An improved method allowing for rapid sensitive and standardized detection
of a target nucleic acid from a pathogenic microorganism or virus or
normal or abnormal gene in a sample is provided. The method involves
hybridizing a target nucleic acid to several non-overlapping
oligonucleotide probes that hybridize to adjacent regions in the target
nucleic acid, the probes being referred to capture/amplification probes
and amplification probes, respectively, in the presence of paramagnetic
beads coated with a ligand binding moiety. Through the binding of a ligand
attached to one end of the capture/amplification probe and the specific
hybridization of portions of the probes to adjacent sequences in the
target nucleic acid, a complex comprising the target nucleic acid, the
probes and the paramagnetic beads is formed. The probes may then ligated
together to form a contiguous ligated amplification sequence bound to the
beads, which complex may be denatured to remove the target nucleic acid
and unligated probes. Alternatively, separate capture and amplification
probes may be used which form continuous full-length or circular probes,
and may be directly detected or amplified using a suitable amplification
technique, e.g., PCR, RAM or HSAM for detection. The detection of the
ligated amplification sequence, either directly or following amplification
of the ligated amplification sequence, indicates the presence of the
target nucleic acid in a sample. Methods for the detection of the ligated
amplification sequence, including hybridization signal amplification
method and ramification-extension amplification method, are also provided.
