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

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Description:

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

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

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

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.

For additional guidance on the transport of infectious substances in other languages, please click on the link below. 

http://www.who.int/ihr/capacity-strengthening/infectious-substances/en/

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. 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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