RT-qPCR analysis of efflux pump genes

Different serotypes of 10 NTS isolates were selected as representatives (AR1, AR6, AR7, AR9 and AR10) for measuring the relative expression of efflux pump genes (acrB, acrD, acrF, mdsB, mdtB, mdfA, emrB, mdtJ, macB, tolC, ompF, ompC, ompA and ompS2). Of these, five NTS isolates were incubated at 37°C and 180 rpm until the density of 1.5 × 10⁸ CFU/mL. The total RNA of five NTS isolates was extracted by Bacterial RNA Kit (Omega, Guangzhou, China) and then RNA was reverse-transcribed to cDNA using a cDNA synthesis kit (Takara). cDNA was amplified by RT-PCR using Taq SYBR Green qPCR Premix (Innovagene, Changsha, China) as described in the manufacturer’s instructions on Nucleic Acid Amplification. After a 10 min activation of the modified Taq polymerase at 95°C, 40 cycles of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C were performed. Data acquisition was done at 60°C and 72°C. The relative gene expression for pump gene transcripts was calculated against the internal control 16S rRNA gene. The relative expression of efflux pump genes in five NTS isolates was calculated against S. Typhimurium ATCC 14028 (expression = 1), which served as the control to evaluate overexpressed pump genes. The primer sequences were listed in Table S3. The 2^−ΔΔCT method was used to calculate the relative expression level of pump genes.⁴³ All reactions were performed in triplicate.