title: Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.
abstract: OBJECTIVE
  To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.
  
  
  METHODS
  The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.
  
  
  RESULTS
  The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus.
  
  
  CONCLUSIONS
  A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.
authors: Department of Emergency Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA. syang10@jhmi.edu
authors: Samuel
authors: S
authors: Yang
authors: Richard E
authors: RE
authors: Rothman
authors: Justin
authors: J
authors: Hardick
authors: Marcos
authors: M
authors: Kuroki
authors: Andrew
authors: A
authors: Hardick
authors: Vishal
authors: V
authors: Doshi
authors: Padmini
authors: P
authors: Ramachandran
authors: Charlotte A
authors: CA
authors: Gaydos
mesh_terms: D000465:Algorithms; D001419:Bacteria / Q000145:classification* / Q000235:genetics; D023482:Bioterrorism; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004636:Emergency Service, Hospital; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D019914:Taq Polymerase