Abi prism 7500 fast apparatus

In multiplex real-time PCR 1, primers specific for the p16S–23S rRNA ITS-, precA-, and pNDM-1-positive strains were combined. In multiplex real-time PCR 2, primers specific for the pOXA-23-, pOXA-40-, pOXA-51-, and pOXA-58-positive strains were combined. The SYBR-Green-I based real-time PCR assays were performed on an ABI Prism 7500 FAST apparatus (Applied Biosystems, Foster City, CA, USA) using the SYBR Premix Ex Taq™ kit (TaKaRa Bio Inc., Dalian, China), as recommended by the manufacturers. The qPCR mixture contained 1 μL of extracted DNA, 10 μL of SYBR Premix Ex Taq qPCR (2×), 0.4 μL of ROX Reference Dye II (50×), pairs of primers (optimized to final concentrations of 0.2 mM for Ab16S–23S rRNA ITS, to 0.05 mM for recA, and 0.025 mM for NDM in multiplex real-time PCR 1; the primers were optimized to 0.075 mM for OXA-23-like, 0.05 mM for OXA-40-like, 0.05 mM for OXA-51-like, and 0.025 mM for OXA-58-like in multiplex real-time PCR 2), and sterile water to a final reaction volume of 20 μL. Each run was performed with the seven positive controls and a water blank as the negative control. The optimal cycling conditions were 5 min at 95°C; 35 cycles of 20 s at 95°C, 45 s at 55°C, and 30 s at 72°C; and a melting curve step, gradually increasing from 65°C by 0.1°C /s to 95°C (with fluorescence data acquisition every 1s). All real-time reactions were performed in triplicate.

RNA derived from lung tissues was extracted with Trizol reagent (Gibco BRL, Grand Island, NY, USA). Reverse transcription was performed with 1000 ng of total RNA with SYBR Premix Ex Taq^TM (TaKaRa, Japan). Quantitative Real-time PCR (RT-qPCR) was conducted using ABI Prism 7500 FAST apparatus (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The primer sequences are listed in Table 1. The 2^-ΔΔCt method was used to quantify mRNA expression relative to β-actin.

Total RNA was extracted from lung tissues or cultured cells using the RNAiso plus (Takara, Japan). Reverse transcription reactions were performed with 500 ng of total RNA and a PrimeScript™ RT Reagent Kit (TaKaRa, Japan) according to the manufacturer's protocol. RT-qPCR was performed using the ABI Prism 7500 FAST apparatus (Applied Biosystems, Foster City, CA, USA). The primer sequences are shown in Table 1. The 2^–ΔΔCt method was used to quantify mRNA expression relative to β-actin.