Taq dna polymerase mastermix

In order to identify LAB species present in pancetta and prosciutto, 16S rRNA gene amplification and sequencing were performed for 106 selected isolates using the following primers: LucF, 5′-CTTGTTACGACTTCACCC-3′ and LucR, 5′- TGCCTAATACATGCAAGT-3′ (Eurofins MWG, Ebersberg, Germany) [20 (link)]. PCR amplification of 16S rRNA genes was conducted using Taq DNA polymerase mastermix (Qiagen, Hilden, Germany) with the following PCR conditions: initial denaturation at 94 °C for 10 min, 30 cycles of 94 °C for 30 s, 40 °C for 30 s, 72 °C for 1 min and 30 s followed by a final extension at 72 °C for 10 min. PCR amplifications were performed with Applied Biosystems™ 2720 Thermal Cycler (Thermo Fisher). The amplicons were purified using the GenElute™ PCR Clean-Up Kit (Sigma Aldrich) according to the manufacturer’s instruction and subjected to Sanger sequencing (Eurofins MWG). The generated sequences were analyzed by comparative sequence analysis (BLASTN) against available sequence data on the National Center for Biotechnology Information (NCBI) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 October 2020).

The selected isolates were subjected to PCR genomic fingerprinting with the single oligonucleotide primer (GTG)₅, 5′-GTGGTGGTGGTGGTG-3′ [22 (link)]. PCR amplifications containing Taq DNA polymerase mastermix (Qiagen, Manchester, UK) were performed with Applied Biosystems™ 2720 Thermal Cycler (Thermo Fisher) with the following conditions: initial denaturation at 95 °C for 7 min; 30 cycles of 90 °C for 30 s, 40 °C for 1 min and 65 °C for 8 min; and a final extension at 65 °C for 16 min. The PCR products were applied to a 1% agarose gel containing ethidium bromide for 1 h at a constant voltage of 110 V in 1 × TAE buffer (40 mM Tris–Acetate, 1 mM EDTA, pH 8.0). The PCR profiles were visualized by UV (ultraviolet) transillumination and a digital image was captured using GeneSnap software (Syngene, MD, USA).