Abstract
Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
Highlights
Recognition of hazardous biological materials is essential to all biodefence and biosafety strategies
We have developed a system for environmental monitoring of pathogens based on homogenous amplified single-molecule detection, or rolling circle amplification (RCA) [17,18,19,20]
Proximity ligation assay employs two antibodies equipped with oligonucleotides that template a DNA circularization reaction upon proximal coincident binding to the same target molecule. We have developed these molecular probing mechanisms to achieve faster detection with retained sensitivity. To achieve this we have developed a protocol for onbead padlock probing and rolling circle amplification (RCA)
Summary
Recognition of hazardous biological materials is essential to all biodefence and biosafety strategies. The demands on pathogen detection strategies for biodefence applications are high. High sensitivity is important as false negative events will bring the full devastating effects on society that the system was installed to prevent. Pathogen detection and identification has to be done as rapidly as possible to maximize the effect of protective measures. PCR-based applications are not very suitable for continuous sample processing such as required for surveillance of important sites in the community such as airports, subways, and other hubs of human communication. The current immunoassays are subjected to limitations in specificity, due to cross-reactivity with nonpathogenic naturally occurring close relatives [13,14], and sensitivity when analysing samples from environmental matrices [15,16] and are problematic to use in biowarfare applications
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