Quick test-paper detection method for porcine-derived material adulteration
A detection method and pig-derived technology, which can be used in measuring devices, analyzing materials, and analyzing materials through chemical reactions, etc., can solve problems such as harming national interests and consumer interests, wrong detection, missed detection, and lack of detection methods.
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Embodiment 1
[0021] Example 1 Establishment of gold nanoparticle probes and test strips combined with porcine-derived component-specific nucleic acid sequence DNA
[0022] Using 10nm gold nanoparticles with negatively charged surface, the concentration is 3nmol / L. The specific synthesis and processing steps are as follows: Measure 100mL of 0.1g / L HAuCl 4 Add the solution to the Erlenmeyer flask, stir and heat until boiling, quickly add 2mL, 10g / L trisodium citrate solution after 5 minutes, continue heating for 30 minutes, after cooling, put it into a dialysis bag with a molecular weight of 12000 Daltons, and use ultrapure water Dialysis was performed for 2 days, during which time the water was changed 3 times. Take 1mL gold nanoparticle solution to 1.5mL EP tube, which is the gold nanoparticle solution with a concentration of 3nmol / L, add A 260 =0.27, that is, 30 μL of DNA probe with a concentration of 10 μg / mL, shake and mix well, and place the EP tube in a 37° C. water bath to incubate ...
Embodiment 2
[0024] Example 2 Extraction and processing of nucleic acid fragments in samples
[0025] Take the meat containing suspected pork components, cut it into pieces, accurately weigh 0.1g and place it in a 1.5mL EP tube, add 1mL tissue lysate (10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 0.4 mg / mL proteinase K), cover the lid, boil the EP tube in boiling water for 20min, centrifuge the EP tube at 5000rpm for 10min, and absorb the supernatant. Add an equal volume of extract (phenol: chloroform: isoamyl alcohol = 25:24:1) to the supernatant, mix well, let stand for 5 minutes, centrifuge at 5000rpm for 10 minutes, absorb the supernatant of the upper aqueous phase, add etc. Volume ice absolute ethanol, shake gently, let stand for 15min, carefully absorb the white floc formed by the precipitated DNA'' with a suction tip, rinse twice with 70% ethanol, add 100μL ultrapure water to dissolve, and extract the DNA'' samples were tested by UV to confirm A 260 / A 280 The value...
Embodiment 3
[0026] Embodiment 3 test paper method detects
[0027] Pipette 30 μL of the DNA'' sample obtained from the extraction process, add it to 1 mL of the gold nanoparticle probe system, incubate at 37°C for 15 minutes, insert the end of the glass fiber membrane of the test strip into the probe system, and the probe solution will be absorbed by capillary action. , start to flow to the upper end of the test strip, if the DNA'' sample does not contain pork DNA, the gold nanoparticles modified with specific pig-derived component DNA will specifically bind to the complementary sequence DNA' on the nitrocellulose membrane, A red visible band appears; on the contrary, when the DNA'' sample contains pork DNA, the DNA will specifically bind to the specific nucleic acid sequence DNA that was originally adsorbed on the surface of the gold nanoparticles, and the gold nanoparticle probe will no longer bind to it. Complementary DNA' on the test strip is combined, and no visible bands will appear...
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