Pgem t cloning vector system kit

DNA was extracted from pelleted samples following the noncommercial IHMS Protocol Q recommended by the International Human Microbiome Consortium (Costea et al., 2017) (link). Quantitative microbiological analysis of DNA samples was carried out by qPCR using SYBR® green methodology in a ViiA7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Standards, primers, amplicon size, and annealing temperature for Akkermansia, Bacteroides, Bifidobacterium, Blautia coccoides-Eubacterium rectale Cluster XIVa, Clostridium leptum subgroup specific cluster IV, Enterobacteriaceae, Enterococcus, Faecalibacterium, Lactobacillus, Prevotella, Roseburia, and Ruminococcus Cluster IV have been described previously (Lozano-Ojalvo et al., 2019) (link). Additional primers and qPCR conditions were selected for the analysis of Alistipes (Roager, Licht, Poulsen, Larsen, & Bahl, 2014) (link), Atopobium (Matsuki, Watanabe, Fujimoto, Takada, & Tanaka, 2004) (link), Bilophila (Baldwin et al., 2016) (link) and sulphate-reducing bacteria (SRB) based on the dissimilatory sulphite reductase (dsr) gene (Kondo, Nedwell, Purdy, & Silva, 2004) (link). Standards for these groups were derived from the targeted cloned genes using the pGEM-T cloning vector system kit (Promega, Madison, WI, USA) as described previously (Barroso et al., 2013) (link).

Total bacteria and different groups and genera of bacteria that are representative in human saliva (Table 1) were quantified by qPCR using SYBR green methodology (Kappa Biosystems, Woburn, MA, USA) with the IQ5 Multicolor Real-Time PCR Detection System and data analyses (Bio-Rad Laboratories Inc., Hercules, USA). DNA from Escherichia coli DH5α, Lactobacillus plantarum IFPL935, Bifidobacterium lactis Bb12 and Streptococcus thermophilus ATCC 19987 was used for quantification of total bacteria, Lactobacillus, Bifidobacterium and Streptococcus, respectively. For the rest of the groups analyzed (Actinomyces, Fusobacterium, Haemophilus, Neisseria, Veillonella and Prevotella), samples were quantified using standards derived from targeted cloned genes using the pGEM-T cloning vector system kit (Promega, Madison, WI, USA), as described previously by Barroso et al. 29