Express qpcr supermix

PCR reactions were prepared using 96-wells real-time PCR plates (Eppendorf Hamburg, Germany) and a Mastercycler ep gradient S (Eppendorf Hamburg, Germany). 12.5 μL of Express qPCR Supermix with premixed ROX (Invitrogen, France), 5 μL of DNA extracts with three appropriate dilutions, 200 nM of the forward and reverse primers [23 (link)], 50 nM of the TaqMan probe, and water were added to obtain a final volume of 25 μL for all analyses.
An initial incubation of 20 s at 95°C and 40 cycles of denaturation (95°C, 15 s; 60°C, 1 min) were performed. One standard curve was generated at each assay, using 10-fold dilutions in sterilized water (Aguettant Laboratory, Lyon, France) of PCR products from known environmental clones. Clones DF10 and LC103 [29 (link)] were used as standards for Archaea and Bacteria, respectively. One measurement at two or three dilutions was obtained per sample for each primer set.
The amplification efficiency in 16S rRNA standard curves for Bacteria and Archaea ranged from 86 to 96%. The detection limit was 1.6 × 10⁵ and 8.4 × 10⁴ copies per ml of sludge in average for Bacteria and Archaea respectively.

Viremia in experimentally inoculated animals was quantified at different days post-infection using quantitative PCR (qPCR). Genomic DNA was extracted from 200 μL of EDTA anti-coagulated blood using a Zymo Quick-DNA miniprep DNA extraction kit (Ref: D3025, Zymo research, Irvine, CA, USA). For the detection of ASFV genome copies in tissues, approximately 0.01 g to 0.025 g of splenic, submandibular, and gastrohepatic lymph tissue was weighed and DNA extracted using the Qiagen DNeasy Blood and Tissue Kits (Cat# 69506, Qiagen, Hilden, Germany). qPCR was performed using QuantStudio^TM 5 system (Applied Biosystems, Waltham, MA, United States). Each reaction was conducted in duplicates in a 10 μL reaction mixture containing 2.23 μL nuclease-free water, 5 μL EXPRESS qPCR Supermix (Invitrogen, Waltham, MA, USA), 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 0.15 μL of TaqMan^® probe (10 μM), 0.02 μL of ROX reference dye, and 2 μL template DNA. The plasmid standard dilutions, primers, and qPCR conditions are described by Abkallo et al. [19 (link)]. Data, in eds file format, were exported and analysed on a QuantStudio™ design and analysis software (Applied Biosystems, United States). Results were analysed on GraphPad Prism (version 6) for Windows (GraphPad Software, San Diego, CA, USA).