Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae.
A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.
Eremeeva
Marina E
ME
School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland 21201-1559, USA. mge6@cdc.gov
Dasch
Gregory A
GA
Silverman
David J
DJ
D000818:Animals; D001483:Base Sequence; D002138:Calibration; D002522:Chlorocebus aethiops; D017931:DNA Primers; D006801:Humans; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012284:Rickettsia rickettsii / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012373:Rocky Mountain Spotted Fever / Q000382:microbiology*; D013698:Templates, Genetic; D013987:Ticks / Q000382:microbiology; D014709:Vero Cells
D017931:DNA Primers