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J Microbiol Methods. 2009 Sep;78(3):307-11. doi: 10.1016/j.mimet.2009.07.001. Epub 2009 Jul 23.

Prion protein detection via direct immuno-quantitative real-time PCR.

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Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1.


We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32x10(2) prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06x10(4) to 3.24x10(2) epitopes (R(2)=0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.

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