Research Project
2231469
The strategy for highly parallelized screening of blood borne pathogens is proposed. Constituent steps are simultaneous collection of all suspect pathogens on magnetic beads coated with corresponding antibodies. The polymerase chain reaction (PCR) is then used to amplify different length, targeted segments of the proposed pathogens genomes. The primers carry PG and/or EC isotopic labels incorporated. The 5' (non-priming) end of the primers utilized has an additional Tag sequence with is a source identifier of the donor/patient. Pools of PCR products from different sources are pooled for subsequent analytical steps. A part of each pool of forty is put through the protocol for Direct Sequencing (DS) of genomes of a few megabase complexity. Undegraded PCR fragments are size markers added back to it. After a subsequent size fractionation, the presence of a PCR band corresponding in size to a genome segment of the pathogens is revealed by the high sensitivity MultiPhoton Detector (MPD) used to quantify PG and EC isotopes. MPD allows simultaneous but individual quantitation of multiple co-resident EC and PG labels. Whenever a suspect band is identified, the established DS protocols can be implemented to verity that the PCR band in fact encodes the pathogens sequence. An oligonucleotide probing with specific for the source Tag then identifies the donor/patient carrying the pathogen. The constituent steps have all been performed in other R&D. The two most critical features to be validated in this Phase I with non-hazardous biologicals are the degree of any artifact limitations during the PCR applications and dynamic range limitations during the readout of the fractionated DNAs.
