Quantitative PCR Assay for Bifidobacterium Species

Primers and Taqman^® probes were designed to target the B. longum subsp. infantis sialidase gene (locus tag ‘Blon2348’ from strain ATCC 15697, NCBI Reference Sequence: NC_011593.1) and the B. longum subsp. longum sugar kinase gene (locus tag ‘BL0274’ from strain NCC 2705, NCBI Reference Sequence: NC_004307.2) using ‘primer 3’ (Untergasser et al., 2012 (link)). Primers and probes were obtained from IDT (Singapore) and are described in Table 1. The primer/probe combinations were tested for reaction efficiency and specificity using genomic DNA (gDNA) purified from bifidobacterial type cultures (the gold standard cultures for species) of species reported to be detected in infant feces: B. adolescentis (DSM 20083^T), B. animalis subsp. lactis (DSM 10140^T), B. angulatum DSM 20098^T, B. bifidum (DSM 20456^T), B. breve (ATCC 15700^T), B. catenulatum (DSM 20224^T), B. dentium (ATCC 27534^T), B. longum subsp. infantis (DSM 20088^T), B. longum subsp. longum (ATCC 15707^T), B. pseudocatenulatum (DSM 20438^T), and B. pseudolongum (ATCC 25526^T), (Grönlund et al., 2011 (link); Makino et al., 2013 (link); Huda et al., 2014 (link); Bäckhed et al., 2015 (link); Vazquez-Gutierrez et al., 2015 (link); Martin et al., 2016 (link)). A Life Technologies ViiA™7 real time PCR system and MicroAmp Fast optical 96-well or 384-well plates with optical adhesive film (Applied Biosystems, Carlsbad, CA, USA) were used. All reactions were carried out in a final volume of 15 μl containing 1 × TaqMan^® Fast PCR mastermix (Applied Biosystems), 300 nM of each primer and 100 nM TaqMan^® probe. For specificity testing, template DNA was diluted to 5 ng∕μl, and 2 ng was added to each reaction. The thermocycling profile consisted of an initial activation of the polymerase at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Fluorescence levels were measured after the 60 °C annealing/extension step. Standard curves (to measure reaction efficiency) were generated using gDNA extracted from bifidobacterial strains Bifidobacterium longum subsp. longum (ATCC 15707^T) and Bifidobacterium longum subsp. infantis (DSM 20088^T) using the bead-beating phenol/chloroform/ethanol protocol described previously (Tannock et al., 2013 (link)). The standard DNA was quantified spectrophotometrically using a NanoDrop 1,000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and diluted in 10-fold steps from 5 × 10⁶ to 5 × 10¹ genomes/reaction, calculated using target gene copies per genome obtained from genome sequence information (NCBI). All reactions were carried out in duplicate and were run twice on separate plates. No-template controls were also included on each plate. Reactions in which duplicate Ct values varied by more than 0.5 Ct’s were also repeated.