Faststart universal probe master kit

The qPCR assay was performed under standard conditions, as indicated by the manufacturer, in a ViiA 7 Dx Real-Time PCR System (ABI Foster City, CA, USA). They were amplified in a 20-µL reaction volume using a FastStart Universal Probe Master Kit (04914058001, Roche, Penzberg, Germany) containing 2 µL of DNA, 10 µL of master mix, 400 nM of each primer (M-F and M-R), and 400 nM of probe (M-Probe) (Table 1). The following PCR program was used for amplification: 2 min at 50 °C, then 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were tested three times in separate runs and assayed in triplicate for each run, as were the standard, and positive and negative controls. In addition, the specificity of the qPCR assay was verified in vitro using our local collection of 51 strains including 13 colistin-resistance isolates carrying the mcr-1 gene (Table 2). The number of GC was defined as the average of the three independent runs data obtained.

Conventional PCR was carried out in a 25 μL mixture containing 1 μL DNA, 2.5 μL of 10×EX Taq Buffer, 0.2 mmoL/L of dNTPs, 0.4 μmoL/L of each of the forward and reverse primers, and 1.25 U of EX Taq, with sterile water added to create a final volume of 25 μL. PCR amplification with SCBV-F/SCBV-R primers was performed by following a thermal cycling program of 94°C for 5 min, 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 10 min. Conventional PCR amplification with SCBIMV-qPCR or SCBMOV-qPCR primers was performed following a thermal cycling program of 94°C for 2 min, 35 cycles of 94°C for 30 s, 54°C (IM-QF2/IM-QR2) or 60°C (MOR-QF2/MOR-QR2) for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The FastStart Universal Probe Master Kit (Roche Applied Science, Mannheim, Germany) was used for the fluorescence qPCR assay. qPCR was carried out in a 25 μL mixture containing 1 μL DNA (100 ng/μL), 12.5 μL TaqMan Fast Universal PCR Master Mix, 2.25 μL (10 pmol) of each of forward and reverse primers, and 1 μL (10 pmol) TaqMan probe, with sterile water added to create a final volume of 25 μL. The qPCR optimum cycling conditions consisted of 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min.

Total RNA was extracted from FTECs using the RNeasy Mini Kit (#74104, Qiagen, Germantown, MD, USA). Double-stranded cDNA was synthesized using a reverse transcription kit (#4368813, ThermoFisher Scientific). Real-time quantitative polymerase chain reaction (RT-PCR) was performed by the FastStart Universal Probe Master kit (#04913957001, Roche) with Roche Universal Probe #2 and Probe #30 (#04684982001 and #04687639001, Roche) using a StepOne PCR system (Applied Biosystems, Foster, CA, USA). Relative RNA quantitation was performed using ∆∆CT calculations. Primer sequences are listed in Supplementary Table S2.