Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification

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

Sequences of the oligonucleotides used in the study. Underlined nucleotides are locked nucleic acids [17].

<p>Nucleotides in italic style are 2′OMe-RNA. The NRP is furthermore labeled by biotin in the 3′end.</p

Comparison between confocal microscopy and the dedicated instrument for detection of RCPs.

<p>The quantitative response of the same dilution series of EC DNA was measured using the dedicated instrument as well as the confocal setup used in Jarvius <i>et al</i> 2006. Filled symbols: dedicated instrument, open symbols: Zeiss 510 Meta confocal microscope.</p

Detection scheme of the bio-monitoring system.

<p>A) Collection of environmental samples is realized using the Airborne Sample Analysis Platform (ASAP) equipment. Aerosolized particulates stick to a filter, and the content is extracted and analyzed. In contrast to protein detection, detection of nucleic acids requires preparation of the filter content prior to exposure to the molecular procedures. The molecular procedures detect the target molecules using dedicated probes and reacted probes are then amplified. The amplification products are analyzed using a dedicated prototype instrument. B) The molecular procedures of DNA (left) and protein (right) detection. Detection of nucleic acids is achieved by padlock probes that are specifically circularized if correctly hybridized to the correct target in the presence of DNA ligase. Padlock and capture probes are added to the samples along with DNA ligase (5 min). Reacted padlock probes are captured on magnetic beads and excess probes are eliminated by washing (3 min). Detection of proteins is initiated by capture of the target protein using magnetic beads equipped with antibodies. The addition of a pair of PLA probes, which are antibodies with attached oligonucleotides (3 min), forms a DNA circle guided by two connector oligonucletides, and a DNA ligase (5 min). Unreacted probes are eliminated by washing the circles, and from this step the magnetic beads are treated identically in both the genetic and PLA assays. A first RCA is initiated either by an extra primer or the target itself on the beads to replicate the DNA circles (11 min). The products are then restriction digested (2 min), and the monomers are collected. The monomers can then bind head-to-tail to the excess amount of replication oligonucleotides and formed new DNA circles. The new circles are amplified and labeled with fluorescence-tagged detection probes. The ligation, amplification and labeling are performed in one reaction (8 min). The RCPs are therefore ready for analysis in the detection instrument.</p

Discrimination of EC and PA bacteria based on a single nucleotide difference in the target DNA sequence in two samples containing either 1 ng EC genome or PA genome (approximately 300 000 copies), assayed with probes specific for EC and PA genomes in duplex reactions.

<p>Error bars, ±1 s.d.; n = 2.</p