Ultra-Sensitive Detection of Prion Protein in Blood Using Isothermal Amplification Technology

摘要

The detection of pathologic prion protein that is implicated in transmissible spongiform encephalopathies is necessary to diagnose disease. Presently, the Western blot or ELISA are used to test the brain stem in cattle for the presence of PrPsc after proteinase K digestion of normal, cellular prion (PrPc) before admission of these animals into the food chain. However, infected animals and humans cannot be diagnosed in the pre-clinical stage of infection. Because routinely used prion detection methods can identify PrPsc in brain where quantities are high but cannot detect PrPsc in blood where levels are low, a lack of sensitivity of current assay methods is likely the explanation. To address this, we have exploited a real-time immuno-polymerase chain reaction method (IPCR) that couples serologic detection of protein with the amplification ability of PCR and applied it to the detection of prion protein. The method has been optimized for maximum sensitivity through a number of procedural modification and background reduction strategies. Efforts were extended to develop a new method called RNA polymerase immunodetection (RAPID) that is similar but uses an RNA polymerase to produce RNA transcripts at isothermal temperatures and a magnetic bead solid support. Results have shown the sensitivity of IPCR to be 100attograms/mL of recombinant prion protein, and detection levels using scrapie infected hamster brain homogenates down to 10- 100 infectious units. This sensitivity is unmatched by other assays and approaches the sensitivity believed to be required for pre-symptomatic detection of prion protein in blood. The RAPID method has been developed using a number of the parameters that were optimized in the IPCR method; however, its sensitivity was shown to be only 150pg/mL.