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Total DNA was extracted from the swabs sampled by the nurse from SSc patients and HS by QIAampDNAStool Mini Kit instructions (Qiagen, S.r.l. Italy) and quantified with a Qubit® 2.0 fluorometer (Invitrogen, Thermofisher Scientific, Milano, Italy); the qualityof DNA was checked on 1.5% agarose gel. Quantitative PCR (qPCR) was conducted using the specific primers rpoB1, rpoB1o and rpoB2 for the RNA-polymerase β subunit encoding gene, a valuable alternative as present in a single copy per genome, whichgenerate amplicons of 250-bp, on 40 ng DNA for all the samples; negative control contained only ddH2O. Reactions were performed in a CFX Connect 96 apparatus (BioRad, Hercules, CA, USA) and the results were analyzed by the manufacturer’s software. Amplification was carried out in a 25 μL final volume containing: 7.5 μM of each primer, 1X iTaq™ Universal SYBR®GreenSupermix, sterile ddH2O to reach the appropriate volume. Amplification was performed in 96-well microtiter plates (BioRad). The program cycle was: 95 °C 3 min followed by 35 cycles of 95 °C 1 min, 45 °C 1 min and 72 °C 1 min. After that, a melting curve program was run for which measurements were made at 0.5 °C temperature increments every 10 s within a range of 60–100 °C. A rpoB amplified and purified fragment (froma mix of LABs of a commercial probiotic formulation—Lactoflorene® Plus, Montefarmaco OTC, Milano, Italy) was used as standard [34]. The standard curve was developed by plotting the logarithm of known concentrations (tenfold dilution series in triplicate from 1 × 10−1 to 1 × 10−6 in 25 μL reaction) of the rpoB fragment against the threshold cycle (Ct) values. The qPCR standard curve had an R2 of 0.99–0.97 and an efficiency >85%. Three replicates were carried out for each sample. rpoBsequences were expressed in ng DNA ng−1 of template DNA.

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