Primestar premix dna polymerase

The EHP-PTP2 gene (GenBank No. MT249228), SSU rRNA gene (GenBank No. FJ496359.1) and β-Tubulin gene (GenBank No. KY593130) of EHP were amplified via PCR to confirm the EHP-infected shrimp sample. All PCR primers were designed using Primer Premier 5.0 and are listed in Table 1. The amplification system was 25 µL PrimeSTAR premix DNA polymerase (2×, TaKaRa, Dalian, China), 0.4 µM primers, 1 µL genomic DNA extraction, and water up to 50 µL. The amplification reaction was performed according to the following procedure: 98 °C for 5 min, 35 cycles (98 °C for 30 s, 56 °C for 30 s, 72 °C for 10 s), 72 °C for 10 min.

Insect eggs were collected for extracting genomic DNA (gDNA) using the E.Z.N.A. Tissue DNA Kit according to the manufacturer’s instructions. PCR amplification with the primers targeted the large subunit ribosomal DNA (LSU, Forward primer: 5′-GGGGAAAGAAGACCCTGT-3′, Reverse primer: 5′-TCTGTCACCTCCAATCAA-3′) of NbCQ1. The amplification system was composed of 12.5 µL PrimeSTAR premix DNA polymerase (TaKaRa, R045Q), 0.4 µM primers, 1 µL genomic DNA extraction, and water up to 25 µL. Amplification was performed as the following procedures: pre-denaturation at 98 °C for 2 min, denaturation at 98 °C for 15 s, annealing at 62 °C for 15 s, extension for 35 cycles at 72 °C each for 10 s, and final extension at 72 °C for 10 min. An aliquot of 10μL from each PCR product was run on a 1.5% agarose gel to visualize the PCR product.