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Volume 8, Number 3—March 2002

Dispatch

Rickettsia felis in Ctenocephalides spp. Fleas, Brazil

Riva P. Oliveira*, Márcio A.M. Galvão*Comments to Author , Claudio L. Mafra*, Chequer B. Chamone†, Simone B. Calic†, Sergio U. Silva‡, and David H. Walker§
Author affiliations: *Universidade Federal de Ouro Preto, Minas Gerais State, Brazil; †Fundação Ezequiel Dias, Minas Gerais State, Brazil; ‡Secretaria Municipal de Saúde de Coronel Fabriciano, Minas Gerais State, Brazil; §University of Texas Medical Branch, Galveston, Texas, USA;

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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.

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.

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