Gotaq green master mix 2

Genomic DNA was extracted from the tail of a mouse using extraction buffer (25 mM NaOH, 0.2 mM disodium salt EDTA; pH 12) for 60 min, followed by incubation in neutralization buffer. PCR was performed in a 20-μl volume that included 10 μl GoTaq green master mix ( × 2) (Promega), 0.5 μM Cre primers (with positive control) and 0.5 μM flox primers (Integrated DNA Technologies). Reactions were carried out in a PCR cycler (Biometra PCR Cycler) at 95°C for 3 min, followed by 35 cycles at 95°C for 30 s, a cycle at 55°C for 1 min, and an extension step at 72°C for 5 min. The PCR products were kept at 4°C until electrophoresis in 3% agarose gel. The visualization of the PCR product was done based on its size (Cre-recombinase 100 bp, internal positive control 324 bp, flox 390 bp), and the digital images were captured in a Gel Documentation system (UVITEC Ltd.).

DNA was extracted from the cultures of the WT and ClpB mutant strains, and was also purified from mice feces using the QIAamp^R DNA Stool Mini Kit (Qiagen, Courtaboeuf, France). Bacteria were dissolved in water and boiled at 100 °C during 5 min, after 1 min of centrifugation at 11 000 r.p.m., the supernatant containing the DNA was stored at −20 °C. Using the NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), we designed the following nucleotide primers that amplify 180-base pair DNA region coding for the ClpB protein fragment containing one identified α-MSH-like epitope (Figure 1e), forward: 5′-GCAGCTCGAAGGCAAAACTA-3′ and reverse: 5′-ACCGCTTCGTTCTGACCAAT-3′ (Invitrogen Custom Primers, Cergy Pontoise, France). PCR was performed in a thermocycler with MicroAmp tubes (Eppendorf, Hambourg, Germany). The reaction was carried out in a 50-μl volume containing 25 μl of Go Taq Green Master Mix 2 × (Promega), 1 μl (20 pmol) of each primer, 21 μl of bi-distilled water and 1 μl of bacterial DNA. PCR conditions were as follows: 3 min at 94 °C followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1.5 min. PCR products were visualized on a 1% agarose gel (Sigma), with the expected size of 180 base pair and the specificity validated using ClpB mutant strain.

cDNA products deriving from extracted nucleic acid were also amplified for FCV detection using conventional RT-PCR. The specific primers (ORF1 For and ORF1 Rev) were designed for this study (Table 1). The 25-µl mixture of the RT-PCR reaction was composed of 3 µl of cDNA, 12.5 µl of GoTaq^® Green Master Mix (2×) (Promega, USA), 7.5 µl of nuclease-free water, and 1 µl each of forward and reverse primers (0.4 µM). The thermal cycling started with an initial denaturation (95 °C, 5 min), followed by 40 cycles of denaturation (95 °C, 30 s), primer annealing (59 °C, 30 s), and primer extension (72 °C, 1 min). Then, a final extension was run at 72 °C for 5 min. The positive control of FCV was the commercially synthesized string DNA fragment (Thermo Fisher Scientific^®, MA, USA) (Supplementary Table S1). The negative control of the reaction employed distilled water instead of a genomic template. The positive RT-PCR products (414 bp) were visualized using capillary electrophoresis (QIAxcel DNA Screening Kit, Qiaxcel^®, Qiagen GmbH, Germany). Then, the positive amplicons were purified using a commercial kit (NucleoSpin^® Extract II kit, Macherey Nagel, Germany) according to the manufacturer’s instructions. The purified PCR products were submitted for bidirectional Sanger sequencing (Macrogen©, Incheon, South Korea).