Quantitative analysis of Escherichia, Lactobacillus, and Bifidobacterium by real-time quantitative PCR

The standard curve was established using real-time fluorescence quantitative PCR using a 10-fold gradient diluted plasmid standard as a template. The PCR reaction was performed in a 10 μl system, containing 5 μl Real Master Mix (SYBR Green), 1 μl template, 10 μM upstream primers, 10 μM downstream primers, and 3.6 μl deionised water. PCR amplification was performed at 95°C for 3 min, followed by 94°C for 15 s, annealing at 57°C for 20 s, and extension at 72°C for 30 s for 45 cycles. A standard curve was obtained. Genomic DNA samples of Escherichia, Lactobacillus, and Bifidobacterium were tested. The number of DNA copies in the samples was determined according to the measured Ct value and standard curve. The following primers were used: Escherichia: F: 5′-GTTAATACCTTTGCTCATTGA-3′, R: 5′-ACCAGGGTATCTTAATCCTGTT-3′; Lactobacillus: F: 5′-AGCAGTAGGGAATCTTCCA-3′ R: 5′-CACCGCTACACATGGAG-3′, Bifidobacterium: F: 5′-TCGCGTC(C/T)GGTGTGAAAG-3′ R: 5′-CCACATCCAGC(A/G)TCCAC-3′, 16S: F: 5′-ACGGGGGGCCTACGGGAGGCAGCAG-3′, R: 5′-ATTACCGCGGCTGCTGG-3'.