Amplification of the slpH locus

The slpH sequence from L. helveticus CNRZ32 (Table ​(Table1)1) served as reference sequence for primer design. Based on the reference sequence, the primer pair LHslpF (5′-CAAGGAGGAAAGACCACATGA-3′) and LHslpR (5′-TGTACTTGCCAGTTGCCTTG-3′) that amplifies a 1,116-bp region was designed. Primers were designed using Primer3 (version 0.4.0; Untergasser et al., 2012). PCR was carried out on a Veriti® Thermal Cycler (Thermo Fisher Scientific, Baar, Switzerland) with the following conditions: 95°C for 2 min followed by 30 cycles of 95°C for 20 s, 60°C for 10 s, and 70°C for 30 s, and a final extension of 70°C for 7 min. If not otherwise specified, each PCR (25 μL) contained 50 ng of gDNA for cheese or NWC samples or 1 ng of gDNA for pure cultures, 5 pmol of each primer, 5 nmol of each dNTP, 31.25 nmol Mg₂SO₄, 0.02 U of KOD Hot Start Polymerase (Merck), and 1X buffer for KOD Hot Start Polymerase. PCR products were analyzed using the Agilent DNA 7500 kit on an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany).