The lowest limit of detection of the m-PCR amplified
DNA from each of the targeted species was 20 pg
nucleic acid template (Fig. 2). The detection limits for
bacterial cells, determined by mixing dilutions of pure
cultures, isolating nucleic acids, and using the nucleic
acids as template for m-PCR, were 50 to 25 cells for
Flavobacterium columnare, 28 to 14 cells for Edwardsiella
ictaluri and 1300 to 650 cells for Aeromonas
hydrophila (Fig. 3). To determine the sensitivity of the
m-PCR for detection of bacteria in tissues, 10-fold serial
dilutions of each bacterial species (F. columnare, E.
ictaluri and A. hydrophila) were added to tissue
homogenates from naïve fish. DNA isolated from these
spiked homogenates was used as template for m-PCR.
The detection limits for each bacterial species in 1 g of
each tissue ranged from 3.4 × 102 to 2.5 × 105 cells g–1
tissue (Table 3). Differences in sensitivity varied
among tissues and bacterial species. However, detection
limits in blood were lower than or the same as in
the 2 other tissues for all species.