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M-PCR protocol: A multiplex PCR procedure was used for the simultaneous detection
of S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis. The 16S rRNA gene of
the bacteria was chosen as the target. The procedure used one common lower primer and
four species-specific upper primers to generate PCR products of different sizes (H.
influenzae = 525 bp, S. pneumoniae = 484 bp, M. catarrhalis = 237 bp, and A.otitidis =
264 bp). For each reaction, 2X PCR master mix containing 0.05 unit/µl Taq DNA
Polymerase (recombinant), reaction buffer, 4 mM MgCl2 and 0.4 mM of each dNTP
(dATP, dCTP, dGTP, dTTP) from Promega was used in a 50-μl reaction volume. The
reaction procedure was 3 min of initial denaturation and 38 cycles of 94°C for 30 s, 62°C
for 45 s, and 72°C for 1 min, followed by a 5-min final extension at 72°C. The
amplification products were separated in 3% agarose gel containing ethidium bromide for
1.5-2.0 h and were visualized by UV light illumination. For each reaction, positive (DNA
from pure culture) and negative controls (no DNA) were included. The whole set of
reactions were repeated when discrepancies were found either in positive or negative
controls. The sensitivity of the M-PCR method was also studied for all four pathogens
with a dilution series of pure DNA from these pathogens.
RT-PCR on MEF: Reverse transcription was performed in a 20µl volume; 8µl of DNase
treated RNA samples from MEF was mixed with Random hexamer primer from Promega
and denatured for 5 minutes at 70C, and then added to a 12 µl of master mix including
20U of RNasin (Promega), 5X buffer, 4mmol/L of dNTP and 50U of MMLV reverse
transcriptase (Promega). The mixture was incubated for 55 min at 37C. The
complimentary DNA generated from the RT-PCR reaction was used as a template and
the PCR reaction was performed similarly to that described in the PCR protocol.