Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase

A sensitive, amplified assay for HIV-1 pol region RNA was developed using RNA probes which are replicated by the RNA-dependent RNA polymerase, Q beta replicase. A synthetic target RNA was hybridized in cell lysates prepared with guanidine thiocyanate with an RNA reporter probe and four deoxyoligonucleotide "capture" probes. The RNA reporter probe was a recombinant MDV RNA molecule generated by transcription from a cloned cDNA template. Capture probes are synthetic oligonucleotides that are complementary to the target nucleic acid and that bear 3' poly d(A) tails. The ternary hybrids (of target RNA with capture probe and reporter probe) were captured on oligo d(T)-derivatized paramagnetic particles by hybridization with the d(A) tails of the capture probes. Non-hybridized reporter probes were removed by washing and successively eluting and recapturing the ternary hybrids on fresh particles. After three cycles of elution and capture, the hybrids were eluted in a low ionic strength buffer and the MDV RNA reporter probes were amplified directly by Q beta replicase. Amplified product RNA was detected by fluorescence using propidium iodide. The assay detects one femtogram (600 molecules) of a synthetic target RNA containing the pol region of HIV-1. The complete assay takes about 2.5 hours.