Abstract
Amplifiable hybridization probes-molecules with a probe sequence embedded within the sequence of a replicatable RNA-wiIl promote the development of sensitive clinical assays. To demonstrate their utility, we prepared a recombi-nantRNA that containeda 30-nucleotide-longprobecomple-mentary to a conserved region of the pol gene in human immunodeficiency virus type 1 (HIV-1) mRNA. Test samples were prepared, each containing a different number of HIV-1 transcripts that served as simulated HIV-1 mRNA targets. Hybridizationswere camed out in a solutioncontainingthe chaotropicsalt, guanidinethiocyanate.Probe-target hybrids were isolated by reversibletarget capture on paramagnetic particles.The probes were then released from their targets and amplified by incubation with the RNA-directed RNA polymerase,Q/3 replicase (EC 2.7.7.48). The replicase copied the probesin an exponentialmanner:after each roundof copying,the numberof RNA moleculesdoubled.The amount of RNA synthesizedin each reaction(-50 ng) was sufficient to measure without using radioisotopes.Kinetic analysis of the reactionsdemonstratedthat the numberof HIV-1 targets originallypresent in each sample could be determined by measuringthe time it took to synthesizea particularamount of RNA (the longerthe synthesistook,the fewer the number of targetsoriginallypresent).The resultssuggestthat clinical assays involving replicatable hybridizationprobes will be simple,accurate, sensitive,and automatable.