Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.
Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.
University Medical Center, Department of Virology, Göttingen, Germany.
Milena
M
Euler
Yongjie
Y
Wang
Doris
D
Heidenreich
Pranav
P
Patel
Oliver
O
Strohmeier
Sydney
S
Hakenberg
Matthias
M
Niedrig
Frank T
FT
Hufert
Manfred
M
Weidmann
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D054045:Biological Warfare Agents; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009696:Nucleic Acids / Q000235:genetics / Q000302:isolation & purification; D010949:Plasma / Q000382:microbiology / Q000821:virology; D013696:Temperature; D013997:Time Factors; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification*
D054045:Biological Warfare Agents; D009696:Nucleic Acids