Molecular specific marker primer for identifying indigenous crucian carps and application thereof
A technology of labeled primers and specificity, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of no relevant data, etc., and achieve the effect of high sensitivity and simple method
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Embodiment 1
[0033] (1) Extraction of the genomic DNA of the indigenous crucian carp to be tested
[0034] The tissue samples of indigenous crucian carp were taken, shredded and extracted using a genomic DNA extraction kit, and the DNA obtained was detected by electrophoresis using 2% agarose gel, and the DNA concentration was detected by an ultraviolet spectrophotometer, and stored in a -20°C refrigerator for later use.
[0035] (2) Design and synthesize specific PCR amplification primers
[0036] The specific primers based on indigenous crucian carp were designed and synthesized. The upstream primer JY-D-loop-F: 5'-GTACATATATGTATTATCACCATAT-3'; the downstream primer JY-D-loop-R: 5'- TGGGGAATAATATATTACCCGG-3'.
[0037] (3) PCR amplification of molecular-specific marker primers (JY-D-loop-F / JY-D-loop-R)
[0038] PCR reaction system 25 μL: 10×PCR Buffer 2.5 μL, dNTPS 0.5 μL, Taq (5 U / μl) 0.2 μL, MgCl 21.5 μL, JYF primer (10 μM) 1 μL, JYR primer (10 μM) 1 μL, DNA template (about 50 ng / μl)...
Embodiment 2
[0044]The method in Example 1 was used to detect three kinds of frozen fish pieces of indigenous crucian carp, silver carp, and squid. The sampler recorded and numbered the species according to the specific information, and carried out the experiment when the experimenter did not know the name of the species. The results showed that only one Specific DNA bands with a molecular weight of about 150 bp could be amplified from the species of fish nuggets, but no specific bands could be amplified from the other two fish nuggets, and the test results were completely consistent with the information recorded by the sampler. The results of gel electrophoresis were as follows figure 2 Therefore, it can be shown that the nucleic acid molecular primer of the present invention has extremely high specificity, and can be used to quickly identify indigenous crucian carp.
Embodiment 3
[0046] Two groups of muscle tissue samples were selected from fish tissue samples and preserved in 95% alcohol. The selected muscle tissues belong to two groups of different species of fish, one group is indigenous crucian carp (5 samples), and the other group is other species (5 samples), the sampler shall record and number the species according to the specific information, The experiment operator conducted the experiment without knowing the species of the two groups of samples. Experiments were carried out using the same experimental method as in Example 1, and the PCR product sequencing results showed that 5 samples of one group could amplify specific DNA bands of about 150 bp, which were tissue samples of native crucian carp, and 5 samples of another group It is a tissue sample of non-indigenous crucian carp, and the experimental results are completely consistent with the information recorded by the sampler. The results of gel electrophoresis were as follows image 3 sho...
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