Tuesday, March 6, 2012

Sensitive detection of pathogens for biosecurity analysis

Göransson et al. report a new method of finding pathogens as part of a biosecurity study that is able to detect pathogens down to the single-molecule/organism level. The method combines a padlock probe approach, using Ampligase® Thermostable DNA Ligase, with rolling-circle amplification (RCA).

The authors used an environmental air sampling unit to trap particulate material on a membrane, followed by a rapid extraction of the DNA using magnetic beads. After clean-up, the DNA-containing solution was placed into an "on-bead" padlock probe/proximity ligation assay (PLA) catalyzed by Ampligase enzyme. Reacted probes were then subjected to two further rounds of RCA, first on beads and then in solution. Probes were then tagged with fluorescent dye and detected using an optical system with sensitivity down to 30 bacteria or 5 spores. The authors have improved the performance of the system by reducing the time required for the RCA step and are working improve the sensitivity of the process.

The combination of improved RCA and sensitive detection represent a significant improvement over previous methods for pathogen detection. The method can also be adapted to detect proteins.

ResearchBlogging.orgGöransson, J. et al. (2012). Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031068


Austin J Bouck said...

When you say this system could be adapted to detect proteins, does that mean that it could detect allergens? And how long does this entire process take?

epibio said...

The authors used their detection system for spores, so presumably it would work with protein allergens. You would need to have a suitable antibody for bead-based capture (the scheme is shown in Figure 1 of the paper).