Sea cucumber peptide identification and detection kit and detection method thereof
A technology for detection kits and detection methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problems of low molecular weight of peptides and the inability to determine the source of peptides, etc., and achieve simple and fast operation, simple identification and detection. Effect
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Embodiment 1
[0033] A sea cucumber peptide identification and detection kit, comprising the following components: a mixture of phenol and chloroform; absolute ethanol; specific primers; a PCR reaction mixture; trichloroacetic acid; a mixture of copper sulfate and potassium sulfate;
[0034] The sequence of the specific primer is:
[0035] Upstream primer: 5'- ACTAATACCTCCCCTCCTT -3'
[0036] Downstream primer: 5'-ATAAACTTCTGGGTGTCC-3'.
[0037] The volume ratio of phenol to chloroform in the phenol and chloroform mixture is 1:1.
[0038] The trichloroacetic acid is 9%-15% trichloroacetic acid.
[0039] The mass ratio of copper sulfate and potassium sulfate in the mixture of copper sulfate and potassium sulfate is 1:30.
[0040] The PCR reaction mixture is: containing Mg 2+ Mix 10×PCR buffer, dNTP, and 5U / μL TaqE at a volume ratio of 5:4:0.5.
[0041] The kit is used for: identifying sea cucumber peptides by PCR method and identifying and distinguishing sea cucumber peptides from other...
Embodiment 2
[0043] A detection method utilizing the sea cucumber peptide identification and detection kit described in Example 1: specifically comprising the following steps:
[0044] (1) Identification of sea cucumber peptides
[0045] a. Extraction of DNA from sea cucumber peptide samples:
[0046] Take 0.1g of sea cucumber peptide sample in a 1.5mL EP tube, add 600μL of phenol and chloroform mixture, gently invert for 10min, 12000rpm, centrifuge for 10min; take 500μL of supernatant, repeat the previous step twice, take supernatant 400μL, add 800μL absolute ethanol and shake well to precipitate DNA; 12000rpm, centrifuge for 10min;
[0047] b. PCR amplification of the COI gene fragment in sea cucumber peptide sample DNA:
[0048] Use the DNA in the extracted sea cucumber peptide sample as a template to amplify the COⅠ fragment by PCR. The 50 μL PCR reaction system is: 2 μL of DNA template, 2 μL of upstream and downstream primers, 9.5 μL of PCR reaction mixture, and 34.5 μL of water; ...
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