Cfx96tm real time pcr thermocycler

For all Campylobacter isolates, the presence of the following genes was individually assessed in each isolate: tet(O), tet(A), bla_OxA-61, the cluster of cmeA, cmeB, and cmeC, and the occurrence of Thr-86 to Ile mutations (C-to-T transition) in the quinolone resistance-determining region (QRDR) of the gyrA gene. The above established resistance to three different antibiotic classes: tetracycline, ampicillin, the CmeABC (efflux system) pump system, and quinolones, respectively. DNA extraction was carried out using the DNeasy^® UltraClean^® Microbial Kit (Qiagen Inc., Toronto, ON, Canada), according to the manufacturer’s instructions. The extracted DNA of each isolate was stored at −20 °C until further investigations, where it was then used as the template DNA in the PCR. The primer sequences and PCR conditions are listed in Table 2. The final amplification reaction volume was 20 µL, containing 2 µL of template DNA preparation from each Campylobacter isolate, 0.5 µL of each primer, 10 µL of (1X) QuantiNova SYBR Green PCR Master Mix (Qiagen, Hilden, Germany), and 7 µL of RNase-free water. The final reaction mixture volume was adjusted to 20 μL. Amplification was performed in a CFX96TM Real-Time PCR thermocycler (BioRad, CA, USA) with reaction conditions as suggested by Poudel et al. [36 (link)] and Hungaro et al. [35 (link)]. Simplex PCR assays for each gene were performed.

The detection and quantification of NoV GI and NoV GII genomes were performed by real-time quantitative reverse transcription PCR (RT-qPCR) according to ISO 15216-1:2017, and the reactions were carried out using the Biorad CFX96TM Real-Time PCR thermocycler (Biorad, Hercules, CA, USA). The number of RNA copies per μL of each sample was calculated by matching the sample Cq value to the standard curves (one for each target) created with the tenfold serial dilution of a dsDNA standard for NoV GI and NoV GII. Therefore, the final concentrations were expressed as genomic copies per gram (g.c./g) and were calculated based on the volume of the analyzed extract. According to ISO 15216-1:2017, the Cq value of Mengovirus was obtained in spiked samples and was compared with extracted samples by viral stock to evaluate extraction efficiency. Furthermore, to evaluate the inhibition of RT-qPCR, the Cq value was obtained in samples spiked with 1 µL of external control RNA for both GI and GII and was compared with that obtained in samples without the addition of external control. Results with extraction efficiency > 1% and RT-qPCR inhibition ≤ 75% were considered valid. The LOQ was established by the European Union Reference Laboratory (EURL). The LOQ for NoV GI was calculated as 140 g.c./g and 130 g.c./g for NoV GII.