Kapa sybr fast qpcr master mix universal

A quantitative PCR (q-PCR) assay was done using species-specific rpoD primers for V. parahaemolyticus detection and tdh, trh1, and trh2 primers for V. parahaemolyticus pathogenic strain detection (Table S4) (Nemoto et al., 2009 (link); Messelhäusser et al., 2010 (link); Yamazaki et al., 2010 (link); Nemoto et al., 2011 (link)). A 20-µL reaction mixture consisted of 10 µL of 2× KAPA SYBR FAST qPCR Master Mix Universal (Sigma-Aldrich, St.Louis, MO, USA), 10 mM each forward primer and backward primer, and 1 µL aliquot of 20 ng/µL of a gDNA template. A final volume was adjusted using UltraPure™ DW (Invitrogen, Grand Island, Germany). The qPCR amplification was carried out in a Rotor-Gene Q R10116106 (Qiagen Hilden, Germany). A positive control, a negative control, and a standard curve were included in each run.

Total RNA was isolated either with RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) or with TRIzol through standard procedures, followed by reverse transcription via QuantiTect Reverse Transcription kit (Qiagen, Venlo, The Netherlands). RT-PCR was performed with Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA). Quantitative PCR (qPCR) was performed using KAPA SYBR FAST qPCR Master Mix Universal (Sigma-Aldrich, St. Louis, MO, USA) in a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). Data were evaluated with the 2^−ΔΔCT method. GAPDH was used as reference gene. (Primers used for RT and qPCR were listed in Supplementary Material Table S1).
The heatmap of gene expression and PCA analysis results were generated with web tool ClustVis.

Total RNA was isolated with TRIzol (Thermo Fisher Scientific) following the manufacturer’s instructions. cDNA was synthesized from 500 ng RNA using a QuantiTect Reverse Transcription kit (QIAGEN). qPCR was performed using KAPA SYBR FAST qPCR Master Mix Universal (Sigma Aldrich) and measured using BioRad CFX (BioRad). Data were evaluated with the 2-ΔΔCT method⁵⁹ (link) with GAPDH as reference gene. Primers for qPCR are indicated in Table S6.