NOTIFy (non-toxic lyophilized field)-FISH for the identification of biological agents by Fluorescence in situ Hybridization
Fig 5
Identification of biological agents on skin surface after sampling by tape, in powder samples and in a spore suspension using the NotiFy-algorithm.
E. coli was detected on porcine skin in the first hybridization by the Gam42a probe (labeled in Cy5 and FLUOS, turquoise color in the overlay) and by the probe Esco864 (labeled in Cy5) in the second hybridization. Additional bacteria were detected in the first hybridization by the Bet42a probe (labeled in Cy3 and FLUOS, yellow color in the overlay) and DAPI or by DAPI alone, but could not be identified by the algorithm. DAPI-signals for bacteria other than E. coli were also observed in the second hybridization. In powder samples, Y. pestis was identified by the Gam42a probe (labeled in Cy5 and FLUOS, turquoise color in the overlay) in the first hybridization and by the probe Ypest1531LNA (labeled in Cy3, red color in the overlay) in the second hybridization. Autofluorescent powder material can be observed in all three channels. B. anthracis spores were detected after pretreatment to break the spore coat with the probe LGC354B (labeled in Cy5 and Cy3, violet color in the overlay) in the first and with the Bac1157 probe (labeled in Cy3) in the second hybridization. Not all spores were accessible to FISH-probes after the pretreatment, resulting in only DAPI signals but no FISH signals for some of the spores. Bars 10 μm.