Standard black sea bream DNA bar code detecting sequence and application thereof
A standard detection and barcoding technology, applied in recombinant DNA technology, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., can solve problems such as lack of data, achieve high repeatability, shorten identification time, and good stability.
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Embodiment 1
[0042] 1. Collection, storage and processing of samples to be tested
[0043] Part of the muscle tissue of the black sea bream was cut from the soaked specimen, stored in 95% alcohol and stored in a -20°C refrigerator.
[0044] 2. DNA extraction
[0045] The DNA in the samples was extracted by the phenol-chloroform method and stored at -20°C for future use.
[0046] 3. Synthesis of PCR primers
[0047] This method selects 2 pairs of universal primers:
[0048] FishF2_t1:
[0049] 5'-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3' (SEQ ID NO: 2);
[0050] FishR2_t1:
[0051] 5'-CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3' (SEQ ID NO: 3);
[0052] VF2_t1:
[0053] 5'-TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3' (SEQ ID NO: 4);
[0054] FR1d_t1:
[0055] 5'-CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3' (SEQ ID NO: 5). Wherein, the base at R is A or G, the base at Y is C or T, and the establishment of R or Y is used to amplify unknown bases.
[0056] 4. PCR reaction...
Embodiment 2
[0069] Two groups of muscle tissue samples were selected from fish tissue samples (incomplete, unable to carry out biological identification), and preserved in 95% alcohol. The selected muscle tissue belonged to two groups of different species of fish, one group was black sea bream (5 samples), and the other group was other species (5 samples), and the sampler recorded and numbered the species according to the specific information. The experiment operator conducted the experiment without knowing the species of the two groups of samples. Adopt the same experimental method as Example 1 to carry out the experiment, PCR product sequencing results show that 5 samples of one group are tissue samples of black sea bream, and 5 samples of another group are tissue samples of non-black sea bream, and the experimental results are the same as sampling The records are exactly the same.
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