iii) Real-time quantitative PCR (RTQ-PCR)
Total DNA was extracted from a 50 μl aliquot of the sample. In brief, cells were incubated in 50 mM phosphate buffer pH 6.7 containing 20 mM diethyl pyrocarbonate, 1 mg lysozyme ml-1, 1 mg mutanolysin ml-1 and 2 mg Proteinase K ml-1 at 56°C for 40 min and lysed with 1% (w/v) SDS, prior to DNA extraction and purification using QIAmp DNA Mini kit (QIAGEN, Clifton Hill, Vic) according to the manufacturer's protocol. Genomic DNA of the type strains, S. pneumoniae ATCC 6305, H. influenzae ATCC 10211 and M. catarrhalis ATCC 25238, was extracted from overnight cultures using the QIAmp DNA mini kit, as described above, except that diethyl pyrocarbonate was not included. DNA concentrations were determined with the PicoGreen double-stranded DNA quantification kit (Invitrogen-Molecular Probes, Mulgrave, Vic) and Luminescence spectrophotometer model LS 50B (Perkin Elmer, Melbourne, Vic).
Total bacterial loads were determined using pre-optimised concentrations of the universal forward (5'-TCCTACGGGAGGCAGCAGT-3'; 300 nM) and reverse (5'-GGACTACCAGGGTATCTAATCCTGTT-3'; 300 nM) primers and probe (5'- [6-FAM]-CGTATTACCGCGGCTGCTGGCAC- [TAMRA]-3'; 175 nM) for the 16S rRNA gene, as previously described [
15]. Purified genomic DNA of
Streptococcus mitis strain NCTC 1226, in the range of 100 fg to 1 ng, was used to standardise the values. DNA quantities determined by RTQ-PCR were converted to cells (ml of sample)
-1, in order to represent the number of bacterial cells per nasal swab, by assuming that 1 pg of DNA was equivalent to 447.4 cells, i.e. a genome size of 2 Mb.
Total
S. pneumoniae loads were determined using pre-optimised concentrations of the forward (5'-TCTTACGCAATCTAGCAGATGAAGC-3'; 100 nM) and reverse (5'-GTTGTTTGGTTGGTTATTCGTGC-3'; 100 nM) primers and probe (5'- [6-FAM]-TTTGCCGAAAACGCTTGATACAGGG- [TAMRA]-3'; 200 nM) for the autolysin gene,
lytA, as described previously [
16], except that the primers were modified for compatible
T
m
s. Purified genomic DNAof
S. pneumoniae ATCC 6305, in the range of 20 fg to 200 pg, was used to standardise the values. DNA loads were converted to cells (ml of sample)
-1 assuming that 1 pg of DNA was equivalent to 447.4 cells, i.e. a genome size of 2 Mb.
Total
H. influenzae loads were determined using pre-optimised concentrations of the forward (5'-CTGGWGCAATGGCAGAAGTG-3'; 100 nM) and reverse (5'-TCTTTACGCACGGTGTAAGGATG-3'; 200 nM) primers and probe (5'- [6-FAM]-AATATGCCGATGGTGTTGGYCCAGGTT- [TAMRA]-3'; 100 nM) for the outer membrane protein D gene,
glpQ, which shows limited sequence variation among
H. influenzae type b and non-encapsulated strains [
17]. Purified genomic DNA of
H. influenzae ATCC 10211, in the range of 20 fg to 200 pg, was used to standardise the values. DNA quantities were converted to cells (ml of sample)
-1 assuming that 1 pg of DNA was equivalent to 497.1 cells, i.e. a genome size of 1.8 Mb.
Total
M. catarrhalis loads were determined using pre-optimised concentrations of the forward (5'- GTGAGTGCCGCTTTTACAACC-3'; 300 nM) and reverse (5'-TGTATCGCCTGCCAAGACAA-3'; 300 nM) primers and probe (5'- [6-FAM]-TGCTTTTGCAGCTGTTAGCCAGCCTAA- [TAMRA]-3'; 200 nM) for the outer membrane protein gene,
copB, as described previously [
18]. Purified genomic DNA of
M. catarrhalis ATCC 25238, in the range of 20 fg to 200 pg, was used to standardise the values. DNA quantities were converted to cells (ml of sample)
-1 assuming that 1 pg of DNA was equivalent to 497.1 cells, i.e. a genome size of 1.8 Mb.
RTQ-PCR was performed with the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Scoresby, Vic) using optical grade 96-well plates in a reaction volume of 25 μl using the TaqMan PCR Core Reagent Kit (Applied Biosystems) containing the forward and reverse primers and the fluorogenic probe. The RTQ-PCR conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. Reactions were performed in triplicate and the mean values calculated. Data analysis was performed using the Sequence Detection Software version 1.6.3 supplied by Applied Biosystems. Samples that were negative by RTQ-PCR for S. pneumoniae, H. influenzae or M. catarrhalis DNA were checked qualitatively by PCR in 25 μl reactions containing 1 × Platinum PCR SuperMix (Invitrogen), 2 μl of the sample DNA and 200 nM each of the specific primers. PCR was performed using the GeneAmp PCR System 9700 (Perkin Elmer) with an initial denaturation step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A 10 μl aliquot of the PCR reaction was subjected to electrophoresis on a 2% (w/v) agarose gel containing 1 μg ethidium bromide ml-1 and the DNA bands visualized by UV illumination.