Rickettsia felis in Ctenocephalides spp. Fleas, Brazil
Figure 2. Detection of the Rickettsia specific 17-kDa gene by polymerase chain reaction amplification in DNA extracted from ticks and fleas. The vectors were first placed in 1.5-mL microcentrifuge tubes containing 200 µL of 10 mM phosphate-buffered saline, pH 7.4, and were crushed with a micropestle. The suspensions were lysed in 0.5% sodium dodecyl sulfate and incubated with 100 µg/mL proteinase K at 37°C for 1 hour in the case of fleas or overnight in the case of ticks. The lysed suspensions were extracted twice with an equal volume of phenolchloroform, followed by a single chloroform extraction. The extracted DNA was amplified with primer 1 (5′-GCTCTTGCAACTTCTATGTT-3′) and primer 2 (5′-CATTGTTCGTCAGGTTGGCA-3′) as described by Webb et al. (10) for amplification of a 434-bp fragment from the rickettsial 17-kDa protein gene. PCR was performed at 30 cycles for 1 minute at 94°C, 5 minutes at 48°C, and 2 minutes at 72°C. The PCR products were then separated by electrophoresis in 1% agarose gel and stained with ethidium bromide. Lanes 1-3: DNA from cat fleas, Lanes 4-6: DNA from dog fleas, Lane 7: 17- kDa gene Rickettsia felis DNA (Positive Control), Lanes 8-14: DNA from ticks.