Molecular Detection by Real-Time PCR and Speciation by Conventional PCR

Molecular detection by real-time PCR and speciation of Rickettsial species.

• Endemic typhus (murine typhus)
• Typhus group rickettsiosis
• Epidemic typhus

Whole blood – minimum 1.0 mL. Tissue – biopsy of eschar.

Whole blood – EDTA tube. Tissue or Swab – submit in a sterile collection tube.

Store blood samples refrigerated until shipped for testing. Store tissue samples frozen until shipped. Ship whole blood at 4oC and ship tissues frozen.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Suspected rickettsial infection.

Completed Rickettsia and Related Zoonotic Diseases requisition including sender name, address and telephone number. Patient name or identifier (referring lab #), date of birth, test(s) requested, clinical history and symptom onset date. Type of specimen and date collected. If possible, include the antibiotics administered and travel history.

Positive samples are speciated using conventional PCR and sequencing.

Testing is performed, in whole or in part, using a lab-developed test which has not been fully validated/verified due to a lack of well-characterized panel.

Real-time PCR is performed using assays specific for the 17 kDa antigen gene and citrate synthase gene of the Rickettsia genus. If a sample is positive by real-time PCR, conventional PCR and sequencing of the outer membrane protein A gene will be performed to determine the exact spotted fever group species. For the specific detection of R. prowazekii, an assay specific for the citrate synthase gene of this species is performed.

15 calendar days.

1. Jiang J, TC Chan, JJ Temenak, GA Dasch, WM Ching, AL Richards. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 2004; 70(4): 351-356.
2. Stenos, J, SR Graves, NB Unsworth. A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group rickettsia. Am J Trop Med Hyg 2005; 73(6):1083-1085.
3. Henry KM, J Jiang, PJ Rozmajzl, AF Azad, KR Macaluso, AL Richards. Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever. Mol Cell Probes 2007; 21(1):17-23.
4. Wikswo ME, R Hu, GA Dasch, L Krueger, A Arugay, K Jones, B Hess, S Bennett, V Kramer, ME Eremeeva. Detection and identification of spotted fever group rickettsiae in Dermacentor species from Southern California. J Med Entomol; 45(3): 509- 516.
5. Svraka S, JM Rolain, Y Bechah, J Gatabazi, D Raoult. Rickettsia prowazekii and real-time polymerase chain reaction. Emerg Infect Dis 2006; 12(3): 428-432.

Pathogen Safety Data Sheets: Infectious Substances – Rickettsia prowazekii