Itaq supermix with rox

For each sample, 50 mg leaf tissue was ground in liquid nitrogen. Genomic DNA was then isolated using Qiagen DNeasy Plant Mini kit (Qiagen, Germany) following the manufactures protocol (Tables 1 and 2). All real-time PCR reactions were performed on the StepOnePlus real-time PCR system (Applied Biosystems, CA, USA) following the procedures described in our previously published paper [22 ]. Primers used for the detection of D. destructiva were DdITS_F1 and DdITS_R1, along with the probe DdITS_Probe1 [22 ]. The following conditions were used to carry out the real-time PCR reaction: 3 min of 95°C, followed by 45 cycles of 15 s at 95°C, and 40 s at 60°C. Each reaction consisted of 10 μl iTaq Supermix with ROX (Bio-Rad, CA, USA), 250 nM probe, 500 nM of each primer, and 4 μl template DNA for a total volume of 20 μl. A standard curve was also constructed using genomic DNA from D. destructiva isolate MD235, which is a type culture of this species [1 ]. A Ct value of less than 32 was counted as positive detection of D. destructiva. Each sample was tested in triplicate. The real-time PCR products were further subjected to purification using QIAquick PCR purification kit (Qiagen, CA), following the manufacturers protocol. The purified amplicons were sequenced by GeneWiz, Inc. (South Plainfield, NJ, USA) with primers DdITS_F1 and DdITS_R1 to confirm the identity of the amplified sequences.

To have a similar amount of lung sample tested by PCR for the presence of W. chondrophila, DNA extraction was always done on the same part of each lung, the other parts being used for histology, immunohistochemistry and cytokines measurement. Briefly, waddlial DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions, and quantified by quantitative real-time PCR assay as described previously [16 (link)]. Briefly, the reaction was performed using iTaq supermix with ROX (Bio-Rad, Reinach, Switzerland), 200 nM of forward primer (WadF4, 5′- GGCCCTTGGGTCGTAAAGTTCT-3′), 200 nM of reverse primer (WadR4, 5′CGGAGTTAGCCGGTGCTTCT-3′), and 100 nM of probe (WadS2, 5′-FAM-CATGGGAACAAGAGAAGGATG- BHQ-3′). Amplification and detection of PCR products were performed with the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland) using the following cycling conditions: 2 min at 50°C, 3 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C.

Gene expression was quantified by quantitative reverse-transcription PCR (qRT-PCR). Infected Vero cells were grown in 24-well plates at 37 °C. Infected cells were scrapped at 24, 32, 48, and 72 hours post infection, and 500 µL of cell suspensions was mixed with 1 mL of RNA Protect (Qiagen, Venlo, Netherlands), vortexed for 5 min, and then incubated for 5 min, at room temperature. The samples were then centrifuged for 5 min at 10,000× g. The supernatant was removed and the pellet was kept at –80 °C. Remaining DNA was eliminated, using the Ambion DNA-free kit™ (Life technologies). The retrotranscription was performed, using the GoScriptTM Reverse Transcription System (Promega). The qRT-PCR was performed, using iTaq supermix with ROX (BioRad, Hercules, CA). W. chondrophila primers, WadF4 and WadR4 targeting the 16S rRNA [12 (link)], and primers specific for the genes of interest (Table S1). Cycling conditions were 3 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, and 1 min at 60 °C on a StepOne Plus Realtime PCR System (Applied Biosystems, Carlsbad, CA).