Detection of nucleic acids by cycling probe technology on magnetic particles: High sensitivity and ease of separation

Cycling Probe Technology (CPT) is a signal amplification system that allows detection of nucleic acid target sequences without target amplification. C PT employs a sequence specific chimeric probe, typically DNA-RNA-DNA, which hybridizes to a complementary target DNA sequence and becomes a substrate for RNase H. Cleavage occurs at the RNA internucleotide linkages and result s in dissociation of the probe from the target, thereby making it available for the next probe molecule. This communication describes the use of oligo nucleotides attached to solid supports for target capture and release follo wed by solution and solid phase cycling. Through the attachment of chimeric probes to Sera-Mag(TM) magnetic particles (SMP) a simple and effective met hod of separating the cleaved probe from non-cycled probe has been develope d. By capturing the target DNA on particles and separating it from the extr aneous non-specific DNA we are able to dramatically reduce background and t hus discriminate between samples of Methicillin Resistant (MRSA) and Methic illin Sensitive (MSSA) Staphylococcus Aureus. We conjugated oligonucleotide probes to SMPs (similar to 1 um) and Nylon beads (NB) which were coated wi th ID Biomedical's proprietary coating materials (R, patent pending). The g eneral structure of the constructs is shown below: [GRAPHICS]