New assays for Pathogens Relevant to Blood Transfusion

The strategy for highly parallel screening of blood borne pathogens is proposed. Constituentsteps are simultaneous collection of all suspect pathogens on magnetic beads coated with correspondingantibodies. The polymerase chain reaction (PCR) is then used to amplify different length, targetedsegments of the proposed pathogens genomes. The primers carry PG and/or EC isotopic labelsincorporated. The 5' (non-priming) end of the primers utilized has an additional Tag sequence with isa source identifier of the donor/patient. Pools of PCR products from different sources are pooled forsubsequent analytical steps. A part of each pool of forty is put through the protocol for DirectSequencing (DS) of genomes of a few megabase complexity. Undegraded PCR fragments are sizemarkers added back to it. After a subsequent size fractionation, the presence of a PCR bandcorresponding in size to a genome segment of the pathogens is revealed by the high sensitivityMultiPhoton Detector (MPD) used to quantify PG and EC isotopes. MPD allows simultaneous butindividual 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 thepathogens sequence. An oligonucleotide probing with specific for the source Tag then identifies thedonor/patient carrying the pathogen. The constituent steps have all been performed in other R&D. Thetwo most critical features to be validated in this Phase I with non-hazardous biologicals are the degreeof any artifact limitations during the PCR applications and dynamic range limitations during the readoutof the fractionated DNAs.