Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR

• Published in: Journal of microbiological methods Vol. 83; no. 1; pp. 42 - 47
• Main Authors: Walker, Roblena E., Petersen, Jeannine M., Stephens, Kenyatta W., Dauphin, Leslie A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.2010; Elsevier
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Bioterrorism; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Francisella tularensis; Francisella tularensis - genetics; Francisella tularensis - isolation & purification; Fundamental and applied biological sciences. Psychology; Humans; Microbiology; Polymerase Chain Reaction - methods; Real-time PCR; Swabs; Tularemia; Tularemia - microbiology; Real-time PCR; Bioterrorism; Swabs; Tularemia; Francisella tularensis; Samplings; Method; Real time; Infection; Polymerase chain reaction; Bacteriosis; Bacteria; Detection
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2010.07.015

Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5′nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.