Evaluation of a PCR Assay for Quantitation of Rickettsia rickettsii and Closely Related Spotted Fever Group Rickettsiae

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.Rickettsia rickettsii is an obligately intracellular bacterium. In nature, R. rickettsii circulates in a zoonotic cycle that includes transovarial and transstadial maintenance by its arthropod hosts, ixodid ticks, and horizontal transmission to its vertebrate hosts, which include a wide range of small and medium-sized mammals. Following an inopportune tick bite, R. rickettsii is transmitted to humans and causes Rocky Mountain spotted fever, a severe febrile disease, which is associated with proinflammatory and procoagulant changes and development of a systemic vasculitis.Because of the intracellular habitat of rickettsiae and their slow generation time, the use of traditional techniques to quantify the number of viable rickettsiae in a sample is laborious, inaccurate, and tedious. In principle, the absolute number of rickettsial particles can be determined by microscopic observation of smears prepared from cosuspension with a standardized bacterial suspension following staining with chemical or fluorescent dyes (3, 19) or fluorescein-labeled antibodies (20). Quantitation by this method may be inconsistent because of the variable distribution of microorganisms in the smear and because direct counts of bacterial particles by microscopic observation suffer from subjectivity and lack of reproducibility. The assay is also a time-intensive procedure that may not work well for infected animal tissues. Alternatively, the number of viable rickettsiae can be estimated by measurement of metabolically 35 S-labeled rickettsiae or 32 P-labeled rickettsiae (21, 22), by titration of infected samples in susceptible animals and embryonated chicken eggs (2, 5), and by tissue culture procedures, including plaque assays (23, 24). However, only infectious and/or metabolically active rickettsiae can be measured by using these biological approaches. These last techniques are expensive, require special facilities for work with radioactive materials or for housing of infected animals, and cannot be used with samples contaminated with other ...

Beta Interferon-Mediated Activation of Signal Transducer and Activator of Transcription Protein 1 Interferes with Rickettsia conorii Replication in Human Endothelial Cells

“…In the final preparatory step, DNA was eluted in 100 l of Ultra-Pure distilled water (Gibco), followed by determination of its concentration in final preparations on a Nanodrop spectrophotometer (ND-1000; Thermo Scientific). The primer pair RR190.547F and RR190.701R, previously described for the quantification of SFG rickettsiae, was used to evaluate R. conorii replication in HMECs (5). Briefly, the PCR mixture (total volume, 20 l) contained 5 ng of total DNA, primers at a final concentration of 10 M, and iQ SYBR green Supermix (Bio-Rad).…”

“…DNA template was initially denatured at 95°C for 3 min, followed by 95°C for 30 s, 57°C for 20 s, and 65°C for 1 min for 45 cycles and a final extension cycle at 72°C for 5 min. R. conorii copy number was calculated using a standard curve, generated by the pCR2.1-TOPO plasmid containing the RR190.547/ RR190.701 PCR amplicon (5), and the R. conorii DNA copy number in each well was normalized to the amount of total DNA.…”

Detection and Identification of Ehrlichia Species in Blood by Use of PCR and Electrospray Ionization Mass Spectrometry

“…The same PCR-EIA format and procedure were used to detect R. rickettsii by targeting a 154-bp fragment of the R. rickettsii-specific rickettsial outer membrane protein A (rOmpA) gene. RR190.547F, RR190.701R, and RR190.588F were used as the forward primer, reverse primer, and hybridization probe, respectively, as described previously (16).…”

Molecular Typing of Isolates ofRickettsia rickettsiiby Use of DNA Sequencing of Variable Intergenic Regions

“…R. rickettsii isolates (Table 1) were cultivated in Vero cells (strain C1008; green monkey kidney cells) as described elsewhere (17). DNAs for isolates Brazil-A, 84JG, Hlp#2-A, Bitterroot, Colombia, Lost Horse Canyon, Morgan, PriceT, and Sheila Smith were prepared by phenol-chloroform extraction from partially purified R. rickettsii cells as described previously (16). DNAs for the remaining isolates were isolated from infected cell cultures by using a QIAamp DNA Mini kit from QIAGEN (Valencia, CA).…”