Bacterial DNA from the 84 feces samples and 192 bacterial isolates was obtained
using, respectively, the commercial QIAmp DNA Stool Mini Kit and QIAmp DNA Mini Kit
(QIAGEN), according to the manufacturer's instructions. DNA was stored at −80 °C
until use.
The presence of B. fragilis in the fecal samples was detected by PCR
amplification with 16S rRNA primers. DNA from the bacterial isolates was amplified
with 16S-23S rRNA primers. All DNA samples positive for B. fragiliswere screened for the presence of ETBF and/or NTBF sequences. The oligonucleotide
pairs and amplification conditions used are described inTable 1 .
All PCR assays were performed as follows: 1X PCR buffer, 50 mM MgCl2, 0.2
mM dNTP mix, 0.4 mM each primer, 0.5 U of Platinum Taq polymerase
(Invitrogen), and 1 ng of DNA. PCR products were analyzed by 1% agarose gel
electrophoresis, stained with 0.5 μg/mL of ethidium bromide and photographed under UV
light.
B. fragilis ATCC 43858 (ETBF pattern I, bft+) and ATCC 25285 (NTBF pattern III, bft-) were used, respectively, as positive and negative controls. The other
strains used as controls were: B. thetaiotaomicron ATCC 29741,
B. vulgatus ATCC 8482, B. caccae ATCC 43185,
B. ovatus ATCC 8483, B. eggerthii ATCC 27754,
B. uniformis ATCC 8492, B. stercoris ATCC 43183,
Parabacteroides distasonis ATCC 8503, and P.
merdae ATCC 43184.
using, respectively, the commercial QIAmp DNA Stool Mini Kit and QIAmp DNA Mini Kit
(QIAGEN), according to the manufacturer's instructions. DNA was stored at −80 °C
until use.
The presence of B. fragilis in the fecal samples was detected by PCR
amplification with 16S rRNA primers. DNA from the bacterial isolates was amplified
with 16S-23S rRNA primers. All DNA samples positive for B. fragiliswere screened for the presence of ETBF and/or NTBF sequences. The oligonucleotide
pairs and amplification conditions used are described in
All PCR assays were performed as follows: 1X PCR buffer, 50 mM MgCl2, 0.2
mM dNTP mix, 0.4 mM each primer, 0.5 U of Platinum Taq polymerase
(Invitrogen), and 1 ng of DNA. PCR products were analyzed by 1% agarose gel
electrophoresis, stained with 0.5 μg/mL of ethidium bromide and photographed under UV
light.
B. fragilis ATCC 43858 (ETBF pattern I, bft+) and ATCC 25285 (NTBF pattern III, bft-) were used, respectively, as positive and negative controls. The other
strains used as controls were: B. thetaiotaomicron ATCC 29741,
B. vulgatus ATCC 8482, B. caccae ATCC 43185,
B. ovatus ATCC 8483, B. eggerthii ATCC 27754,
B. uniformis ATCC 8492, B. stercoris ATCC 43183,
Parabacteroides distasonis ATCC 8503, and P.
merdae ATCC 43184.