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Comparative Study
. 2003 Dec;41(12):5466-72.
doi: 10.1128/JCM.41.12.5466-5472.2003.

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae

Marina E Eremeeva  1 , Gregory A Dasch, David J Silverman
Affiliations
Comparative Study

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae

Marina E Eremeeva et al. J Clin Microbiol. 2003 Dec.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Standard calibration curve for SQ-PCR for Rickettsia with plasmid pCR2.1-TOPO containing the cloned Rr190.547F-Rr190.701R fragment of R. rickettsii Bitterroot. CF RFU, curve-fit relative fluorescence units.
FIG. 2.
FIG. 2.
Effect of added Vero cell DNA on detection and quantitation of the R. rickettsii rOmpA fragment, using the Rr190.547F and Rr190.701R primer pair. Serial dilutions of two independently isolated and cloned standard plasmid preparations (plasmid P1 and P2) were made in distilled water with or without Vero cell DNA to give a final assay concentration of 0.4 μg/ml. PCR conditions were described in Materials and Methods.
FIG. 3.
FIG. 3.
Detection of R. rickettsii growth in Vero cells by SQ-PCR. Vero cell monolayers were infected with 2.0 rickettsial PFU per cell (squares) or 0.2 rickettsial PFU per cell (diamonds). Each value is the average ± standard error of three replicates. Some error bars are too small to be seen.
See this image and copyright information in PMC

References

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