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. Syndromic panels for infectious and emerging infectious diseases have been suggested to be of value in point-of-care (POC) diagnostics for developing countries and for biodefense (1). Since the introduction of molecular diagnostics and in particular real-time PCR, ample proof of its sensitivity and specificity has been generated. Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2-4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5-7).In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8-12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets. Nonspecific intercalating fluorophores or fluorescent primers have been used for real-time detection in LAMP, SDA, HDA, and RCA, and specific detection probe formats have been developed for NASBA, RCA, HDA,.In isothermal and exponential RPA, the phage recombinase UvsX and its cofactor UvsY form a nucleoprotein complex with oligonucleotide primers to scan for homologous sequences in a DNA template. Recognition of a specific homologous sequence leads to the initiation of strand invasion of the com...
dSeveral real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. Because of its extraordinary infectiousness, the zoonotic pathogen Francisella tularensis causing tularemia was in the past the subject of state-run biowarfare research programs and therefore is included on the CDC category A list of biothreat agents. It causes disease in a vast range of animals, with relevant disease transmission to humans by direct contact or via vectors such as deer flies, horse flies, mosquitoes, and hard ticks. Infection due to inhalation of aerosols can occur through contact with infected hares. There are three F. tularensis subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. tularensis subsp. mediasiatica, which can be found in several environments and geographical regions. The first two subspecies and Francisella novicida cause the bulk of human infections (7,19). Infection by Francisella hispaniensis has also been described, and results of phylogenetic analysis suggest that F. novicida should be regrouped as a fourth subspecies of F. tularensis (3,12).In all scenarios dealing with intentional release of biothreat agents, timely diagnosis is regarded as essential to identifying and containing outbreaks of infectious disease (4). Many efforts have been made to reduce the assay time of PCR-based nucleic acid detection. In spite of engineering constraints regarding temperature cycling needed for the PCR assays, short protocols and miniaturized cyclers or chip platforms are being developed (5, 6, 18).In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing even simpler point-of-care systems. One example is the ESEQuant Tube Scanner device (Qiagen Lake Constance GmbH, Stockach, Germany). This device contains a sophisticated fluorescence sensor which slides back and forth under a set of eight tubes, collecting fluorescence signals over time and allowing for real-time documentation of increasing fluorescence signals. A combined threshold and signal slope analysis is used for signal interpretation, which can be confirmed by second-derivative analysis (11; also ESEQuant Tube Scanner software [Qiagen]). The recombinase polymerase amplification (RPA) assay is an isothermal amplification method which can be combined with a sequencespecific fluorescent probe for real-time detection. In RPA the phage-derived recombinase UvsX, assisted by its cofactor UvsY, aggregates with oligo...
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