2.1. DNA Extraction and Quantification of Lactobacillus

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.