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

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Reference Details

Description:

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

Test Category:
Molecular Detection
Pathogen:
Spotted fever rickettsiae
Illnesses and Diseases:
  • Spotted fever rickettsiosis
  • African tick bite fever
  • Rocky Mountain spotted fever (RMSF)
  • Mediterranean spotted fever
  • Rickettsial pox
Specimen:

Whole blood – minimum 1.0 mL.  Tissue – biopsy of eschar.  Swab – cutaneous swab of skin lesion after removal of eschar scab.

Collection Method:

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

Specimen Processing, Storage and Shipping:

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

Transportation of Dangerous Goods:

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.

 

Patient Criteria:

Suspected rickettsial infection.

Accompanying Documentation:

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.

Comments:

Positive samples are speciated using conventional PCR and sequencing.

Methods and Interpretation of Results:

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. typhi, an assay specific for the outer membrane protein B gene is performed.

Turnaround Time:

15 calendar days.

Contact:
Phone #: (204) 789-7037
Fax: (204) 789-2018
References:
  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.
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