500 μL of extraction buffer lysis solution (0.05M Tris-HCl (tris-hydroxymethylaminomethane-hydrochloric acid), 0.1M EDTA (ethylenediaminetetraacetic acid), 0.2M sodium chloride, pH 7.6 sterile double distilled water) , fully oscillating;
[0056] B. Add proteinase K 60 μL, 10% SDS 2 μL, invert and mix. 57°C water bath for 10h until the raw material is digested into a supernatant, centrifuged at 4000rpm for 2min, and the supernatant is taken;
[0057] C. Add 500 μL equal volume of Tris-phenol, invert and mix for 10 min, centrifuge at 12000 rpm for 10 min, and take the supernatant;
[0058] D. Repeat the previous step;
[0059] E. Add Tris-phenol, chloroform and isoamyl alcohol (volume ratio is 25:24:1) in the supernatant, invert and mix for 10m...
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