Platinum direct pcr universal master mix

DNA samples extracted using the lysis buffer provided in the Platinum Direct PCR Universal Master Mix (Invitrogen, Waltham, MA, USA) were diluted (1:10) using nuclease-free water and used for the PCR reaction. The PCR amplification potential of the DNA extracts obtained using three kits was tested using two PCR master mixes, i.e., 2 × Platinum Direct PCR Universal Master Mix (Invitrogen, Waltham, MA, USA) and Apex 2 × RED Taq Master Mix (Genesee Scientific, CA, USA). The PCR reactions were performed in a 20 µl reaction volume. A 20 µl reaction mixture consisted of 10 µl of PCR master mix, 0.40 µM of forward and reverse primers (WS-197F-FAM and WS-313R-biotin), 0.23 µM of shrimp IAC primers (IAC-Shrimp HRM-3F and IAC-Shrimp HRM-1R), 2 µl of diluted DNA and 7.37 µl of nuclease-free water to reach a final reaction volume of 20 µl. Each PCR reaction was performed in duplicate. The PCR amplification was carried out using a miniaturized Watson PCR instrument (IEH Laboratories & Consulting Group, Seattle, WA, USA). The PCR amplification profile consisted of an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 20 s, and a final extension at 72 °C for 5 min.

Stored tissue samples were defrosted at 4 °C. About 0.5–1 mm of tissue was collected using fine forceps and placed into a microcentrifuge tube. We tested the applicability of the DNeasy® Blood & Tissue kit (QIAGEN, Valencia, CA, USA), Extracta™ DNA Prep for PCR (Quanta Biosciences, Beverly, MA, USA), and lysis buffer from Platinum Direct PCR Universal Master Mix (Invitrogen, Waltham, MA, USA) for rapid isolation of shrimp tissue DNA. DNA isolation was performed following the manufacturer's recommendations. The DNA concentration was measured using the NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).