Prion protein detection via direct immuno-quantitative real-time PCR |
| |
| Authors: | Tim Reuter Brandon H Gilroyed Trevor W Alexander Gordon Mitchell Aru Balachandran Stefanie Czub Tim A McAllister |
| |
| Institution: | aAgriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta, Canada T1J 4B1;bAnimal Diseases Research Institute, Canadian Food Inspection Agency, Ottawa, Ontario, Canada K2H 8P9;cAnimal Diseases Research Institute, Canadian Food Inspection Agency, National and OIE BSE Reference Laboratories, Lethbridge, Alberta, Canada T1J 3Z4 |
| |
| Abstract: | 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 2.32 × 102 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 1.06 × 104 to 3.24 × 102 epitopes (R2 = 0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies. |
| |
| Keywords: | Prion detection Immuno-quantitative PCR Real-time PCR Transmissible spongiform encephalopathies PVDF membrane |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
