Amplification assays for the detection of biothreat agents

37 38 Word count abstract: 173 words 39 Word count author's summary: 200 40 Word count text: 3147 words 41 42 2 43 ABSTRAC 44 45 46 Syndromic panels for infectious disease have been suggested to be of value in point 47 of care diagnostics for developing countries and for biodefense. To test the 48 performance of isothermal Recombinase Polymerase Amplification (RPA) assays we 49 developed a panel of ten RPAs for biothreat agents. The panel included RPA assays 50 for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and 51 Recerse Transcriptase Recombinase Polymerase Amplification (RT-RPA) assays for 52 Rift Valley fever virus, Ebola virus, Sudan virus and Marburg virus. Their analytical 53 sensitivities ranged from 16-21 molecules detected (probit analysis) for the majority 54 of RPA and RT-RPA assays. A magnetic bead based total nucleic acid extraction 55 method was combined with the RPA assays and tested using inactivated whole 56 organisms spiked into plasma. The RPA showed comparable sensitivities to real time 57 RCR assays in these extracts. 58 The run times of the assays at 42°C ranged from 6-10 minutes and they showed no 59 cross detection of any of target genomes of the panel nor of the human genome. The 60 RPA assays therefore seem suitable for implementation of syndromic panels onto 61 microfluidic platforms. 62 3 Introduction 63