Ultra-Sensitive Detection of Prion Protein in Blood Using Isothermal Amplification Technology
Abstract
The detection of the pathologic prion protein that is Implicated in transmissible spongiform encephalopathies (TSEs) is necessary to diagnose the disease. Presently, the Western Blot or ELISA are used to test the brain stem in cattle for the presence of pathologic prion (PrP(exp Sc)) after Proteinase K (PK) digestion of normal, cellular prion (PrP(exp c)) before admission of these animals Into the food chain. An animal in the end stages of disease (40 -72 weeks after infection) will be detected by these methods; however, an infected animal will not be detected by these methods during preclinical stages of prion infection (1-20 weeks). The RNA-polymerase immunodetection method (RAPID) is a technique whereby the exponential amplification ability of the PCR is coupled to the detection of proteins by antibodies in an enzyme linked immunosorbent assay (ELISA) format using magnetic beads. It is similar to the immuno-PCR method except that the final step of nucleic acid amplification is by RNA polymerase during isothermal incubation. For the IPCR, the final step of nucleic acid amplification is by Taq polymerase using a 2- temperature cycle Incubation. As a starting platform using microwell plates as the solid format, we have been able to show that real-time Immuno-PCR (IPCR) detects recombinant hamster PrP(exp c) down to 0.1-1.0 femtogram/mL concentrations. Recombinant hamster PrP(exp c), as well as PK-digested scrapie infected hamster brain homogenates diluted from 10(exp -1) to 10(exp-6) exhibited a quantitative dose response. The methods we use In real-time IPCR will now be modified for use with a magnetic bead solid format and RNA polymerase isothermal amplification.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2004
- Accession Number
- ADA427814
Entities
People
- Neil T. Constantine
Organizations
- University of Maryland, Baltimore