Method for authenticating pork, beef, mutton and products thereof
A technology for mutton and its products, which is applied in the identification of mutton and its products, cattle, and pigs. It can solve the problems that the average efficiency cannot reach the theoretical value, and achieve the effect of simple method, high sensitivity and improved sensitivity.
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Embodiment 1
[0033] 1) Extract sample DNA: take 0.1g sample, cut the meat sample with scissors, grind it in a mortar, transfer it to a 2mL centrifuge tube, add 1mL CTAB extract (CTAB 20g / L, NaCl 1.4mol / L, Tris 0.1mol / L, Na2EDTA0.02mol / L, PH=8.0) and 20μL proteinase K (20mg / mL), fully shake the centrifuge tube, 65 ℃ water bath for 1h, shake from time to time; Centrifuge at 12000g for 15min; transfer the supernatant to a new centrifuge tube, add an equal volume of phenol and chloroform / isoamyl alcohol mixed solvent (the volume ratio of chloroform to isoamyl alcohol is 24:1), shake the centrifuge tube fully, and Centrifuge at 12,000 g for 10 min; after the mixture is separated, take the supernatant and transfer it to a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol mixed solvent (the volume ratio of chloroform to isoamyl alcohol is 24:1), shake and centrifuge After the tube, centrifuge at 12000g for 10min; take the supernatant and transfer it to a new centrifuge tube, ...
Embodiment 2
[0037] Embodiment 2PCR amplification result
[0038] Taking pork, beef, goat meat and sheep meat as samples respectively, the above-mentioned samples are detected respectively with the method of embodiment 1, and the results are as follows: figure 1 shown.
[0039] The results showed that a single DNA band could be obtained after PCR amplification of the four samples, and the DNA amplification bands of pigs, cattle, and sheep had different sizes, which were consistent with the expected fragment sizes (as shown in Table 1).
[0040] Table 1 Oligonucleotide sequences used for species-specific mitochondrial fragment amplification
[0041]
Embodiment 3
[0042] Embodiment 3 sensitivity analysis
[0043] Different dilutions of DNA from raw pork samples were used as templates (concentrations were 128ng / μL, 12.9ng / μL, 1.0ng / μL, 0.5ng / μL, 128pg / μL, 64pg / μL, 42pg / μL, 32pg / μL, 25pg / μL), carry out PCR amplification according to the method of Example 1. The result is as figure 2 shown.
[0044] The results showed that when the 25 μL reaction system contained 25 pg of raw pork genomic DNA, after 35 cycles of amplification, EB staining could be imaged under ultraviolet light after electrophoresis, and obvious target bands could be seen.
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