A nested primer and its application for early warning of Enterococcus hepatica infection in cultured prawns in my country
A technology for Enterobacter hepatis and early warning, applied in DNA/RNA fragments, recombinant DNA technology and other directions, can solve the problems of lack of molecular detection technology, high instrument requirements, and increased detection cost, and achieves good application prospects, good specificity, Sensitive effect
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[0020] EMISF / EMISR primers and nested primers (EMISF / EMISR and EMISnF / EMISnR) were used to amplify DNA templates of hepatopancreas from 4 shrimp ponds for PCR amplification.
[0021] External primer (EMISF / EMISR) amplification system and conditions: the amplification system is 25 μL in total, consisting of 1 μL tissue DNA template, 0.5 μL forward external primer EMISF (10 μM), 0.5 μL reverse external primer EMISR (10 μM), 12.5 μL GreenMaster Mix (promega) and 10.5 μL DNase-free water. Amplification conditions were as follows: pre-denaturation at 95°C for 4 min, followed by 25 cycles of 95°C for 40 s, 52°C for 30 s, and 72°C for 35 s, and finally extension at 72°C for 5 min. A portion of the PCR product was used as a template for internal primer amplification.
[0022] Internal primer (EMISnF / EMISnR) amplification system and conditions: Amplification system total 25 μL, PCR product amplified by 1 μL external primer, 0.4 μL forward internal primer EMISnF (10 μM), 0.4 μL revers...
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