Fig. 2

PCR methodologies with goat lymph node samples. a Attempt at detection of Brucella transcripts by endpoint RT-PCR. The BMEI1305 primers were used to amplify cDNA templates (0.5 µl each) derived from long-term samples (lanes 1–4), short-term samples (lanes 5–8; one sample reflected a short-term goat that was not included in the coincidence cloning profiling), or a B. melitensis culture cDNA sample as a positive control (lane 9), for 40 cycles. The culture cDNA sample was prepared in parallel to the lymph node cDNA samples, using equivalent starting amounts of total DNA; the red arrow indicates the amplified band of interest. NC = negative control with no template. Numbers presented to the left of each gel (a–c) are sizes of DNA ladder bands in base pairs. b Assessment of impacts of template dilution on amplification. As in (a), the BMEI1305 primers were used to amplify cDNA templates, in this case for 35 cycles. For lanes 2–7, 0.5 µl aliquots of serially-diluted B. melitensis culture-derived cDNA were added to each reaction; dilutions are indicated above each lane. In lanes 8 and 9, 0.5 µl of diluted bacterial cDNA was mixed with 0.5 µl of undiluted host cDNA in each reaction, with the bacterial dilution indicated above each lane. NC = negative control with no template. c Additional assessment of impacts of host cDNA template on PCR amplification, with reactions as described in (b) for 35 cycles. Dilution factors above each lane indicate the dilution factor of the culture-derived cDNA added to the reaction. Since multiple background bands were observed in the BMEI1305 reactions, we also completed negative control PCR reactions with the NdvB (lane 7) and DksA (lane 9) primers to demonstrate the absence of contamination in reactions. “+ control” reactions for NdvB (lane 8) and DksA (lane 10) contained 0.5 µl of culture-derived cDNA template