Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument

• Published in: Journal of microbiological methods 2015-08, Vol.115, p.89-93
• Main Authors: Mölsä, Markos, Hemmilä, Heidi, Katz, Anna, Niemimaa, Jukka, Forbes, Kristian M., Huitu, Otso, Stuart, Peter, Henttonen, Heikki, Nikkari, Simo
• Format: Article
• Language: English
• Subjects: Bacillus anthracis; Bacillus anthracis - genetics; Bacillus anthracis - isolation & purification; Biological Warfare Agents; Biothreat; DNA, Bacterial - genetics; Francisella tularensis; Francisella tularensis - genetics; Francisella tularensis - isolation & purification; Mobile Applications; PikoReal; Portable; Rapid identification; Real-time PCR; Real-Time Polymerase Chain Reaction - instrumentation; Real-Time Polymerase Chain Reaction - methods; Yersinia pestis; Yersinia pestis - genetics; Yersinia pestis - isolation & purification
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2015.05.026

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95min, as the pre-PCR preparation, including DNA extraction, required an additional 45min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods. •We compared the performance of the portable qPCR to the standard qPCR, for the detection of three biothreat pathogens.•The PikoReal system performed considerably faster than the standard platform, with equal sensitivity and specificity.•We evaluated this system under field conditions by screening 1035 wild rodent samples for F. tularensis.•A novel confirmatory qPCR assay for the detection of F. tularensis, targeting the ISFtu2 gene was also developed.