Volume 7, Number 7—June 2001
Waterborne Outbreak of Tularemia Associated with Crayfish Fishing
|Crayfish, Batch A
||Crayfish, Batch B
Batch A = crayfish collected from a patient's house; Batch B = crayfish collected from the river; SP = sewage plant; R = river; LNA = lymph node aspirate; B = branchia; S = stomach; G = gonads; H = hepatopancreas; W = washed pellet; I = intestines.
aSeveral annealing temperatures were tested, from 60°C to 68°C for the 25 cycles of the first round. The second round consisted of 45 cycles with an annealing temperature of 60°C. The rest of the parameters were as described (21). All reagents were from Perkin Elmer (Foster City, CA); the cycling was done in a PCT-100 thermocycler (MJ Research Inc., Watertown, MA). The PCR products, with a size of 1550 bp and 950 bp for the first and second rounds of amplification, respectively, were visualized in 1% low-melt agarose gels (Pronadisa, Alcobendas, Madrid, Spain) stained with ethidium bromide (Sigma-Aldrich). Cross-contamination was avoided by using standard methods. Half the samples studied were kept unprocessed and frozen in a different area. A positive result was confirmed by processing the rest of the sample. To assess the sensitivity of our system, 10 L of serial twofold dilutions of an aqueous suspension of bacteria, ranging from 104 CFU to 102 mL was added to 1-mL volumes of distilled water. These samples were subjected to DNA extraction and PCR as above. Negative controls (autoclaved distilled H2O) were included in all extractions at a ratio of a negative control for each five samples. The specificity was checked against DNA from Salmonella Typhimurium, Escherichia coli, Legionella pneumophila, Yersinia enterocolitica, and Proteus vulgaris.